Use of prime editing in correcting mutations in cftr

Abstract

The present disclosure provides methods of editing CFTR using a prime editor (e.g., for correcting an F508del mutation in a CFTR protein) and a pegRNA. Such methods may be useful for treating cystic fibrosis. The present disclosure also provides pegRNAs, ngRNAs, dsgRNAs, systems, and compositions for editing CFTR and treating cystic fibrosis. Polynucleotides, vectors, cells, and kits for editing CFTR and treating cystic fibrosis are also provided herein.

Description

USE OF PRIME EDITING IN CORRECTING MUTATIONS IN CFTR RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application, U.S.S.N.63 / 619,412, filed January 10, 2024, and U.S. Provisional Application, U.S.S.N.63 / 669,163, filed July 9, 2024, each of which is incorporated herein by reference . GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers P01HL152960, RM1HG009490, R35GM118062, K99HL163805, and U24 HG010423, awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] Prime editing (PE) enables the replacement of targeted DNA nucleotides with any specified replacement of up to hundreds of nucleotides, thereby enabling a wide variety of substitutions, insertions, and deletions in the genomes of living systems1–4. The mechanism of prime editing is inherently resistant both to bystander editing (unwanted editing outcomes at the target site)1and to off-target editing1,3,5–17. In contrast with nuclease-mediated gene editing, prime editors do not require the creation of double-stranded DNA breaks (DSBs), minimizing undesirable outcomes such as uncontrolled insertions and deletions (indels)2–4,18, large deletions19,20, p53 activation21–23, retrotransposon insertion24, and chromosomal defects19,25–28. PE does not require co-delivery of donor DNA template, is active in mitotic and non-mitotic cells1,5,29–34, and has been successfully performed in vivo in mice5,29,32–38and in non-human primates39.

[0004] Prime editors combine a programmable nickase such as Streptococcus pyogenes Cas9 (SpCas9) H840A nickase with a reverse transcriptase (RT) such as an engineered Moloney murine leukemia virus RT1. A PE guide RNA (pegRNA) guides the prime editor protein to its spacer-specified genomic target and also contains a 3′ extension with a primer binding site (PBS) complementary to the nicked target DNA and an RT template (RTT) encoding the desired edited sequence1,40. When bound to its programmed target sequence, the prime editor•pegRNA complex nicks the target site to create an accessible 3′ end of ssDNA. This target DNA 3′ end hybridizes to the pegRNA's 3′ primer binding site (PBS), creating a 1 / 370 B1195.70196WO00 13363760.2primer•template complex that initiates reverse transcription using the RTT to create a 3′ DNA flap containing the edited DNA sequence1. The 3′ flap of edited DNA can displace the original sequence and be ligated into the genome, creating a DNA heteroduplex of edited and unedited DNA strands. This editing intermediate is then resolved into a permanent edit on both strands by DNA repair or replication41. A nickase, reverse transcriptase, and pegRNA constitute a ‘PE2’ editing system. A ‘PE3’ system adds an additional nicking guide RNA (ngRNA) that nicks the unedited strand of the DNA heteroduplex intermediate to enhance editing efficiency by directing mismatch repair to remake the unedited strand using the edited strand as a template1(FIG.1A). Additional prime editing strategies to correct pathogenic mutations and treat diseases are needed. SUMMARY OF THE INVENTION

[0005] Prime editing strategies can be developed to correct pathogenic mutations, such as the 3-base pair CTT deletion (F508del) in the CFTR (cystic fibrosis transmembrane conductance regulator) gene that results in the loss of phenylalanine 508 in the CFTR protein. This deletion is the most common cause of cystic fibrosis (CF)42, an autosomal recessive disorder that affects more than 160,000 people worldwide43. In people with CF, CFTR mutations impair the anion channel activity of CFTR that conducts Cl−and HCO3−transport across the apical membranes of epithelia lined secretory organs including the pancreas, gastrointestinal tract, and respiratory tract44–48. While over 2,000 CFTR variants have been identified, and more than 700 are verified to cause CF, one mutation, CFTR F508del, is present in 85% of CF patients42,49. The CFTR F508del protein misfolds, and the majority of the protein undergoes proteasomal degradation47,50–52. If trafficked to the cell membrane, the CFTR F508del channel is functional, albeit with a reduced open probability (Po)53. These molecular defects have been the target of several breakthrough small-molecule therapies that have greatly enhanced clinical outcomes for CF patients54–58. While effective and impactful, current small-molecule therapies require daily administration for life at an annual cost of approximately $300,00043,59,60. The development of a prime editing strategy to precisely correct the CFTR F508del CTT deletion could offer a path to a durable, one-time treatment for the most common CF-causing mutation.

[0006] The present disclosure describes the development of a PE approach to efficiently correct the CFTR F508del mutation (e.g., in primary CF patient airway epithelial cells) and rescue CFTR-dependent anion channel activity. Initial attempts to correct this mutation with 2 / 370 B1195.70196WO00 13363760.2the originally reported PE2 and PE3 systems yielded minimal editing in all cell types, revealing that this mutation was an unusually challenging one to correct by prime editing. By systematically optimizing prime editing guide RNAs (pegRNAs) and applying several recent advances in PE technology, F508del correction efficiencies of up to 51% in an immortalized human bronchial epithelial cell model and 25% in primary CF patient airway epithelial cells were achieved. Optimized PE restored CFTR channel function to greater than 50% of wild type levels in primary CF patient airway epithelial cells.

[0007] In one aspect, the present disclosure provides prime editing guide RNAs (pegRNAs) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence: TCTGTATCTATATTCATCAT (SEQ ID NO: 1), ACCATTAAAGAAAATATCAT (SEQ ID NO: 2), ATTATGCCTGGCACCATTAA (SEQ ID NO: 3), CTGTATCTATATTCATCATA (SEQ ID NO: 4), TTCATCATAGGAAACACCAA (SEQ ID NO: 5), CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), or CTGTATCTATATTCATCAT (SEQ ID NO: 8). In some embodiments, the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence of GTCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTAAAGA AAATATCATCTTTGGTGTTTCCTATGATGAATATAGATACGCGGTTCTATCTAGTTAC GCGTTAAACCAACTAGAA (SEQ ID NO: 250), TCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC TAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTAAAG AAAATATCATCTTTGGTGTGAGTTACGATGAATATAGATACGCGGTTCTATCTAG TTACGCGTTAAACCAACTAGAA (SEQ ID NO: 744), or CTGTATCTATATTCATCATGTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGCCTGGCACCATTAAA GAAAATATCATCTTTGGTGTGTCATACGATGAATATAGATACGCGGTTCTATCTAGTT ACGCGTTAAACCAACTAGAA (SEQ ID NO: 251).

[0008] In another aspect, the present disclosure provides nicking guide RNAs (ngRNAs) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 3 / 370 B1195.70196WO00 13363760.295%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence TTCACTTCTAATGGTGATTA (SEQ ID NO: 745), TCACTTCTAATGGTGATTAT (SEQ ID NO: 746), AATGGTGATTATGGGAGAAC (SEQ ID NO: 747), GGGAGAACTGGAGCCTTCAG (SEQ ID NO: 748), GGAGAACTGGAGCCTTCAGA (SEQ ID NO: 749), GAGGGTAAAATTAAGCACAG (SEQ ID NO: 750), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), ATTAAAGAAAATATCATCTT (SEQ ID NO: 751), CAAAGCATGCCAACTAGAAG (SEQ ID NO: 752), or CACTACCCAAATTATATATT (SEQ ID NO: 753).

[0009] In another aspect, the present disclosure provides dead single guide RNAs (dsgRNAs) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence CTCCCTCCAAGTTGTCCACG (SEQ ID NO: 764), CTGGAGCCTTCAGA (SEQ ID NO: 765), ATTTTACCCTCTGA (SEQ ID NO: 766), GCCAGGCATAATCC (SEQ ID NO: 767), TCCTGGATTATGCC (SEQ ID NO: 768), TCTTTAATGGTGCC (SEQ ID NO: 769), TTACCTCTTCTAGT (SEQ ID NO: 770), ATGCCAACTAGAAG (SEQ ID NO: 771), GCCAAATATATAATT (SEQ ID NO: 772), or CCGCGCGCGCGAAAAAGCCG (SEQ ID NO: 773).

[0010] In another aspect, the present disclosure provides methods of prime editing a CFTR gene comprising contacting a nucleic acid sequence encoding the CFTR gene with a prime editor and any of the pegRNAs disclosed herein. In some embodiments, the prime editor is a PE6 prime editor. In certain embodiments, the prime editor is a PE6c prime editor. In some embodiments, the method further comprises providing any of the ngRNAs disclosed herein. In some embodiments, the method further comprises providing any of the dsgRNAs disclosed herein. In some embodiments, the step of contacting corrects an F508del mutation in the CFTR protein. In some embodiments, the step of contacting results in the insertion of the sequence 5′-CTT-3′ into the CFTR gene.

[0011] In another aspect, the present disclosure provides compositions comprising any of the pegRNAs, ngRNAs, and / or dsgRNAs described herein. In some embodiments, the composition further comprises a prime editor.

[0012] In another aspect, the present disclosure provides systems comprising any of the pegRNAs, ngRNAs, and / or dsgRNAs described herein. In some embodiments, the system further comprises a prime editor. 4 / 370 B1195.70196WO00 13363760.2

[0013] In another aspect, the present disclosure provides polynucleotides encoding any of the pegRNAs, ngRNAs, and / or dsgRNAs described herein.

[0014] In another aspect, the present disclosure provides vectors encoding any of the pegRNAs, ngRNAs, and / or dsgRNAs described herein.

[0015] In another aspect, the present disclosure provides cells comprising any of the pegRNAs, ngRNAs, dsgRNAs, or compositions described herein.

[0016] In another aspect, the present disclosure provides kits comprising any of the pegRNAs, ngRNAs, dsgRNAs, or compositions described herein.

[0017] In another aspect, the present disclosure provides pharmaceutical compositions comprising any of the pegRNAs, ngRNAs, dsgRNAs, or compositions described herein.

[0018] In another, the present disclosure provides for the use of any of the pegRNAs, ngRNAs, dsgRNAs, compositions, polynucleotides, vectors, pharmaceutical compositions, and / or cells disclosed herein in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cystic fibrosis).

[0019] In another aspect, any of the pegRNAs, ngRNAs, dsgRNAs, compositions, polynucleotides, vectors, pharmaceutical compositions, and / or cells disclosed herein are for use in medicine.

[0020] The foregoing concepts, and additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0022] FIGs.1A-1E show that PE2 and PE3 systems correct CFTR F508del. FIG.1A shows a schematic of PE2 and PE3 prime editing systems. Factors that influence PE2 and PE3 editing efficiency include (1) pegRNA spacer sequence, (2) pegRNA primer binding site (PBS) length, (3) pegRNA reverse transcriptase template (RTT) length, and (4) nicking sgRNA (ngRNA) spacer sequence. FIG.1B shows the quantification of PE2 correction of 5 / 370 B1195.70196WO00 13363760.2CFTR F508del in HEK293T cells using NGG2 pegRNAs with different combinations of PBS and RTT lengths. FIGs.1C-1D show PE3 correction of CFTR F508del in HEK293T cells using the NGG2 PBS13 RTT29 pegRNA (FIG.1C) or NGG2 PBS14 RTT41 pegRNA (FIG. 1D) in combination with several ngRNAs. X-axis labels identify different ngRNAs by their nicking position relative to the pegRNA nick (in base pairs). The PE2 x-axis label specifies an editing condition with no ngRNA. FIG.1E shows adenine base editing in HEK293T cells at adenines in NGG1 and NGG2 protospacers. Adenines (A) are numbered 5′ to 3′ starting from the PAM-distal end of the protospacers. For FIGs.1B-1E, data and error bars represent mean and standard deviation, respectively, of three independent biological replicates (shown as black dots).

[0023] FIGs.2A-2E show that prime editing enhancements synergistically enhance correction of CFTR F508del. FIG.2A shows a schematic of the prime editing system with enhancements that improve F508del correction. Enhancements include (5) epegRNA 3′ structured RNA motifs, (6) co-expression of MLH1dn, (7) translationally silent edits, and (8) engineered and evolved prime editors (PEmax and PE6). FIG.2B shows a heatmap of F508del correction in HEK293T cells using NGG2 epegRNAs with variable combinations of PBS and RTT lengths, in nucleotides. Edits completed with PE4max. FIG.2C shows silent edit installation strategies (SE0-SE4). Correction of the F508del CTT deletion alone shown as SE0. Sequences shown correspond to SEQ ID NOs: 802 (Pathogenic), 803 (SE0), 804 (SE1), 805 (SE2), 806 (SE3), 807 (SE4), and 808 (Translation). FIG.2D shows PE5max correction of F508del in HEK293T cells with the NGG2 PBS13 RTT41 epegRNA encoded with SE0-SE4. FIG.2E shows a comparison of F508del correction with PEmax and PE6 variants a-g in HEK293T cells. All conditions use the NGG2 PBS13 RTT41 SE2 epegRNA, MLH1dn, and the +104 ngRNA. For FIGs.2B, 2D, and 2E, data and error bars represent mean and standard deviation, respectively, of three independent biological replicates (shown as black dots).

[0024] FIGs.3A-3E show that enhanced prime editing systems enable F508del correction in human immortalized airway epithelial cells. FIG.3A shows adenine base editing (ABE) of 16HBEge-F508del cells at NGG2 guided by a sgRNA, pegRNA, or epegRNA. Adenines (A) are numbered 5′ to 3′ starting from the PAM-distal end of the protospacers. FIG.3B shows a schematic of the prime editing system with dsgRNA (9) added to modulate the chromatin state of a target locus. FIG.3C shows a comparison of F508del PE correction in 16HBEge- F508del cells with NGG2-proximal dsgRNAs. dsgRNA position indicates the distance (in 6 / 370 B1195.70196WO00 13363760.2nucleotides) between the nicking site of NGG2 and the putative nicking site of the dsgRNA. Strand indicates the genomic DNA strand to which the dsgRNA binds (NGG2 targets the (–) strand). All conditions use PE6c, the NGG2 PBS13 RTT41 SE2 epegRNA, MLH1dn, and the +104 ngRNA. FIG.3D shows combinatorial improvements in F508del correction with epegRNAs, silent edits, PE6c, and dsgRNA. For epegRNAs, (–) denotes the use of a pegRNA and (+) denotes the use of an epegRNA; for silent edits, (–) denotes the use of SE0 and (+) denotes the use of SE2; for PE6c, (–) denotes the use of PEmax and (+) denotes the use of PE6c; for dsgRNA, (–) denotes the use of a non-targeting dsgRNA and (+) denotes the use of the -40 dsgRNA (see FIG.3C). All conditions use MLH1dn co-expression and the +104 ngRNA. FIG.3E shows the effect of MLH1dn co-expression on F508del correction efficiency and edit-to-indel ratio. All conditions use PE6c, the NGG2 PBS13 RTT41 SE2 epegRNA, the -40 dsgRNA, and the +104 ngRNA. For (FIGs.3A, 3C, 3D, and 3E data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as black dots).

[0025] FIGs.4A-4D show that prime editing corrects CFTR F508del in primary CF patient airway epithelial cells and rescues ion channel function. FIG.4A shows the quantification of F508del correction in CFTR F508del homozygous primary CF patient airway epithelial cells using HTS. Data and error bars represent mean and standard deviation, respectively, and were collected from three independent donors (shown as black dots). FIG.4B shows representative short circuit current (Isc) recordings with the transepithelial voltage held at 0 mV. Three weeks after electroporation of PE reagents, the transepithelial Isc of fully differentiated airway epithelial cells was quantified in response to forskolin and 3-isobutyl-1- methylxanthine (F&I) and CFTR(inh)-172 treatment. All the cells were pre-incubated with 10 µM forskolin and 100 µM IBMX for 24 hours before recording Cl–transport. Non-CF cultures and donor-matched F508 / F508 cultures with ETI pretreatment served as positive controls. FIG.4C shows a summary of the change in short circuit current (△Isc) in response to F&I and CFTR(inh)-172 treatment. Data and error bars represent mean and standard error, respectively, and were collected from three independent donors (shown as black dots). P- values, determined by two-way ANOVA, are shown. FIG.4D shows the ratio of editing in treated versus untreated primary airway epithelial cells at off-target loci nominated by CIRCLE-seq. Indels and substitutions (Sub) are shown for sites cleaved in vitro by the epegRNA, ngRNA, and dsgRNA used in the prime editing strategy. For the epegRNA and dsgRNA, the top 31 CIRCLE-seq nominated off-target sites are shown as one of the top 32 7 / 370 B1195.70196WO00 13363760.2identified sites was CFTR F508del. For the ngRNA, the top 32 CIRCLE-seq nominated off- target sites are shown. All data points are the ratios of the mean of three replicates for treated and untreated samples. The thick horizontal line indicates a ratio of editing in treated versus untreated cells of 1.5. Characteristics of off-target sites for which this ratio exceeds 1.5 are shown in the table to the right. Observed editing frequency is the mean of three independently edited primary airway epithelial cell lines. For FIGs.4B-4C, ETI=elexacaftor+tezacaftor+ivacaftor.

[0026] FIG.5 shows optimization of PE2 strategy to install the CFTR F508del mutation in HEK293T cells. To identify a PE2 strategy to install the CFTR F508del CTT deletion into HEK293T cells (bottom sequence schematic), a panel of 18 pegRNAs with a PBS length of 14 nt and variable RTT lengths was transfected into HEK293T cells (top data plot). One of the most efficient pegRNA designs (PBS14 and RTT30) was selected to generate a monoclonal HEK293T cell line homozygous for the CFTR F508del mutation. RTT lengths are listed in nucleotides (nt). Data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as black dots). Sequences shown correspond to SEQ ID NO: 809 (top) and 810 (bottom).

[0027] FIG.6 shows CFTR F508del proximal protospacers NGG1 and NGG2. A schematic of the targeted NGG1 and NGG2 protospacers around the CFTR F508del CTT deletion is provided. The non-PAM containing strand of NGG1 contains a TTTT sequence that may act as an RNA Polymerase III transcriptional terminator that prevents complete pegRNA PBS transcription from a U6 promoter during pegRNA reverse transcription. Sequences shown correspond to SEQ ID NO: 811 (top) and 812 (bottom).

[0028] FIG.7 shows low PE2 correction of CFTR F508del with NGG1 pegRNAs. A panel of 24 pegRNAs with variable PBS and RTT lengths was transfected into CFTR F508del HEK293T cells. PBS and RTT lengths are listed in nucleotides. Data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as black dots).

[0029] FIGs.8A-8H show PE4max correction of CFTR F508del in HEK293T cells with epegRNAs at several targets with NGA and NGG PAMs. FIG.8A shows a schematic of the targeted NGA1-3 and NGG1-4 protospacers around the CFTR F508del CTT deletion. Sequences shown correspond to SEQ ID NO: 811 (top) and 812 (bottom). FIGs.8B-8H show PE4max CFTR F508del correction using epegRNAs with variable PBS lengths and variable RTT lengths targeted to the protospacers NGA1 (FIG.8B), NGA2 (FIG.8C), 8 / 370 B1195.70196WO00 13363760.2NGA3 (FIG.8D), NGG1 (FIG.8E), NGG3 (FIG.8F), NGG4 (FIG.8G), NGG2 (FIG.8H). PBS and RTT lengths are listed in nucleotides. For FIGs.8B-8H, data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as black dots).

[0030] FIG.9 shows the correction of CFTR F508del in HEK293T cells with several ngRNAs using PE5max and an epegRNA. Using the epegRNA NGG2 PBS13 RTT41, a PE5max experiment was performed against a panel of ngRNAs to identify the most efficient strategy to correct CFTR F508del. X-axis labels identify different ngRNAs by their nicking position relative to the epegRNA nick (in base pairs). The PE4max x-axis label specifies an editing condition with no ngRNA. Data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as black dots).

[0031] FIG.10 shows that silent edits co-installed with the F508del corrective CTT insertion disrupt NGG2’s PAM and recode several F508del mutation proximal codons. Schematics of the silent edits (shown as boxed base pairs) co-installed with the F508del corrective CTT insertion (shown as underlined base pairs) as part of silent edit strategies SE1-SE4. The undisrupted PAM of NGG2 is shown as double-underlined base pairs in the SE0 schematic. Sequences shown correspond to the SEQ ID NOs indicated in the figure.

[0032] FIGs.11A-11B show prime and base editing in 16HBEge-F508del cells via mRNA nucleofection. FIG.11A shows the effect of epegRNA 3′ modifications on prime editing efficiency at CFTR F508del. Both epegRNAs consist of NGG2 PBS13 RTT41 SE2, and both have three phosphorothioate (mS) modifications in the place of phosphodiester bonds on their 5′ end. One epegRNA incorporates 3 mS modifications on its 3′ end and the other incorporates 2 phosphonoacetate (mP) modifications on its 3′ end. FIG.11B shows base editing at CFTR F508del proximal protospacers. Deamination efficiencies for all target bases within the first 12 bases of the guide RNA protospacer sequence (indexed from the PAM- distal end of the protospacer) are shown. For FIGs.11A-11B, data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as black dots).

[0033] FIGs.12A-12B show scaffold integration in prime edited primary CF patient airway epithelial cells and 16HBEge-F508del cells. FIG.12A shows scaffold integration in prime edited primary CF patient airway epithelial cells. The frequency of HTS reads containing CTT insertion and a defined number of scaffold-incorporated bases, indexed from the 3′ end of the pegRNA scaffold, is shown. FIG.12B shows scaffold integration in prime edited 9 / 370 B1195.70196WO00 13363760.216HBEge-F508del cells. Scaffold integration frequencies are shown as described in FIG. 12A for both PEmax and PE6c edited cells. For both FIGs.12A-12B, the epegRNA used consists of NGG2 PBS13 RTT41 SE2 with 2x 3′ phosphonoacetate modifications.

[0034] FIGs.13A-13B show partial incorporation of silent edits in prime edited primary CF patient airway epithelial cells. FIG.13A shows occurrences of all possible combinations of SE2 edit incorporation, both with and without concomitant CTT insertion, are shown. Primary CF patient airway epithelial cells were treated with PE6c. The epegRNA used consists of NGG2 PBS13 RTT41 SE2 with 2x 3′ phosphonoacetate modifications. FIG.13B shows a schematic showing the location of Cas9 PAM and protospacer edits within SE2. Sequences shown correspond to SEQ ID NOs: 824 (SE0) and 825 (SE2).

[0035] FIGs.14A-14C show analysis of off-target editing in primary CF patient airway epithelial cells. FIG.14A shows indel and substitution quantification at the top 32 human genomic sites identified by CIRCLE-seq for epegRNA NGG2 PBS13 RTT41 SE2. FIG.14B shows indel and substitution quantification at the top 32 human genomic sites identified by CIRCLE-seq for ngRNA +104. FIG.14C shows indel and substitution quantification at the top 32 human genomic sites identified by CIRCLE-seq for dsgRNA –40.

[0036] FIGs.15A-15D show pegRNA, ngRNA, and petRNA optimization in HEK293T cells in the context of other prime editing enhancements. FIG.15A shows screening of epegRNA PBS and RTT lengths using SE2 silent edits, PE6c, MLH1dn, the +104 ngRNA, and the -40 dsgRNA. FIG.15B shows screening of ngRNA using PE6c, MLH1dn, epegRNA NGG2 PBS13 RTT41 SE2, and the -40 dsgRNA. FIG.15C shows screening of petRNA using SE2 silent edits, PE6c, MLH1dn, the +104 ngRNA, and the -40 dsgRNA. FIG.15D shows editing efficiencies using petRNAs at previously reported positive control target sites. For FIG.15C and FIG.15D, an nCas9 (based on PEmax) and an MCP-RT (using a PE6c-based reverse transcriptase) were used for petRNA editing, as previously described94. For FIGs.15A-15D, data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as black dots).

[0037] FIGs.16A-16D show CFTR F508del correction efficiency in HEK293T cells by epegRNAs designed using computational tools. CFTR F508del correction mediated by the top 24 ranked epegRNAs designed by (FIG.16A) DeepPrime without the co-installation of silent edits (SE0), (FIG.16B) PRIDICT without the co-installation of silent edits (SE0), (FIG.16C) DeepPrime with the co-installation of SE2 silent edits, and (FIG.16D) PRIDICT with the co-installation of SE2 silent edits. All conditions use PE6c, MLH1dn, the +104 10 / 370 B1195.70196WO00 13363760.2ngRNA, and the -40 dsgRNA is shown. For FIGs.16A-16D, data and error bars represent mean and standard deviation, respectively, collected from three independent biological replicates (shown as black dots). n.d.: No data.

[0038] FIG.17 shows that co-installation of translationally silent edits improves editing efficiency for CFTR.F508∆ correction. Examples of different silent edits introduced into serine codon sequence are shown. Sequences shown correspond (top-bottom) to SEQ ID NOs: 826, 827 (SE0), 828 (TCC), 829 (AGC), 830 (TCT), 831 (TCG), 832 (TCA), 833 (SE2), and 834 (amino acid).

[0039] FIG.18 shows CFTR.F508∆ screen A: Screen of 72 different silent edit strategies (here shown with all PBS13 / RTT41 and all 19 bp protospacer). Top-performing pegRNA (OP253) shows 1.56x-fold higher editing than SE2.

[0040] FIG.19 shows CFTR.F508∆ screen A: Improved silent edit strategies can recode codons both before and after the position of the therapeutic edit (F at position 508). Sequences shown correspond (top-bottom) to SEQ ID NOs: 826, 827 (SE0), 833 (SE2), 832 (OP253), 835 (OP356), 836 (OP286), 837 (OP356), 829 (OP198), 830 (OP282), 838 (OP116), 839 (OP272), and 834 (amino acid).

[0041] FIGs.20A-20B show that silent edits can improve editing efficiency in the context of different (e)pegRNA parameters like PBS, RTT, and protospacer length. FIG.20A shows that silent edits improve efficiency for epegRNAs with different PBS and RTT lengths. FIG. 20B shows that silent edits improve editing efficiency in the context of both 19bp and 20pb epegRNA protospacers.

[0042] FIG.21 shows CFTR.F508∆ screen B: Screen of 36 different silent edit strategies (some with varied PBS and RTT lengths, all 19 bp protospacer sequence). Sequences shown correspond (top-bottom) to SEQ ID NOs: 826, 827 (SE0), 833 (SE2), 832 (OPO253), 840 (OPR025), and 834 (amino acid).

[0043] FIG.22 shows CFTR.F508∆ screen C: Top silent edits from screens A and B with different pegRNA PBS / RTT lengths. Sequences shown correspond (top-bottom) to SEQ ID NOs: 826, 827 (SE0), 833 (SE2), 832 (OPO253), and 840 (OPR025). DEFINITIONS

[0044] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms 11 / 370 B1195.70196WO00 13363760.2used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise. Cas9

[0045] The term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 domain, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and / or the gRNA binding domain of Cas9). A “Cas9 domain,” as used herein, is a protein fragment comprising an active or fully or partly inactive cleavage domain of Cas9 and / or the gRNA binding domain of Cas9. A “Cas9 protein” is a full length Cas9 protein. A Cas9 nuclease is also referred to sometimes as a casn1 nuclease or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 domain. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves a linear or circular dsDNA target complementary to the spacer. The strand in the target DNA not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821(2012), the contents of which are incorporated herein by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., 12 / 370 B1195.70196WO00 13363760.2White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816- 821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease comprises one or more mutations that partially impair or inactivate the DNA cleavage domain.

[0046] A nuclease-inactivated Cas9 domain may interchangeably be referred to as a “dCas9” protein (for nuclease-“dead” Cas9). Methods for generating a Cas9 domain (or a fragment thereof) having an inactive DNA cleavage domain are known (see, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell.28;152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science.337:816-821(2012); Qi et al., Cell.28;152(5):1173-83 (2013)). In some embodiments, a Cas9 protein comprises one or more mutations to inactivate the nuclease activity of only one of the HNH subdomain or the RuvC1 subdomain.

[0047] In some embodiments, proteins comprising fragments of a Cas9 protein are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9, or fragments thereof, are referred to as “Cas9 13 / 370 B1195.70196WO00 13363760.2variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, at least about 99.8% identical, or at least about 99.9% identical to wild type Cas9 (e.g., SpCas9). In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid changes compared to wild type Cas9 (e.g., SpCas9). In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9 (e.g., SpCas9). In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9 (e.g., SpCas9). CFTR

[0048] The term “cystic fibrosis transmembrane conductance regulator” or “CFTR” refers to a membrane protein that acts as an ion channel, as well as the CFTR gene that encodes it. Mutations in CFTR, including the CTT deletion in the CFTR gene resulting in an F508del mutation in the CFTR protein as described herein, cause cystic fibrosis.

[0049] The sequence of the human CFTR protein is provided below: MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWD RELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEE RSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISI GQLVSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLA LFQAGLGRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQ TELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAV TRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFG ELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAG 14 / 370 B1195.70196WO00 13363760.2KTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKA CQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVL TEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPD FSSKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKR KNSILNPINSIRKFSIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGP TLQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQET GLEISEEINEEDLKECFFDDMESIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAA SLVVLWLLGNTPLQDKGNSTHSRNNSYAVIITSTSSYYVFYIYVGVADTLLAMGFFR GLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLLPLTIF DFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTSQQLKQLESEGRSPIF THLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQMRIEMIFVI FFIAVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFID MPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGN AILENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEGEIQIDGVSWDSITLQQWRK AFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVD GGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVIL CEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKC KSKPQIAALKEETEEEVQDTRL (SEQ ID NO: 790) Cystic Fibrosis

[0050] The term “cystic fibrosis” or “CF” refers to a genetic disorder that primarily affects the lungs. CF results in the accumulation of thick mucus in various organs, including the lungs, resulting in difficulty breathing and frequent lung infections. Cystic fibrosis is caused by mutations in the CFTR gene, including the CTT deletion resulting in an F508del mutation in the CFTR protein as described herein. Dead single guide RNA (dsgRNA)

[0051] The terms “dead guide RNA,” “dgRNA,” “dead single guide RNA,” and “dsgRNA” refer to 5′-truncated guide RNAs that enable Cas9 to bind to a target sequence but do not support DNA cleavage. DsgRNAs may have, for example, only 14-16 nucleotides of protospacer complementarity. DsgRNAs may be useful, e.g., for manipulating the accessibility of a target site to be edited by facilitating the unfolding of genomic DNA. DsgRNAs are further described in Kiani, S. et al., Cas9 gRNA engineering for genome editing, activation and repression. Nat. Methods 12, 1051–1054 (2015), which is incorporated herein by reference. 15 / 370 B1195.70196WO00 13363760.2Fusion protein

[0052] The term “fusion protein” as used herein refers to a hybrid polypeptide that comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C- terminal) protein, thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. A protein may comprise different domains, for example, a Cas9 protein fused to a polymerase such as a reverse transcriptase (i.e., a prime editor). Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which is incorporated herein by reference. Guide RNA (“gRNA”)

[0053] As used herein, the term “guide RNA” is a particular type of guide nucleic acid which is commonly associated with a Cas protein (e.g., a Cas9 protein), directing the Cas protein to a specific sequence in a DNA molecule that includes complementarity to the protospacer sequence of the guide RNA. For example, a gRNA may direct a Cas protein (e.g., as part of a prime editor) to a target site in the CFTR gene. However, this term also embraces the equivalent guide nucleic acid molecules that associate with Cas protein equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and which otherwise program the Cas protein equivalent to localize to a specific target nucleotide sequence. The Cas protein equivalents may include other napDNAbps from any type of CRISPR system (e.g., type II, V, VI), including Cpf1 (a type-V CRISPR-Cas system), C2c1 (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system), and C2c3 (a type V CRISPR-Cas system). Further Cas-equivalents are described in Makarova et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299), which is incorporated herein by reference. Exemplary sequences and structures of guide RNAs are provided herein.

[0054] Functionally, guide RNAs associate with a Cas protein, directing (or programming) the Cas protein to a specific sequence in a DNA molecule that includes a sequence complementary to the protospacer sequence for the guide RNA. A gRNA is a component of 16 / 370 B1195.70196WO00 13363760.2the CRISPR / Cas system. The sequence specificity of a Cas DNA-binding protein is determined by gRNAs, which have nucleotide base-pairing complementarity to target DNA sequences. The native gRNA comprises a 20 nucleotide (nt) Specificity Determining Sequence (SDS), or spacer, which specifies the DNA sequence to be targeted, and is immediately followed by an 80 nt scaffold sequence, which associates the gRNA with the Cas protein. In some embodiments, an SDS of the present disclosure has a length of 15 to 100 nucleotides, or more. For example, an SDS may have a length of 15 to 90, 15 to 85, 15 to 80, 15 to 75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, or 15 to 20 nucleotides. In some embodiments, the SDS is 20 nucleotides long. For example, the SDS may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. At least a portion of the target DNA sequence is complementary to the SDS of the gRNA. For a Cas protein to successfully bind to the DNA target sequence, a region of the target sequence is complementary to the SDS of the gRNA sequence and is immediately followed by the correct protospacer adjacent motif (PAM) sequence. In some embodiments, an SDS is 100% complementary to its target sequence. In some embodiments, the SDS sequence is less than 100% complementary to its target sequence and is, thus, considered to be partially complementary to its target sequence. For example, a targeting sequence may be 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% complementary to its target sequence. In some embodiments, the SDS of template DNA or target DNA may differ from a complementary region of a gRNA by 1, 2, 3, 4, or 5 nucleotides.

[0055] In some embodiments, the guide RNA is about 15-120 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence (e.g., a target sequence in CFTR). In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 nucleotides long. In some embodiments, the guide RNA comprises a sequence of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides that is complementary to a target sequence. Sequence complementarity refers to distinct interactions between adenine and thymine (DNA) or uracil (RNA), and between guanine and cytosine. 17 / 370 B1195.70196WO00 13363760.2Linker

[0056] The term “linker,” as used herein, refers to a molecule linking two other molecules or moieties. The linker can be an amino acid sequence in the case of a linker joining two components of a fusion protein. For example, a napDNAbp (e.g., a Cas9 protein) can be fused to a polymerase (e.g., a reverse transcriptase) by an amino acid linker sequence. The linker can also be a nucleotide sequence in the case of joining two nucleotide sequences together (e.g., in a gRNA). In other embodiments, the linker is a non-peptidic linker. In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-200 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45- 50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. MLH1dn

[0057] “MLH1dn” refers to a dominant negative variant of the DNA-mismatch repair (MMR) enzyme MLH1. MLH1dn is an inhibitor of the DNA mismatch repair pathway and can help increase the efficiency of prime editing.

[0058] In some embodiments, the dominant negative MLH1 can include, for example, MLH1 Δ754-Δ756, which has the following amino acid sequence:

[0059] MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEG GLKLIQIQDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVT ITTKTADGKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNPSE EYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELI EIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKN THPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQ TLLPGLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKP LSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEMSE KRGPTSSNPRKRHREDSDVEMVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINEQGH EVLREMLHNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLR LSEPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEE GNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISE ESTLSGQQSEVPGSIPNSWKWTVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYK VF[- - -] (SEQ ID NO: 791), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at 18 / 370 B1195.70196WO00 13363760.2least 99%, or up to and including 100% sequence identity thereto (wherein the [- - -] indicates deleted amino acid residue(s) relative to the parent or wildtype MLH1 sequence). napDNAbp

[0060] As used herein, the term “nucleic acid programmable DNA binding protein” or “napDNAbp,” of which Cas proteins such as Cas9 and variants thereof are examples, refers to a protein that uses RNA:DNA hybridization to target and bind to specific sequences in a DNA molecule. Each napDNAbp is associated with at least one guide nucleic acid (e.g., guide RNA), which localizes the napDNAbp to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid, or a portion thereof (e.g., the protospacer of a guide RNA). In other words, the guide nucleic-acid “programs” the napDNAbp (e.g., Cas9, or a variant thereof) to localize and bind to a complementary sequence.

[0061] Without being bound by theory, the binding mechanism of a napDNAbp–guide RNA complex, in general, includes the step of forming an R-loop whereby the napDNAbp induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the napDNAbp. The guide RNA protospacer then hybridizes to the “target strand.” This displaces a “non-target strand” that is complementary to the target strand, which forms the single strand region of the R-loop. In some embodiments, the napDNAbp includes one or more nuclease activities, which then cut the DNA, leaving various types of lesions. For example, the napDNAbp may comprise a nuclease activity that cuts the non-target strand at a first location, and / or cuts the target strand at a second location. Depending on the nuclease activity, the target DNA can be cut to form a “double-stranded break” whereby both strands are cut. In other embodiments, the target DNA can be cut at only a single site, i.e., the DNA is “nicked” on one strand. Nickase

[0062] As used herein, a “nickase” refers to a napDNAbp (e.g., a Cas9 protein) that is capable of cleaving only one of the two complementary strands of a double-stranded target DNA sequence, thereby generating a nick in that strand. In some embodiments, the nickase cleaves a non-target strand of a double stranded target DNA sequence. In some embodiments, the nickase comprises an amino acid sequence with one or more mutations in a catalytic domain of a canonical napDNAbp (e.g., a Cas9 protein), wherein the one or more mutations reduces or abolishes nuclease activity of the catalytic domain. In some embodiments, the nickase is a Cas9 that comprises one or more mutations in a RuvC-like domain relative to a 19 / 370 B1195.70196WO00 13363760.2wild type Cas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that comprises one or more mutations in an HNH-like domain relative to a wild type Cas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that comprises an aspartate-to-alanine substitution (D10A) in the RuvC1 catalytic domain of Cas9 relative to a canonical SpCas9 sequence or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is a Cas9 that comprises an H840A, N854A, and / or N863A mutation relative to a canonical SpCas9 sequence, or to an equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the term “Cas9 nickase” refers to a Cas9 with one of the two nuclease domains inactivated. This enzyme is capable of cleaving only one strand of a target DNA. In some embodiments, the nickase is a Cas protein that is not a Cas9 nickase.

[0063] In some embodiments, the napDNAbp of a prime editor is a Cas9 nickase (nCas9) that nicks only a single strand. In other embodiments, the napDNAbp can be selected from the group consisting of: Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Cas12f1, Cas12j (CasΦ), and Argonaute and optionally has a nickase activity such that only one strand is cut. In some embodiments, the napDNAbp is selected from Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Cas12f1, Cas12j (CasΦ), and Argonaute and optionally has a nickase activity such that one DNA strand is cut preferentially to the other DNA strand. Nicking Guide RNA (ngRNA)

[0064] In some embodiments, a guide RNA is a “nicking guide RNA.” Nicking guide RNAs may be used to nick the non-edited strand of a target nucleic acid molecule, which may facilitate incorporation of the edit by cellular DNA repair mechanisms. Nuclear localization sequence (NLS)

[0065] The term “nuclear localization sequence” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., international PCT application, PCT / EP2000 / 011690, filed November 23, 2000, published as WO / 2001 / 038547 on May 31, 2001, the contents of which are incorporated herein by reference for its disclosure 20 / 370 B1195.70196WO00 13363760.2of exemplary nuclear localization sequences. In some embodiments, a prime editor comprises one or more NLS as described herein. Nucleic acid molecule

[0066] The term “nucleic acid,” as used herein, (also referred to as a “polynucleotide”) refers to a polymer of nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyl uridine, C5 propynyl cytidine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, O(6) methylguanine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1- methyl adenosine, 1-methyl guanosine, N6-methyl adenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, 2´-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5ʹ N phosphoramidite linkages). pegRNA

[0067] As used herein, the terms “prime editing guide RNA,” “PEgRNA,” “pegRNA,” or “extended guide RNA” refer to a specialized form of a guide RNA that has been modified to include one or more additional sequences for implementing the prime editing as described herein. As described herein, the prime editing guide RNAs comprise one or more “extended regions,” also referred to herein as “extension arms,” of nucleic acid sequence. The extended regions may comprise, but are not limited to, single-stranded RNA or DNA. Further, the extended regions may occur at the 3′ end of a traditional guide RNA. In other arrangements, the extended regions may occur at the 5′ end of a traditional guide RNA. In still other arrangements, the extended region may occur at an intramolecular region of the traditional guide RNA, for example, in the gRNA core region which associates and / or binds to the napDNAbp. The extended region comprises a “DNA synthesis template” or “reverse transcriptase template” that encodes (by the polymerase / reverse transcriptase of the prime editor) a single-stranded DNA which, in turn, has been designed to be (a) homologous with the endogenous target DNA to be edited, and (b) which comprises at least one desired nucleotide change (e.g., a transition, a transversion, a deletion, or an insertion) to be introduced or integrated into the endogenous target DNA. The extended region may also 21 / 370 B1195.70196WO00 13363760.2comprise other functional sequence elements, such as, but not limited to, a “primer binding site” and a “linker” sequence, or other structural elements, such as, but not limited to, aptamers, stem loops, hairpins, toe-loops (e.g., a 3′ toeloop), or an RNA-protein recruitment domain (e.g., MS2 hairpin). As used herein, the “primer binding site” comprises a sequence that hybridizes to a single-strand DNA sequence having a 3′ end generated from the nicked DNA of the R-loop.

[0068] In certain embodiments, the pegRNAs have a 3ʹ extension arm, a spacer, and a gRNA core. The 3ʹ extension arm further comprises in the 5ʹ to 3ʹ direction a DNA synthesis template, a primer binding site, and a linker. The DNA synthesis template may also be referred to more broadly as the “DNA synthesis template” where the polymerase of a prime editor described herein is not an RT, but another type of polymerase.

[0069] In certain other embodiments, the pegRNAs have a 5ʹ extension arm, a spacer, and a gRNA core. The 5ʹ extension further comprises in the 5ʹ to 3ʹ direction a DNA synthesis template, a primer binding site, and a linker. The DNA synthesis template may also be referred to more broadly as the “DNA synthesis template” where the polymerase of a prime editor described herein is not an RT, but another type of polymerase.

[0070] In still other embodiments, the pegRNAs have in the 5ʹ to 3ʹ direction a spacer, a gRNA core, and an extension arm. The extension arm is at the 3ʹ end of the pegRNA. The extension arm further comprises in the 5ʹ to 3ʹ direction a homology arm, an edit template, and a primer binding site. The extension arm may also comprise an optional modifier region at the 3ʹ and 5ʹ ends, which may be the same sequences or different sequences. In addition, the 3ʹ end of the pegRNA may comprise a transcriptional terminator sequence. These sequence elements of the pegRNAs are further described and defined herein.

[0071] In still other embodiments, the pegRNAs have in the 5ʹ to 3ʹ direction an extension arm, a spacer, and a gRNA core. The extension arm is at the 5ʹ end of the pegRNA. The extension arm further comprises in the 3ʹ to 5ʹ direction a primer binding site, an edit template, and a homology arm. The extension arm may also comprise an optional modifier region at the 3ʹ and 5ʹ ends, which may be the same sequences or different sequences. The pegRNAs may also comprise a transcriptional terminator sequence at the 3ʹ end. These sequence elements of the pegRNAs are further described and defined herein.

[0072] In some embodiments, the spacer sequence of the pegRNA is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length. In certain 22 / 370 B1195.70196WO00 13363760.2embodiments, the spacer sequence of the pegRNA is about 20 nucleotides in length. In some embodiments, the primer binding site is about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, or about 17 nucleotides in length. In certain embodiments, the primer binding site is about 9, about 10, about 11, about 12, about 13, about 14, or about 15 nucleotides in length. In some embodiments, the homology arm of the pegRNA is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length. In some embodiments, the DNA synthesis template is from about 14 to about 21 nucleotides in length.

[0073] In some embodiments, a pegRNA is an “engineered pegRNA” (“epegRNA”). Relative to a pegRNA, an epegRNA comprises an additional structured motif, for example, attached to its 3′ end. Such additional structured motifs may stabilize the pegRNA or otherwise prevent it from being degraded. Suitable structured motifs include, but are not limited to, toe-loops, hairpins, stem-loops, pseudoknots, aptamers, G-quadruplexes, tRNAs, riboswitches, and ribozymes. In some embodiments, a 3′ structured motif comprises evopreq1. In some embodiments, the 3′ structured motif in the pegRNAs disclosed herein comprises the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 248), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 248).

[0074] pegRNAs are further described, e.g., in International Patent Application No. PCT / US2020 / 023721, filed March 19, 2020, which published as WO 2020 / 191239; International Patent Application No. PCT / US2021 / 031439, filed May 7, 2021, which published as WO 2021 / 226558; International Patent Application No. PCT / 2021 / 052097, filed September 24, 2021, which published as WO 2022 / 067130; International Patent Application No. PCT / US2022 / 012054, filed January 11, 2022, which published as WO 2022 / 150790; International Patent Application No. PCT / US2022 / 078655, filed October 25, 2022, which published as WO 2023 / 076898; and International Patent Application No. PCT / US2022 / 074628, filed August 5, 2022, which published as WO 2023 / 015309; the contents of each of which is incorporated by reference herein.

[0075] In the pegRNAs described herein, and any other RNA sequences provided herein, T’s and U’s may be used interchangeably in the sequences. A person of ordinary skill in the art will understand that T’s in any provided pegRNA or other RNA sequence should be construed as U’s. 23 / 370 B1195.70196WO00 13363760.2PE1

[0076] As used herein, “PE1” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a wild type MMLV RT having the following structure: [NLS]- [Cas9(H840A)]-[linker]-[MMLV_RT(wt)] + a desired pegRNA, wherein the PE fusion has the following amino acid sequence: MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAK VDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFD LAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEI TKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYV TEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVE DRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNF MQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLV SDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD VRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIV WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW DPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDF LEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDAT LIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTL NIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQY PMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVED IHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLT WTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALL QTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREF LGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTK PFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAG KLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTET EVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSE GKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTS TLLIENSSPSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 792) KEY: NUCLEAR LOCALIZATION SEQUENCE (NLS) CAS9(H840A) 33-AMINO ACID LINKER 24 / 370 B1195.70196WO00 13363760.2M-MLV reverse transcriptase PE2

[0077] As used herein, “PE2” refers to a PE complex comprising a fusion protein comprising Cas9(H840A) and a variant MMLV RT having the following structure: [NLS]- [Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] + a desired pegRNA, wherein the PE fusion has the amino acid sequence of: MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAK VDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFD LAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEI TKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYV TEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVE DRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNF MQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLV SDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYD VRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIV WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW DPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDF LEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDAT LIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTL NIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQY PMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVED IHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLT WTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALL QTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREF LGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTK PFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAG KLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLL PLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTET EVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTS EGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDT STLLIENSSPSGGSKRTADGSEFEPKKKRKV (SEQ ID NO: 793) KEY: 25 / 370 B1195.70196WO00 13363760.2NUCLEAR LOCALIZATION SEQUENCE (NLS) CAS9(H840A) 33-AMINO ACID LINKER

[0078] M-MLV reverse transcriptase PE3

[0079] As used herein, “PE3” refers to a prime editing composition comprising a PE2 prime editor and further comprising a second-strand nicking guide RNA that complexes with PE2 and introduces a nick in the non-edit DNA strand in order to induce preferential replacement of the edit strand. PE3b

[0080] As used herein, “PE3b” refers to a prime editing composition comprising PE2 and further comprising a second-strand nicking guide RNA that complexes with PE2 and introduces a nick in the non-edit DNA strand, wherein the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing the second strand nicking guide RNA with a spacer sequence that comprises complementarity to, and only hybridizes with, the edited strand after installation of the desired nucleotide edit(s), but not the endogenous target DNA sequence. Using this strategy, mismatches between the nicking guide RNA spacer and the unedited target DNA should disfavor nicking by the sgRNA until after the editing event on the PAM strand takes place. PE4

[0081] As used herein, “PE4” refers to a prime editing composition comprising a PE2 and further comprising an MLH1 dominant negative protein variant (i.e., wild-type MLH1 with amino acids 754-756 truncated, which may be referred to herein as “MLH1 Δ754-756” or “MLH1dn”). The MLH1 dominant negative protein variant may be expressed in trans in some embodiments. In some embodiments, a PE4 system comprises a fusion protein comprising a PE2 protein and an MLH1 dominant negative protein joined via an optional linker. PE5 and PE5b

[0082] As used herein, “PE5” refers to a prime editing composition comprising a PE3 prime editor and further comprising an MLH1 dominant negative protein variant (i.e., wild-type MLH1 with amino acids 754-756 truncated, which may be referred to as “MLH1 Δ754-756” or “MLH1dn”). The MLH1 dominant negative variant may be expressed in trans in some 26 / 370 B1195.70196WO00 13363760.2embodiments. In some embodiments, a PE5 system comprises a fusion protein comprising a PE2 protein and an MLH1 dominant negative protein joined via an optional linker. “PE5b” refers to a prime editing composition comprising a PE3 and an MLH1 dominant negative protein, wherein the second-strand nicking guide RNA is designed for temporal control such that the second strand nick is not introduced until after the installation of the desired edit. This is achieved by designing the second strand nicking guide RNA with a spacer sequence that comprise complementarity to, and hybridize with, only the edited strand after installation of the desired nucleotide edit(s), but not the endogenous target DNA sequence. PE6

[0083] The term “PE6” refers to a suite of prime editors (PE6a, PE6b, PE6c, PE6d, PE6e, PE6f, and PE6g) comprising improved reverse transcriptase and / or Cas9 variants. The improved reverse transcriptase and Cas9 domains of the PE6 variants can also be combined with each other to offer cumulative benefits. For example, a PE6 prime editor comprising an improved reverse transcriptase variant of PE6a and an improved Cas9 variant of PE6e is referred to herein as the prime editor “PE6a-e” (or “PE6e-a”). Any possible combination of PE6 prime editors is contemplated by the present disclosure including, for example, PE6a-e, PE6a-f, PE6a-g, PE6b-e, PE6b-f, PE6b-g, PE6c-e, PE6c-f, PE6c-g, PE6d-e, PE6d-f, and PE6d-g.

[0084] Any of the PE6 prime editors may also comprise the architecture of the PEmax protein as provided herein. In some embodiments, any of the PE6 prime editors provided herein may further comprise additional amino acid mutations, e.g., any of those included in PEmax as provided herein.

[0085] In some embodiments, a PE6 protein comprises a reverse transcriptase of the following amino acid sequence (the RT domain of “PE6a”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence: GRPYVTLNLNGMFMDKFKPYSKSNAPITTLEKLSKALSISVEELKAIAELSLDEKYTL KKIPKIDGSKRIVYSLHPKMRLLQSRINERIFKELVVFPSFLFGSVPSKNDVLNSNVKR DYVSCAKAHCGAKTVLKVDISNFFDNIHRDLVRSVFEEILHIKDEALDYLVDICTKDD FVVQGALTSSYIATLCLFAVEGDVVRRAQRKGLVYTRLVDDITVSSKISNYDFSQMQ SHIERMLSEHNLPINKHKTKIFHCSSEPIKVHGLIVDYDSPRLPSDKVKRIRASIHNLKL LAAKNNTKTSVAYRKEFNRCMGRVNELGRVGHEKYESFKKQLQAIKPMPSNRDVA 27 / 370 B1195.70196WO00 13363760.2VIDAAIKSLELSYSKGNQNKHWYKRKYDLTRYKMIILTRSESFKEKLECFKSRLASLK PL (SEQ ID NO: 795)

[0086] In some embodiments, a PE6 protein comprises a reverse transcriptase of the following amino acid sequence (the RT domain of “PE6b”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence: ISSSKHTLSQMNKVSNIVKEPELPDIYKEFKDITADTNTEKLPKPIKGLEFEVELTQEN YRLPIRNYPLTPVKMQAMNDEINQGLKGGIIRESKAINACPVIFVPRKEGTLRMVVDY RPLNKYVKPNVYPLPLIEQLLAKIQGSTIFTKLDLKSAYHQIRVRKGDEHKLAFRCPR GVFEYLVMPYGISTAPAHFQYFINTILGEAKESHVVCYMDDILIHSKSESEHVKHVKD VLQKLKNANLIINQAKCEFHQSQVKFIGYHISEKGLTPCQENIDKVLQWKQPKNRKE LRQFLGSVNYLRKFIPKTSQLTHPLNKLLKKDVRWKWTPTQTQAIENIKQCLVSPPVL RHFDFSKKILLETDVSDVAVGAVLSQKHDDDKYYPVGYYSAKMSKAQLNYSVSDK EMLAIIKSLEHWRHYLESTIEPFKILTDHRNLIGRITNESEPENKRLARWQLFLQDFNF EINYRPGSANHIADALSRIVDETEPIPKDNEDNSINFVNQISI (SEQ ID NO: 796)

[0087] In some embodiments, a PE6 protein comprises a reverse transcriptase of the following amino acid sequence (the RT domain of “PE6c”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence: ISSSKHTLSQMNKVSNIVKEPELPDIYKEFKDITADTNTEKLPKPIKGLEFEVELTQEN YRLPIRNYPLTPVKMQAMNDEINQGLKGGIIRESKAINACPVIFVPRKEGTLRMVVDY RPLNKYVKPNVYPLPLIEQLLAKIQGSTIFTKLDLKSAYHQIRVRKGDEHKLAFRCPR GVFEYLVMPYGIKTAPAHFQYFINTILGEAKESHVVCYMDDILIHSKSESEHVKHVKD VLQKLKNANLIINQAKCEFHQSQVKFLGYHISEKGLTPCQENIDKVLQWKQPKNQKE LRQFLGQVNYLRKFIPKTSQLTHPLNKLLKKDVRWKWTPTQTQAIENIKQCLVSPPV LRHFDFSKKILLETDVSDVAVGAVLSQKHDDDKYYPVGYYSAKMSKAQLNYSVSD KEMLAIIKSLEHWRHYLESTIEPFKILTDHRNLIGRITNESEPENKRLARWQLFLQDFN FEINYRPGSANHIADALSRIVDETEPIPKDNEDNSINFVNQISI (SEQ ID NO: 797)

[0088] In some embodiments, a PE6 protein comprises a reverse transcriptase comprising the following amino acid sequence (the RT domain of “PE6d”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence: 28 / 370 B1195.70196WO00 13363760.2TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTP VSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLR EVNKRVEDIHPNVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWR DPEMGISGQLTWTRLPQGFKNSPTLFCEALHRDLADFRIQHPDLILLQYYDDLLLAAT SELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKE TVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKA YQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSN ARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLD (SEQ ID NO: 798)

[0089] In some embodiments, a PE6 protein comprises a Cas9 protein of the following amino acid sequence (the Cas9 domain of “PE6e”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHE RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFA WMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK VMGRHKPENIVIEMARENQTTQKGQRNSRERMKRIEEGIKELGSQILKEHPVENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIA RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV 29 / 370 B1195.70196WO00 13363760.2AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 799)

[0090] In some embodiments, a PE6 protein comprises a Cas9 protein of the following amino acid sequence (the Cas9 domain of “PE6f”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFRRLEESFLVEEDKKHE RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFA WMTRKSEKTITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMVEER LKTYAHLFDNKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMQLIHDDSLTFKEDIQKAQVSGQGDSLYEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQ LQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSD KNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFI ARQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYK VREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLV VAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG 30 / 370 B1195.70196WO00 13363760.2APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 800)

[0091] In some embodiments, a PE6 protein comprises a Cas9 protein of the following amino acid sequence (the Cas9 domain of “PE6g”), or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the following amino acid sequence: MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFRRLEESFLVEEDKKHE RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFA WMTRKSEKTITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMVEER LKTYAHLFDNKVMKQLKRCRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMQLIHDDSLTFKEDIQKAQVSGQGDSLYEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQ LQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSD KNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFI KRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYK VREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLV VAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 801) 31 / 370 B1195.70196WO00 13363760.2PE7

[0092] The term “PE7” refers to the PE6 prime editors plus a second strand nicking guide RNA. For example, “PE7a” refers to the PE6a prime editor as provided herein, plus a second strand nicking guide RNA. PEmax

[0093] As used herein, “PEmax” refers to a prime editing composition comprising 1) a fusion protein comprising a Cas9 protein variant Cas9(R221K N39K H840A) and a variant MMLV RT having the following structure: [bipartite NLS]-[Cas9(R221K)(N394K)(H840A)]- [linker]-[MMLV_RT(D200N)(T330P)(L603W)]-[bipartite NLS]-[NLS] and 2) a desired PEgRNA, wherein the fusion protein (referred to as the PEmax protein) has the following amino acid sequence: MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLG NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMA KVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST DKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NASGVDAKAILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYID GGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLRKQRTFDNGSIPHQIHLGE LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKV KYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIS GVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKV VDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQIL KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKD DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVY GDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET NGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKL IARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYT STKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKR KVSGGSSGGSTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQA PLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGT NDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPT SQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQ YVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEG QRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTL FNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWR 32 / 370 B1195.70196WO00 13363760.2RPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALV KQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILA EAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGT SAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKN KDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL IENSSPSGGSKRTADGSEFESPKKKRKVGSGPAAKRVKLD (SEQ ID NO: 794) KEY: BIPARTITE SV40 NUCLEAR LOCALIZATION SEQUENCE (NLS), CAS9(R221K N39K H840A) SGGSx2-BIPARTITE SV40NLS-SGGSx2 LINKER M-MLV reverse transcriptase(D200N T306K W313F T330P L603W) Other linker sequence BIPARTITE SV40NLS Other linker sequence c-Myc NLS Prime editing

[0094] As used herein, the term “prime editing” refers to an approach for gene editing using napDNAbps, a polymerase (e.g., a reverse transcriptase), and specialized guide RNAs that include a primer binding site and a DNA synthesis template for encoding desired new genetic information (or deleting genetic information) that is then incorporated into a target DNA sequence. Prime editing is described in Anzalone, A. V. et al., Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019), which is incorporated herein by reference. See also International PCT Application, PCT / US2020 / 023721, filed March 19, 2020, and published as WO 2020 / 191239, which is incorporated herein by reference.

[0095] Prime editing represents a platform for genome editing that is a versatile and precise method to directly write new genetic information into a specified DNA site using a nucleic acid programmable DNA binding protein (“napDNAbp”) working in association with a polymerase (i.e., in the form of a fusion protein or otherwise provided in trans with the napDNAbp), wherein the prime editing system is programmed with a prime editing (PE) guide RNA (“pegRNA”) that both specifies the target site and templates the synthesis of the desired edit in the form of a replacement DNA strand by way of an extension (either DNA or RNA) engineered onto a guide RNA (e.g., at the 5ʹ or 3ʹ end, or at an internal portion of a guide RNA). The replacement strand containing the desired edit (e.g., a single nucleobase substitution) shares the same sequence as the endogenous strand (or is homologous to it) immediately downstream of the nick site of the target site to be edited (with the exception that it includes the desired edit). Through DNA repair and / or replication machinery, the endogenous strand downstream of the nick site is replaced by the newly synthesized 33 / 370 B1195.70196WO00 13363760.2replacement strand containing the desired edit. In some cases, prime editing may be thought of as a “search-and-replace” genome editing technology since the prime editors, as described herein, not only search and locate the desired target site to be edited, but at the same time, encode a replacement strand containing a desired edit that is installed in place of the corresponding target site endogenous DNA strand. The prime editors of the present disclosure relate, in part, to the discovery that the mechanism of target-primed reverse transcription (TPRT) or “prime editing” can be leveraged or adapted for conducting precision CRISPR / Cas-based genome editing with high efficiency and genetic flexibility. TPRT is naturally used by mobile DNA elements, such as mammalian non-LTR retrotransposons and bacterial Group II introns. Cas protein-reverse transcriptase fusions or related systems are used to target a specific DNA sequence with a guide RNA, generate a single strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of an engineered DNA synthesis template that is integrated with the guide RNA. However, while the concept begins with prime editors that use reverse transcriptase as the DNA polymerase component, the prime editors described herein are not limited to reverse transcriptases but may include the use of virtually any DNA polymerase. Indeed, while the application throughout may refer to prime editors with “reverse transcriptases,” it is set forth here that reverse transcriptases are only one type of DNA polymerase that may work with prime editing. Thus, wherever the specification mentions a “reverse transcriptase,” the person having ordinary skill in the art should appreciate that any suitable DNA polymerase may be used in place of the reverse transcriptase. Thus, in one aspect, the prime editors may comprise Cas9 (or an equivalent napDNAbp), which is programmed to target a DNA sequence by associating it with a specialized guide RNA (i.e., pegRNA) containing a spacer sequence that anneals to a complementary sequence (the complementary sequence to an endogenous protospacer sequence) in the target DNA. The pegRNA also contains new genetic information in the form of an extension that encodes a replacement strand of DNA containing a desired nucleotide change which is used to replace a corresponding endogenous DNA strand at the target site. To transfer information from the pegRNA to the target DNA, the mechanism of prime editing involves nicking the target site in one strand of the DNA to expose a 3′-hydroxyl group. The exposed 3′-hydroxyl group can then be used to prime the DNA polymerization of the edit- encoding extension on pegRNA directly into the target site. In various embodiments, the extension—which provides the template for polymerization of the replacement strand containing the edit—can be formed from RNA or DNA. In the case of an RNA extension, the 34 / 370 B1195.70196WO00 13363760.2polymerase of the prime editor can be an RNA-dependent DNA polymerase (such as a reverse transcriptase). In the case of a DNA extension, the polymerase of the prime editor may be a DNA-dependent DNA polymerase. The newly synthesized strand (i.e., the replacement DNA strand containing the desired nucleotide edit) that is formed by the prime editor would be homologous to the genomic target sequence (i.e., have the same sequence as), except for the inclusion of one or more desired nucleotide changes (e.g., a single nucleotide substitution, a deletion, or an insertion, or a combination thereof). The newly synthesized (or replacement) strand of DNA may also be referred to as a single strand DNA flap, which would compete for hybridization with the complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. Resolution of the hybridized intermediate (also referred to as a heteroduplex, comprising the single strand DNA flap synthesized by the reverse transcriptase hybridized to the endogenous DNA strand with the exception of mismatches at positions where desired nucleotide edits are installed in the edit strand) can include removal of the resulting displaced flap of endogenous DNA (e.g., with a 5ʹ end DNA flap endonuclease, FEN1), ligation of the synthesized single strand DNA flap to the target DNA, and assimilation of the desired nucleotide changes as a result of cellular DNA repair and / or replication processes.

[0096] In various embodiments, prime editing operates by contacting a target DNA molecule (for which a change in the nucleotide sequence is desired to be introduced) with a nucleic acid programmable DNA binding protein (napDNAbp) complexed with a prime editing guide RNA (pegRNA). In various embodiments, the prime editing guide RNA (pegRNA) comprises an extension at the 3′ or 5′ end of the guide RNA, or at an intramolecular location in the guide RNA, and encodes the desired nucleotide change (e.g., single nucleotide substitution, insertion, or deletion). First, the napDNAbp / extended gRNA complex contacts the DNA molecule, and the extended gRNA guides the napDNAbp to bind to a target locus. Next, a nick in one of the strands of DNA of the target locus is introduced (e.g., by a nuclease or chemical agent), thereby creating an available 3′ end in one of the strands of the target locus. In certain embodiments, the nick is created in the strand of DNA that corresponds to the R-loop strand, i.e., the strand that is not hybridized to the guide RNA sequence, i.e., the “non-target strand.” The nick, however, could be introduced in either of the strands. That is, the nick could be introduced into the R-loop “target strand” (i.e., the strand hybridized to the protospacer of the extended gRNA) or the “non-target strand” (i.e., the strand forming the single-stranded portion of the R-loop and which is complementary to the target strand). In the 35 / 370 B1195.70196WO00 13363760.2next step, the 3′ end of the DNA strand (formed by the nick) interacts with the extended portion of the guide RNA in order to prime reverse transcription (i.e., “target-primed RT”). In certain embodiments, the 3′ end DNA strand hybridizes to a specific RT priming sequence on the extended portion of the guide RNA, i.e., the “reverse transcriptase priming sequence” or “primer binding site” on the pegRNA. In the next step, a reverse transcriptase (or other suitable DNA polymerase) is introduced that synthesizes a single strand of DNA from the 3′ end of the primed site towards the 5′ end of the prime editing guide RNA. The DNA polymerase (e.g., reverse transcriptase) can be fused to the napDNAbp or alternatively can be provided in trans to the napDNAbp. This forms a single-strand DNA flap comprising the desired nucleotide change (e.g., the single base change, insertion, or deletion, or a combination thereof) and that is otherwise homologous to the endogenous DNA at or adjacent to the nick site. In the next step, the napDNAbp and guide RNA are released. The final two steps relate to the resolution of the single strand DNA flap such that the desired nucleotide change becomes incorporated into the target locus. This process can be driven towards the desired product formation by removing the corresponding 5′ endogenous DNA flap that forms once the 3′ single strand DNA flap invades and hybridizes to the endogenous DNA sequence. Without being bound by theory, the cell’s endogenous DNA repair and replication processes resolve the mismatched DNA to incorporate the nucleotide change(s) to form the desired altered product. The process can also be driven towards product formation with “second strand nicking.” This process may introduce at least one or more of the following genetic changes: transversions, transitions, deletions, and insertions. Prime editor

[0097] The term “prime editor” refers to the polypeptide or polypeptide components involved in prime editing as described herein. In some embodiments, a prime editor comprises a fusion construct comprising a napDNAbp (e.g., Cas9 nickase, and / or any of the Cas9 variants provided herein) and a reverse transcriptase (e.g., any of the reverse transcriptase variants provided herein). In some embodiments, a prime editor is capable of carrying out prime editing on a target nucleotide sequence in the presence of a pegRNA (or “extended guide RNA”). In some embodiments, a prime editor comprises a napDNAbp (e.g., Cas9 nickase) and a reverse transcriptase provided in trans, i.e., the napDNAbp and the reverse transcriptase are not fused. The in trans napDNAbp and the reverse transcriptase may be tethered via a non-peptide linkage, e.g., an MS2 RNA-protein binding RNA sequence and a MS2 coat protein fused to either the napDNAbp or the reverse transcriptase, or may be 36 / 370 B1195.70196WO00 13363760.2unlinked to each other and simply recruited by the pegRNA. In some embodiments, a prime editor composition, system, or complex provided herein comprises a fusion protein or a fusion protein complexed with a pegRNA, and / or further complexed with a second-strand nicking sgRNA. In some embodiments, the prime editor system may also refer to the complex comprising a fusion protein (reverse transcriptase fused to a napDNAbp), a pegRNA, and a regular guide RNA capable of directing the second-site nicking step of the non-edited strand as described herein. Primer binding site

[0098] The term “primer binding site” or “the PBS” refers to the nucleotide sequence located on a pegRNA as component of the extension arm (typically at the 3ʹ end of the extension arm) and serves to bind to the primer sequence that is formed after Cas9 nicking of the target sequence by the prime editor. As detailed elsewhere, when the Cas9 nickase component of a prime editor nicks one strand of the target DNA sequence, a 3ʹ-ended ssDNA flap is formed, which serves a primer sequence that anneals to the primer binding site on the pegRNA to prime reverse transcription. Protein, peptide, and polypeptide

[0099] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein, or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory 37 / 370 B1195.70196WO00 13363760.2Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the contents of which are incorporated herein by reference. Protospacer

[0100] As used herein, the term “protospacer” refers to the sequence (~20 bp) in DNA adjacent to the PAM (protospacer adjacent motif) sequence. The protospacer shares the same sequence as the spacer sequence of the guide RNA. The guide RNA anneals to the complement of the protospacer sequence on the target DNA (specifically, one strand thereof, i.e., the “target strand” versus the “non-target strand” of the target DNA sequence). The skilled person will appreciate that the literature in the state of the art sometimes refers to the “protospacer” as the ~20-nt target-specific guide sequence on the guide RNA itself, rather than referring to it as a “spacer.” Thus, in some cases, the term “protospacer” as used herein may be used interchangeably with the term “spacer.” The context of the description surrounding the appearance of either “protospacer” or “spacer” will help inform the reader as to whether the term is in reference to the gRNA or the DNA target. Protospacer adjacent motif (PAM)

[0101] As used herein, the term “protospacer adjacent motif” or “PAM” refers to a DNA sequence (e.g., an approximately 2-6 nucleotide sequence) that is an important targeting component of a Cas nuclease, e.g., a Cas9. For example, in some embodiments for a Cas9 nuclease, the PAM sequence is on either strand and is downstream in the 5ʹ to 3ʹ direction of the Cas9 cut site. The canonical PAM sequence (i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5ʹ-NGG-3ʹ, wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases. In some embodiments, SpCas9 can also recognize additional non-canonical PAMs (e.g., NAG and NGA).

[0102] Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms. In addition, any given Cas9 nuclease, e.g., SpCas9, may be modified to alter the PAM specificity of the nuclease such that the nuclease recognizes an alternative PAM sequence. Reverse transcriptase

[0103] The term “reverse transcriptase” describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Historically, reverse transcriptase has been used primarily to transcribe mRNA into cDNA, which can then be cloned into a vector for further manipulation. Avian myoblastosis virus (AMV) reverse transcriptase was the first 38 / 370 B1195.70196WO00 13363760.2widely used RNA-dependent DNA polymerase (Verma, Biochim. Biophys. Acta 473:1 (1977)). The enzyme has 5ʹ-3ʹ RNA-directed DNA polymerase activity, 5ʹ-3ʹ DNA-directed DNA polymerase activity, and RNase H activity. RNase H is a processive 5ʹ and 3ʹ ribonuclease specific for the RNA strand for RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). Errors in transcription cannot be corrected by reverse transcriptase because known viral reverse transcriptases lack the 3ʹ-5ʹ exonuclease activity necessary for proofreading (Saunders and Saunders, Microbial Genetics Applied to Biotechnology, London: Croom Helm (1987)). A detailed study of the activity of AMV reverse transcriptase and its associated RNaseH activity has been presented by Berger et al., Biochemistry 22:2365-2372 (1983). Another reverse transcriptase that is used extensively in molecular biology is reverse transcriptase originating from Moloney murine leukemia virus (M-MLV or “MMLV”). See, e.g., Gerard, G. R., DNA 5:271-279 (1986) and Kotewicz, M. L., et al., Gene 35:249-258 (1985). M-MLV reverse transcriptase substantially lacking in RNase H activity has also been described. See, e.g., U.S. Pat. No.5,244,797. The invention contemplates the use of any such reverse transcriptases, or variants or mutants thereof. Silent mutation

[0104] As used herein, the term “silent mutation” refers to a mutation in a nucleic acid molecule that does not have an effect on the phenotype of the nucleic acid molecule, or the protein it produces if it encodes a protein. Silent mutations can be introduced into coding regions of a nucleic acid (i.e., segments of a gene that encode for a protein), or they can be introduced in non-coding regions of a nucleic acid. A silent mutation in a nucleic acid sequence, e.g., in a target DNA sequence or in a DNA synthesis template sequence to be installed in the target sequence, may be a nucleotide alteration that does not result in expression or function of the amino acid sequence encoded by the nucleic acid sequence, or other functional features of the target nucleic acid sequence. When silent mutations are present in a coding region, they may be synonymous mutations. Synonymous mutations refer to substitutions of one base for another in a gene such that the corresponding amino acid residue of the protein produced by the gene is not modified. This is due to the redundancy of the genetic code, allowing for multiple different codons to encode for the same amino acid in a particular organism. When a silent mutation is in a noncoding region or a junction of a coding region and a non-coding region (e.g., an intron / exon junction), it may be in a region that does not impact any biological properties of the nucleic acid molecule (e.g., splicing, 39 / 370 B1195.70196WO00 13363760.2gene regulation, RNA lifetime, etc.). In particular embodiments, a silent mutation may also be a “benign” mutation, for example, where a nucleotide substitution results in one or more alterations in the amino acid sequence encoded, but does not result in detrimental impact on the expression or function of the polypeptide. Silent mutations may be useful, for example, for increasing the length of contiguous changes in a desired nucleotide edit or the number of nucleotide edits made to a target nucleotide sequence using prime editing to evade correction of the edit by the MMR pathway as described herein. In certain embodiments, the number of silent mutations installed may be one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or more. In certain other embodiments involving at least two silent mutations, the silent mutations may be installed within one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 nucleotides from the intended edit site. Spacer sequence

[0105] As used herein, the term “spacer sequence” in connection with a guide RNA refers to the portion of the guide RNA of about 20 nucleotides that contains a nucleotide sequence that shares the same sequence as the protospacer sequence in the target DNA sequence. The spacer sequence anneals to the complement of the protospacer sequence to form a ssRNA / ssDNA hybrid structure at the target site and a corresponding R loop ssDNA structure of the endogenous DNA strand. Subject

[0106] The term “subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex, and at any stage of development. Target site

[0107] The term “target site” refers to a sequence within a nucleic acid molecule that is modified (e.g., edited) by a prime editor as described herein. The target site further refers to 40 / 370 B1195.70196WO00 13363760.2the sequence within a nucleic acid molecule (e.g., a nucleic acid molecule comprising CFTR) to which a complex of, for example, a prime editor and a pegRNA binds. Treatment

[0108] The terms “treatment,” “treat,” and “treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder (e.g., cystic fibrosis), or one or more symptoms thereof, as described herein. As used herein, the terms “treatment,” “treat,” and “treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder (e.g., cystic fibrosis), or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and / or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease (e.g., cystic fibrosis). For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence. Variant

[0109] As used herein, the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant Cas9 is a Cas9 comprising one or more changes in amino acid residues (i.e., “substitutions”) as compared to a wild type Cas9 amino acid sequence. The term “variant” encompasses homologous proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence that display the same or substantially the same functional activity or activities as the reference sequence. Vector

[0110] The term “vector,” as used herein, refers to a nucleic acid that can be modified to encode a gene of interest and that is able to enter a host cell, mutate, and replicate within the host cell, and then transfer a replicated form of the vector into another host cell. Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phage, and conjugative plasmids. Additional suitable vectors will be apparent to those of skill in the art based on the instant disclosure. 41 / 370 B1195.70196WO00 13363760.2DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0111] The present disclosure describes the use of prime editors and pegRNAs for editing the CFTR gene to correct an F508del mutation in the CFTR protein and treat cystic fibrosis. Methods of editing CFTR using a prime editor and a pegRNA are provided herein. Such methods may be useful for treating cystic fibrosis. The present disclosure also provides pegRNAs, ngRNAs, dsgRNAs, systems, and compositions for editing CFTR and treating cystic fibrosis. Polynucleotides, vectors, cells, and kits for editing CFTR and treating cystic fibrosis are also provided herein. Guide RNAs (gRNAs)

[0112] The present disclosure provides pegRNAs, ngRNAs, and dsgRNAs for prime editing a CFTR gene (e.g., a human CFTR gene). The pegRNAs, ngRNAs, and dsgRNAs provided herein may be useful for treating cystic fibrosis disease.

[0113] In some embodiments, the provided pegRNAs target a prime editor to a site in the human CFTR gene. In some embodiments, the gRNAs target a prime editor to a site in the human CFTR gene such that the prime editor corrects an F508del mutation in a CFTR protein by inserting the sequence CTT into the CFTR gene.

[0114] In one aspect, the present disclosure provides prime editing guide RNAs (pegRNAs) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence: TCTGTATCTATATTCATCAT (SEQ ID NO: 1), ACCATTAAAGAAAATATCAT (SEQ ID NO: 2), ATTATGCCTGGCACCATTAA(SEQ ID NO: 3), CTGTATCTATATTCATCATA (SEQ ID NO: 4), TTCATCATAGGAAACACCAA (SEQ ID NO: 5), CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), or CTGTATCTATATTCATCAT (SEQ ID NO: 8). In some embodiments, the pegRNA comprises a spacer comprising the sequence TCTGTATCTATATTCATCAT (SEQ ID NO: 1), ACCATTAAAGAAAATATCAT (SEQ ID NO: 2), ATTATGCCTGGCACCATTAA (SEQ ID NO: 3), CTGTATCTATATTCATCATA (SEQ ID NO: 4), TTCATCATAGGAAACACCAA (SEQ ID NO: 5), CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), or CTGTATCTATATTCATCAT (SEQ ID NO: 8). In some embodiments, the 42 / 370 B1195.70196WO00 13363760.2pegRNA comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence TCTGTATCTATATTCATCAT (SEQ ID NO: 1) or CTGTATCTATATTCATCAT (SEQ ID NO: 8). In certain embodiments, the pegRNA comprises a spacer comprising the sequence TCTGTATCTATATTCATCAT (SEQ ID NO: 1) or CTGTATCTATATTCATCAT (SEQ ID NO: 8). In some embodiments, the pegRNA spacer further comprises a G at its 5' end.

[0115] The pegRNAs provided herein also comprise a backbone scaffold sequence that facilitates binding of the pegRNA to a napDNAbp, for example, a Cas9 protein (e.g., a Cas9 protein as part of a prime editor). In some embodiments, the provided pegRNAs comprise a backbone scaffold sequence for binding to SpCas9. In some embodiments, the pegRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17) or GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 18), or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTAT CAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 20). In certain embodiments, the pegRNA comprises a backbone scaffold comprising the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17) or GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 18), or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTAT CAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 20).

[0116] The pegRNAs provided herein also comprise a reverse transcription template. In some embodiments, the pegRNA comprises an RTT of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the pegRNA comprises an RTT comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the 43 / 370 B1195.70196WO00 13363760.2sequence AGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 21), TAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 22), CATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 23), CACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 24), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 25), GCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 26), CATAGGAAACACCAAAGATG (SEQ ID NO: 27), AAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 28), AAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 29), TTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 30), ATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 31), CCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 32), ACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 33), CTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 34), CCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 35), TCATTGGTGTTTCCTATG (SEQ ID NO: 36), ATCATTGGTGTTTCCTATG (SEQ ID NO: 37), TATCATTGGTGTTTCCTATG (SEQ ID NO: 38), ATATCATTGGTGTTTCCTATG (SEQ ID NO: 39), AATATCATTGGTGTTTCCTATG (SEQ ID NO: 40), AAATATCATTGGTGTTTCCTATG (SEQ ID NO: 41), AAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 42), GAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 43), AGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 44), AAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 45), AAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 46), TAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 47), TTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 48), ATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 49), ACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 50), GCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 51), GGCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 52), TGGCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 53), CATCATAGGAAACACCAAAGATG (SEQ ID NO: 54), ATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 55), 44 / 370 B1195.70196WO00 13363760.2TATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 56), ATCTATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 57), TGTATCTATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 58), GGAAACACCAAAGATGATATTTTCTTTA (SEQ ID NO: 59), ATTCATCATAGGAAACACCAAAGATGATATTTTCTTTA (SEQ ID NO: 60), AAAATATCATCTTTGGTGTTTCCTAT (SEQ ID NO: 61), ACCATTAAAGAAAATATCATCTTTGGTGTTTCCTAT (SEQ ID NO: 62), AAAATATCATCTTTG (SEQ ID NO: 63), ACCATTAAAGAAAATATCATCTTTG (SEQ ID NO: 64), GGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGC (SEQ ID NO: 65), TATTCATCATAGGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGC (SEQ ID NO: 66), GGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGCATAATCCAGGA (SEQ ID NO: 67), ATTCATCATAGGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGCATAAT CCAGGA (SEQ ID NO: 68), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGTTACG (SEQ ID NO: 69), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGTTACG (SEQ ID NO: 70), TGGCACCATTAAAGAAAATATCATCTTCGGTGTGAGTTATG (SEQ ID NO: 71), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGTTATG (SEQ ID NO: 72), TGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 73), TGGCACCATTAAAGAAAATATCATCTTTGGCGTAAGCTACG (SEQ ID NO: 76), TGGCACCATTAAAGAAAATATCATATTTGGTGTTAGCTACG (SEQ ID NO: 77), TGGCACCATTAAAGAAAATATCATATTTGGCGTAAGCTACG (SEQ ID NO: 78), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCAGCTACG (SEQ ID NO: 79), TGGCACCATTAAAGAAAATATCATCTTTGGTGTAAGCTACG (SEQ ID NO: 80), TGGCACCATTAAAGAAAATATCATCTTTGGCGTGAGCTACG (SEQ ID NO: 81), TGGCACCATTAAAGAAAATATCATATTTGGTGTGAGCTACG (SEQ ID NO: 82), TGGCACCATTAAAGAAAATATCATATTTGGTGTCAGCTACG (SEQ ID NO: 83), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCAGCTACG (SEQ ID NO: 84), TGGCACCATTAAAGAAAATATCATATTTGGTGTAAGCTACG (SEQ ID NO: 85), TGGCACCATTAAAGAAAATATCATATTTGGCGTCAGCTACG (SEQ ID NO: 86), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTAGCTACG (SEQ ID NO: 87), 45 / 370 B1195.70196WO00 13363760.2TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCATACG (SEQ ID NO: 88), TGGCACCATTAAAGAAAATATCATATTTGGCGTGAGCTACG (SEQ ID NO: 89), TGGCACCATTAAAGAAAATATCATATTCGGTGTGAGCTACG (SEQ ID NO: 90), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCATACG (SEQ ID NO: 91), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCATACG (SEQ ID NO: 92), TGGCACCATTAAAGAAAATATCATCTTTGGCGTATCATACG (SEQ ID NO: 93), TGGCACCATTAAAGAAAATATCATCTTTGGCGTAAGTTACG (SEQ ID NO: 94), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCGTACG (SEQ ID NO: 95), TGGCACCATTAAAGAAAATATCATCTTCGGCGTCAGCTACG (SEQ ID NO: 96), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCGTACG (SEQ ID NO: 97), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTAGCTACG (SEQ ID NO: 98), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCGTACG (SEQ ID NO: 99), TGGCACCATTAAAGAAAATATCATCTTTGGGGTATCATACG (SEQ ID NO: 100), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCGTACG (SEQ ID NO: 101), TGGCACCATTAAAGAAAATATCATCTTTGGCGTATCGTACG (SEQ ID NO: 102), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCGTACG (SEQ ID NO: 103), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGTTACG (SEQ ID NO: 70), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCGTACG (SEQ ID NO: 104), TGGCACCATTAAAGAAAATATCATCTTCGGTGTATCATACG (SEQ ID NO: 105), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCGTACG (SEQ ID NO: 106), TGGCACCATTAAAGAAAATATCATCTTCGGCGTAAGCTACG (SEQ ID NO: 107), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGCTACG (SEQ ID NO: 108), TGGCACCATTAAAGAAAATATCATCTTTGGCGTTTCGTACG (SEQ ID NO: 109), TGGCACCATTAAAGAAAATATCATCTTTGGTGTAAGTTACG (SEQ ID NO: 110), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCATACG (SEQ ID NO: 111), TGGCACCATTAAAGAAAATATCATATTCGGTGTAAGCTACG (SEQ ID NO: 112), TGGCACCATTAAAGAAAATATCATCTTCGGCGTTAGCTACG (SEQ ID NO: 113), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCATACG (SEQ ID NO: 114), TGGCACCATTAAAGAAAATATCATCTTCGGTGTATCGTACG (SEQ ID NO: 115), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 116), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCAGTTACG (SEQ ID NO: 117), TGGCACCATTAAAGAAAATATCATATTTGGTGTATCATACG (SEQ ID NO: 118), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCATACG (SEQ ID NO: 119), 46 / 370 B1195.70196WO00 13363760.2TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCGTACG (SEQ ID NO: 120), TGGCACCATTAAAGAAAATATCATATTCGGTGTATCGTACG (SEQ ID NO: 121), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCTTACG (SEQ ID NO: 122), TGGCACCATTAAAGAAAATATCATATTTGGTGTATCGTACG (SEQ ID NO: 123), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGCTACG (SEQ ID NO: 124), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCTTACG (SEQ ID NO: 125), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCTTACG (SEQ ID NO: 126), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCTTACG (SEQ ID NO: 127), TGGCACCATTAAAGAAAATATCATATTCGGCGTCAGCTACG (SEQ ID NO: 128), TGGCACCATTAAAGAAAATATCATCTTTGGGGTATCGTACG (SEQ ID NO: 129), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCATACG (SEQ ID NO: 130), TGGCACCATTAAAGAAAATATCATCTTCGGCGTGAGCTACG (SEQ ID NO: 131), TGGCACCATTAAAGAAAATATCATCTTTGGCGTTAGCTACG (SEQ ID NO: 132), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCTCATACG (SEQ ID NO: 133), TGGCACCATTAAAGAAAATATCATCTTTGGAGTTTCGTACG (SEQ ID NO: 134), TGGCACCATTAAAGAAAATATCATATTCGGTGTTAGCTACG (SEQ ID NO: 135), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCTCGTACG (SEQ ID NO: 136), TGGCACCATTAAAGAAAATATCATATTTGGTGTAAGTTACG (SEQ ID NO: 137), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCTTACG (SEQ ID NO: 138), TGGCACCATTAAAGAAAATATCATCTTCGGTGTGTCATACG (SEQ ID NO: 139), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCTCGTACG (SEQ ID NO: 140), TGGCACCATTAAAGAAAATATCATCTTCGGCGTATCGTACG (SEQ ID NO: 141), TGGCACCATTAAAGAAAATATCATATTTGGTGTTAGTTACG (SEQ ID NO: 142), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCTTACG (SEQ ID NO: 143), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCTTACG (SEQ ID NO: 144), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCTTACG (SEQ ID NO: 145), TGGCACCATTAAAGAAAATATCATCTTTGGGGTGAGCTACG (SEQ ID NO: 146), AAGAAAATATCATATTTGGAGTAAGTTACG (SEQ ID NO: 147), AAGAAAATATCATATTTGGAGTAAGCTACG (SEQ ID NO: 148), AAGAAAATATCATATTTGGAGTGAGTTACG (SEQ ID NO: 149), AAAGAAAATATCATATTTGGAGTAAGTTACG (SEQ ID NO: 150), AAGAAAATATCATATTTGGTGTTTCGTACG (SEQ ID NO: 151), AAGAAAATATCATATTTGGTGTTAGCTACG (SEQ ID NO: 152), 47 / 370 B1195.70196WO00 13363760.2AAAGAAAATATCATATTTGGAGTAAGCTACG (SEQ ID NO: 153), AAGAAAATATCATATTTGGAGTAAGTTATG (SEQ ID NO: 154), AAAGAAAATATCATATTTGGAGTGAGTTACG (SEQ ID NO: 155), AAGAAAATATCATATTTGGTGTAAGCTACG (SEQ ID NO: 156), AAGAAAATATCATATTTGGTGTTTCATACG (SEQ ID NO: 157), AAGAAAATATCATATTTGGAGTCTCATACG (SEQ ID NO: 158), AAGAAAATATCATATTTGGAGTATCATACG (SEQ ID NO: 159), AAGAAAATATCATATTCGGAGTTTCATACG (SEQ ID NO: 160), AAGAAAATATCATATTTGGAGTATCGTACG (SEQ ID NO: 161), AAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 162), AAGAAAATATCATATTTGGAGTTAGTTACG (SEQ ID NO: 163), AAGAAAATATCATATTTGGTGTAAGTTACG (SEQ ID NO: 164), AAGAAAATATCATATTCGGTGTTAGCTACG (SEQ ID NO: 165), AAGAAAATATCATATTTGGAGTGAGCTACG (SEQ ID NO: 166), AAGAAAATATCATATTTGGAGTAAGCTATG (SEQ ID NO: 167), AAGAAAATATCATATTCGGAGTAAGTTACG (SEQ ID NO: 168), AAGAAAATATCATATTTGGCGTTAGCTACG (SEQ ID NO: 169), AAGAAAATATCATATTTGGTGTTAGTTACG (SEQ ID NO: 170), AAGAAAATATCATATTTGGGGTAAGTTACG (SEQ ID NO: 171), AAGAAAATATCATATTCGGAGTCTCATACG (SEQ ID NO: 172), AAGAAAATATCATATTTGGAGTGAGCTATG (SEQ ID NO: 173), AAGAAAATATCATATTTGGAGTTAGCTACG (SEQ ID NO: 174), AAGAAAATATCATATTTGGGGTAAGTTATG (SEQ ID NO: 175), AAGAAAATATCATATTCGGAGTGAGTTACG (SEQ ID NO: 176), AAGAAAATATCATATTTGGAGTCAGCTACG (SEQ ID NO: 177), AAGAAAATATCATATTTGGTGTCTCGTACG (SEQ ID NO: 178), AAGAAAATATCATATTTGGAGTCTCGTACG (SEQ ID NO: 179), AAGAAAATATCATATTTGGAGTGTCATACG (SEQ ID NO: 180), AAGAAAATATCATATTTGGAGTTTCATACG (SEQ ID NO: 181), AAGAAAATATCATATTTGGTGTCAGCTACG (SEQ ID NO: 182), AAGAAAATATCATATTTGGTGTGAGCTACG (SEQ ID NO: 183), AAGAAAATATCATATTCGGTGTAAGCTACG (SEQ ID NO: 184), AAGAAAATATCATATTCGGTGTTTCGTACG (SEQ ID NO: 185), 48 / 370 B1195.70196WO00 13363760.2AAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 186), TAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 187), TTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 188), ATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 189), ACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 190), GCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 191), GGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 192), GCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 193), AAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 194), TAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 195), TTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 196), ATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 197), ACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 198), GCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 199), GGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 200), TGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 201), GCCTGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 202), or TGCCTGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 203). In certain embodiments, the pegRNA comprises an RTT comprising the sequence AGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 21), TAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 22), CATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 23), CACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 24), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 25), GCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 26), CATAGGAAACACCAAAGATG (SEQ ID NO: 27), AAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 28), AAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 29), TTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 30), ATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 31), CCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 32), ACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 33), CTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 34), 49 / 370 B1195.70196WO00 13363760.2CCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 35), TCATTGGTGTTTCCTATG (SEQ ID NO: 36), ATCATTGGTGTTTCCTATG (SEQ ID NO: 37), TATCATTGGTGTTTCCTATG (SEQ ID NO: 38), ATATCATTGGTGTTTCCTATG (SEQ ID NO: 39), AATATCATTGGTGTTTCCTATG (SEQ ID NO: 40), AAATATCATTGGTGTTTCCTATG (SEQ ID NO: 41), AAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 42), GAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 43), AGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 44), AAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 45), AAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 46), TAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 47), TTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 48), ATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 49), ACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 50), GCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 51), GGCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 52), TGGCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 53), CATCATAGGAAACACCAAAGATG (SEQ ID NO: 54), ATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 55), TATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 56), ATCTATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 57), TGTATCTATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 58), GGAAACACCAAAGATGATATTTTCTTTA (SEQ ID NO: 59), ATTCATCATAGGAAACACCAAAGATGATATTTTCTTTA (SEQ ID NO: 60), AAAATATCATCTTTGGTGTTTCCTAT (SEQ ID NO: 61), ACCATTAAAGAAAATATCATCTTTGGTGTTTCCTAT (SEQ ID NO: 62), AAAATATCATCTTTG (SEQ ID NO: 63), ACCATTAAAGAAAATATCATCTTTG (SEQ ID NO: 64), GGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGC (SEQ ID NO: 65), TATTCATCATAGGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGC (SEQ ID NO: 66), GGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGCATAATCCAGGA (SEQ ID NO: 67), 50 / 370 B1195.70196WO00 13363760.2ATTCATCATAGGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGCATAAT CCAGGA (SEQ ID NO: 68), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGTTACG (SEQ ID NO: 69), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGTTACG (SEQ ID NO: 70), TGGCACCATTAAAGAAAATATCATCTTCGGTGTGAGTTATG (SEQ ID NO: 71), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGTTATG (SEQ ID NO: 72), TGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 73), TGGCACCATTAAAGAAAATATCATCTTTGGCGTAAGCTACG (SEQ ID NO: 76), TGGCACCATTAAAGAAAATATCATATTTGGTGTTAGCTACG (SEQ ID NO: 77), TGGCACCATTAAAGAAAATATCATATTTGGCGTAAGCTACG (SEQ ID NO: 78), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCAGCTACG (SEQ ID NO: 79), TGGCACCATTAAAGAAAATATCATCTTTGGTGTAAGCTACG (SEQ ID NO: 80), TGGCACCATTAAAGAAAATATCATCTTTGGCGTGAGCTACG (SEQ ID NO: 81), TGGCACCATTAAAGAAAATATCATATTTGGTGTGAGCTACG (SEQ ID NO: 82), TGGCACCATTAAAGAAAATATCATATTTGGTGTCAGCTACG (SEQ ID NO: 83), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCAGCTACG (SEQ ID NO: 84), TGGCACCATTAAAGAAAATATCATATTTGGTGTAAGCTACG (SEQ ID NO: 85), TGGCACCATTAAAGAAAATATCATATTTGGCGTCAGCTACG (SEQ ID NO: 86), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTAGCTACG (SEQ ID NO: 87), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCATACG (SEQ ID NO: 88), TGGCACCATTAAAGAAAATATCATATTTGGCGTGAGCTACG (SEQ ID NO: 89), TGGCACCATTAAAGAAAATATCATATTCGGTGTGAGCTACG (SEQ ID NO: 90), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCATACG (SEQ ID NO: 91), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCATACG (SEQ ID NO: 92), TGGCACCATTAAAGAAAATATCATCTTTGGCGTATCATACG (SEQ ID NO: 93), TGGCACCATTAAAGAAAATATCATCTTTGGCGTAAGTTACG (SEQ ID NO: 94), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCGTACG (SEQ ID NO: 95), TGGCACCATTAAAGAAAATATCATCTTCGGCGTCAGCTACG (SEQ ID NO: 96), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCGTACG (SEQ ID NO: 97), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTAGCTACG (SEQ ID NO: 98), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCGTACG (SEQ ID NO: 99), TGGCACCATTAAAGAAAATATCATCTTTGGGGTATCATACG (SEQ ID NO: 100), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCGTACG (SEQ ID NO: 101), 51 / 370 B1195.70196WO00 13363760.2TGGCACCATTAAAGAAAATATCATCTTTGGCGTATCGTACG (SEQ ID NO: 102), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCGTACG (SEQ ID NO: 103), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGTTACG (SEQ ID NO: 70), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCGTACG (SEQ ID NO: 104), TGGCACCATTAAAGAAAATATCATCTTCGGTGTATCATACG (SEQ ID NO: 105), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCGTACG (SEQ ID NO: 106), TGGCACCATTAAAGAAAATATCATCTTCGGCGTAAGCTACG (SEQ ID NO: 107), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGCTACG (SEQ ID NO: 108), TGGCACCATTAAAGAAAATATCATCTTTGGCGTTTCGTACG (SEQ ID NO: 109), TGGCACCATTAAAGAAAATATCATCTTTGGTGTAAGTTACG (SEQ ID NO: 110), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCATACG (SEQ ID NO: 111), TGGCACCATTAAAGAAAATATCATATTCGGTGTAAGCTACG (SEQ ID NO: 112), TGGCACCATTAAAGAAAATATCATCTTCGGCGTTAGCTACG (SEQ ID NO: 113), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCATACG (SEQ ID NO: 114), TGGCACCATTAAAGAAAATATCATCTTCGGTGTATCGTACG (SEQ ID NO: 115), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 116), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCAGTTACG (SEQ ID NO: 117), TGGCACCATTAAAGAAAATATCATATTTGGTGTATCATACG (SEQ ID NO: 118), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCATACG (SEQ ID NO: 119), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCGTACG (SEQ ID NO: 120), TGGCACCATTAAAGAAAATATCATATTCGGTGTATCGTACG (SEQ ID NO: 121), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCTTACG (SEQ ID NO: 122), TGGCACCATTAAAGAAAATATCATATTTGGTGTATCGTACG (SEQ ID NO: 123), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGCTACG (SEQ ID NO: 124), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCTTACG (SEQ ID NO: 125), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCTTACG (SEQ ID NO: 126), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCTTACG (SEQ ID NO: 127), TGGCACCATTAAAGAAAATATCATATTCGGCGTCAGCTACG (SEQ ID NO: 128), TGGCACCATTAAAGAAAATATCATCTTTGGGGTATCGTACG (SEQ ID NO: 129), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCATACG (SEQ ID NO: 130), TGGCACCATTAAAGAAAATATCATCTTCGGCGTGAGCTACG (SEQ ID NO: 131), TGGCACCATTAAAGAAAATATCATCTTTGGCGTTAGCTACG (SEQ ID NO: 132), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCTCATACG (SEQ ID NO: 133), 52 / 370 B1195.70196WO00 13363760.2TGGCACCATTAAAGAAAATATCATCTTTGGAGTTTCGTACG (SEQ ID NO: 134), TGGCACCATTAAAGAAAATATCATATTCGGTGTTAGCTACG (SEQ ID NO: 135), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCTCGTACG (SEQ ID NO: 136), TGGCACCATTAAAGAAAATATCATATTTGGTGTAAGTTACG (SEQ ID NO: 137), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCTTACG (SEQ ID NO: 138), TGGCACCATTAAAGAAAATATCATCTTCGGTGTGTCATACG (SEQ ID NO: 139), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCTCGTACG (SEQ ID NO: 140), TGGCACCATTAAAGAAAATATCATCTTCGGCGTATCGTACG (SEQ ID NO: 141), TGGCACCATTAAAGAAAATATCATATTTGGTGTTAGTTACG (SEQ ID NO: 142), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCTTACG (SEQ ID NO: 143), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCTTACG (SEQ ID NO: 144), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCTTACG (SEQ ID NO: 145), TGGCACCATTAAAGAAAATATCATCTTTGGGGTGAGCTACG (SEQ ID NO: 146), AAGAAAATATCATATTTGGAGTAAGTTACG (SEQ ID NO: 147), AAGAAAATATCATATTTGGAGTAAGCTACG (SEQ ID NO: 148), AAGAAAATATCATATTTGGAGTGAGTTACG (SEQ ID NO: 149), AAAGAAAATATCATATTTGGAGTAAGTTACG (SEQ ID NO: 150), AAGAAAATATCATATTTGGTGTTTCGTACG (SEQ ID NO: 151), AAGAAAATATCATATTTGGTGTTAGCTACG (SEQ ID NO: 152), AAAGAAAATATCATATTTGGAGTAAGCTACG (SEQ ID NO: 153), AAGAAAATATCATATTTGGAGTAAGTTATG (SEQ ID NO: 154), AAAGAAAATATCATATTTGGAGTGAGTTACG (SEQ ID NO: 155), AAGAAAATATCATATTTGGTGTAAGCTACG (SEQ ID NO: 156), AAGAAAATATCATATTTGGTGTTTCATACG (SEQ ID NO: 157), AAGAAAATATCATATTTGGAGTCTCATACG (SEQ ID NO: 158), AAGAAAATATCATATTTGGAGTATCATACG (SEQ ID NO: 159), AAGAAAATATCATATTCGGAGTTTCATACG (SEQ ID NO: 160), AAGAAAATATCATATTTGGAGTATCGTACG (SEQ ID NO: 161), AAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 162), AAGAAAATATCATATTTGGAGTTAGTTACG (SEQ ID NO: 163), AAGAAAATATCATATTTGGTGTAAGTTACG (SEQ ID NO: 164), AAGAAAATATCATATTCGGTGTTAGCTACG (SEQ ID NO: 165), AAGAAAATATCATATTTGGAGTGAGCTACG (SEQ ID NO: 166), 53 / 370 B1195.70196WO00 13363760.2AAGAAAATATCATATTTGGAGTAAGCTATG (SEQ ID NO: 167), AAGAAAATATCATATTCGGAGTAAGTTACG (SEQ ID NO: 168), AAGAAAATATCATATTTGGCGTTAGCTACG (SEQ ID NO: 169), AAGAAAATATCATATTTGGTGTTAGTTACG (SEQ ID NO: 170), AAGAAAATATCATATTTGGGGTAAGTTACG (SEQ ID NO: 171), AAGAAAATATCATATTCGGAGTCTCATACG (SEQ ID NO: 172), AAGAAAATATCATATTTGGAGTGAGCTATG (SEQ ID NO: 173), AAGAAAATATCATATTTGGAGTTAGCTACG (SEQ ID NO: 174), AAGAAAATATCATATTTGGGGTAAGTTATG (SEQ ID NO: 175), AAGAAAATATCATATTCGGAGTGAGTTACG (SEQ ID NO: 176), AAGAAAATATCATATTTGGAGTCAGCTACG (SEQ ID NO: 177), AAGAAAATATCATATTTGGTGTCTCGTACG (SEQ ID NO: 178), AAGAAAATATCATATTTGGAGTCTCGTACG (SEQ ID NO: 179), AAGAAAATATCATATTTGGAGTGTCATACG (SEQ ID NO: 180), AAGAAAATATCATATTTGGAGTTTCATACG (SEQ ID NO: 181), AAGAAAATATCATATTTGGTGTCAGCTACG (SEQ ID NO: 182), AAGAAAATATCATATTTGGTGTGAGCTACG (SEQ ID NO: 183), AAGAAAATATCATATTCGGTGTAAGCTACG (SEQ ID NO: 184), AAGAAAATATCATATTCGGTGTTTCGTACG (SEQ ID NO: 185), AAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 186), TAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 187), TTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 188), ATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 189), ACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 190), GCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 191), GGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 192), GCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 193), AAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 194), TAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 195), TTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 196), ATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 197), ACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 198), GCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 199), 54 / 370 B1195.70196WO00 13363760.2GGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 200), TGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 201), GCCTGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 202), or TGCCTGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 203). In some embodiments, the pegRNA comprises an RTT comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO:25) or TGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ IDNO: 73). In certain embodiments, the pegRNA comprises an RTT comprising the sequence TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 25) or TGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 73).

[0117] The pegRNAs provided herein also comprise a primer binding site (PBS). In some embodiments, the pegRNA comprises a PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence: ATGAATATAGA (SEQ ID NO: 205), ATGAATATAGAT (SEQ ID NO: 206), ATGAATATAGATA (SEQ ID NO: 207), ATGAATATAGATAC (SEQ ID NO: 208), ATATTTTCTTT (SEQ ID NO: 209), ATGAATATAG (SEQ ID NO: 210), ATGAATATAGATACA (SEQ ID NO: 211), ATGAATATAGATACAG (SEQ ID NO: 212), ATGAATATAGATACAGA (SEQ ID NO: 213), ATATTTTCTTTA (SEQ ID NO: 214), ATATTTTCTTTAA (SEQ ID NO: 215), ATATTTTCTTTAAT (SEQ ID NO: 216), ATGGTGCCAG (SEQ ID NO: 217), ATGGTGCCAGGC (SEQ ID NO: 218), ATGGTGCCAGGCA (SEQ ID NO: 219), ATGGTGCCAGGCAT (SEQ ID NO: 220), ATGGTGCCAGGCATA (SEQ ID NO: 221), ATGGTGCCAGGCATAA (SEQ ID NO: 222), GATGAATATA (SEQ ID NO: 223), GATGAATATAGA (SEQ ID NO: 224), GATGAATATAGAT (SEQ ID NO: 225), GATGAATATAGATA (SEQ ID NO: 226), GATGAATATAGATAC (SEQ ID NO: 227), GATGAATATAGATACA (SEQ ID NO: 228), GTGTTTCCTA (SEQ ID NO: 229), GTGTTTCCTATG (SEQ ID NO: 230), GTGTTTCCTATGA (SEQ ID NO: 231), GTGTTTCCTATGAT (SEQ ID NO: 232), GTGTTTCCTATGATG (SEQ ID NO: 233), GTGTTTCCTATGATGA (SEQ ID NO: 234), ATAATCCAGG (SEQ ID NO: 235), ATAATCCAGGA (SEQ ID NO: 236), 55 / 370 B1195.70196WO00 13363760.2ATAATCCAGGAA (SEQ ID NO: 237), ATAATCCAGGAAA (SEQ ID NO: 238), ATAATCCAGGAAAA (SEQ ID NO: 239), ATAATCCAGGAAAACT (SEQ ID NO: 240), AAACTGAGAA (SEQ ID NO: 241), AAACTGAGAACA (SEQ ID NO: 242), AAACTGAGAACAG (SEQ ID NO: 243), AAACTGAGAACAGA (SEQ ID NO: 244), AAACTGAGAACAGAA (SEQ ID NO: 245), or AAACTGAGAACAGAAT (SEQ ID NO: 246). In some embodiments, the pegRNA comprises a PBS comprising the sequence: ATGAATATAGA (SEQ ID NO: 205), ATGAATATAGAT (SEQ ID NO: 206), ATGAATATAGATA (SEQ ID NO: 207), ATGAATATAGATAC (SEQ ID NO: 208), ATATTTTCTTT (SEQ ID NO: 209), ATGAATATAG (SEQ ID NO: 210), ATGAATATAGATACA (SEQ ID NO: 211), ATGAATATAGATACAG (SEQ ID NO: 212), ATGAATATAGATACAGA (SEQ ID NO: 213), ATATTTTCTTTA (SEQ ID NO: 214), ATATTTTCTTTAA (SEQ ID NO: 215), ATATTTTCTTTAAT (SEQ ID NO: 216), ATGGTGCCAG (SEQ ID NO: 217), ATGGTGCCAGGC (SEQ ID NO: 218), ATGGTGCCAGGCA (SEQ ID NO: 219), ATGGTGCCAGGCAT (SEQ ID NO: 220), ATGGTGCCAGGCATA (SEQ ID NO: 221), ATGGTGCCAGGCATAA (SEQ ID NO: 222), GATGAATATA (SEQ ID NO: 223), GATGAATATAGA (SEQ ID NO: 224), GATGAATATAGAT (SEQ ID NO: 225), GATGAATATAGATA (SEQ ID NO: 226), GATGAATATAGATAC (SEQ ID NO: 227), GATGAATATAGATACA (SEQ ID NO: 228), GTGTTTCCTA (SEQ ID NO: 229), GTGTTTCCTATG (SEQ ID NO: 230), GTGTTTCCTATGA (SEQ ID NO: 231), GTGTTTCCTATGAT (SEQ ID NO: 232), GTGTTTCCTATGATG (SEQ ID NO: 233), GTGTTTCCTATGATGA (SEQ ID NO: 234), ATAATCCAGG (SEQ ID NO: 235), ATAATCCAGGA (SEQ ID NO: 236), ATAATCCAGGAA (SEQ ID NO: 237), ATAATCCAGGAAA (SEQ ID NO: 238), ATAATCCAGGAAAA (SEQ ID NO: 239), ATAATCCAGGAAAACT (SEQ ID NO: 240), AAACTGAGAA (SEQ ID NO: 241), AAACTGAGAACA (SEQ ID NO: 242), AAACTGAGAACAG (SEQ ID NO: 243), AAACTGAGAACAGA (SEQ ID NO: 244), AAACTGAGAACAGAA (SEQ ID NO: 245), or AAACTGAGAACAGAAT (SEQ ID NO: 246). In some embodiments, the pegRNA comprises a PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence ATGAATATAGATA (SEQ ID NO: 207). In certain embodiments, the pegRNA comprises a PBS comprising the sequence ATGAATATAGATA (SEQ ID NO: 207). 56 / 370 B1195.70196WO00 13363760.2

[0118] In some embodiments, the pegRNAs provided herein further comprise a structured motif at the 3' end. In some embodiments, the structured motif is an RNA pseudoknot motif. In some embodiments, the structured motif comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 248). In certain embodiments, the structured motif comprises the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 248).

[0119] In some embodiments, the pegRNA comprises the structure 5'-[spacer]-[backbone scaffold]-[reverse transcription template]-[primer binding site]-[structured motif]-3'. In some embodiments, the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence of any of the pegRNAs in Tables 1 and 2. In some embodiments, the pegRNA comprises a sequence of any of the pegRNAs in Tables 1 and 2. In some embodiments, the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence GTCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTAAAGA AAATATCATCTTTGGTGTTTCCTATGATGAATATAGATACGCGGTTCTATCTAGTTAC GCGTTAAACCAACTAGAA (SEQ ID NO: 250),

[0120] TCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATA AGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATT AAAGAAAATATCATCTTTGGTGTGAGTTACGATGAATATAGATACGCGGTTCTAT CTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 744), or CTGTATCTATATTCATCATGTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGCCTGGCACCATTAAA GAAAATATCATCTTTGGTGTGTCATACGATGAATATAGATACGCGGTTCTATCTAGTT ACGCGTTAAACCAACTAGAA (SEQ ID NO: 251). In certain embodiments, the pegRNA comprises the sequence GTCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTAAAGA 57 / 370 B1195.70196WO00 13363760.2AAATATCATCTTTGGTGTTTCCTATGATGAATATAGATACGCGGTTCTATCTAGTTAC GCGTTAAACCAACTAGAA (SEQ ID NO: 250),

[0121] TCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATA AGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATT AAAGAAAATATCATCTTTGGTGTGAGTTACGATGAATATAGATACGCGGTTCTAT CTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 744), or CTGTATCTATATTCATCATGTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGCCTGGCACCATTAAA GAAAATATCATCTTTGGTGTGTCATACGATGAATATAGATACGCGGTTCTATCTAGTT ACGCGTTAAACCAACTAGAA (SEQ ID NO: 251). In some embodiments, the pegRNA comprises a chemical modification at its 3' end. In certain embodiments, the chemical modification comprises phosphonoacetate (mP).

[0122] In another aspect, the present disclosure provides nicking guide RNAs (ngRNAs) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence TTCACTTCTAATGGTGATTA (SEQ ID NO: 745), TCACTTCTAATGGTGATTAT (SEQ ID NO: 746), AATGGTGATTATGGGAGAAC (SEQ ID NO: 747), GGGAGAACTGGAGCCTTCAG (SEQ ID NO: 748), GGAGAACTGGAGCCTTCAGA (SEQ ID NO: 749), GAGGGTAAAATTAAGCACAG (SEQ ID NO: 750), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), ATTAAAGAAAATATCATCTT (SEQ ID NO: 751), CAAAGCATGCCAACTAGAAG (SEQ ID NO: 752), or CACTACCCAAATTATATATT (SEQ ID NO: 753). In some embodiments, the ngRNA comprises a spacer comprising the sequence TTCACTTCTAATGGTGATTA (SEQ ID NO: 745), TCACTTCTAATGGTGATTAT (SEQ ID NO: 746), AATGGTGATTATGGGAGAAC (SEQ ID NO: 747), GGGAGAACTGGAGCCTTCAG (SEQ ID NO: 748), GGAGAACTGGAGCCTTCAGA (SEQ ID NO: 749), GAGGGTAAAATTAAGCACAG (SEQ ID NO: 750), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), ATTAAAGAAAATATCATCTT (SEQ ID NO: 751), CAAAGCATGCCAACTAGAAG (SEQ ID NO: 752), or CACTACCCAAATTATATATT (SEQ ID NO: 753). In certain embodiments, the ngRNA spacer further comprises a G at its 5' end. 58 / 370 B1195.70196WO00 13363760.2

[0123] In some embodiments, the ngRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17) or GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 761). In certain embodiments, the ngRNA comprises a backbone scaffold comprising the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17) or GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 761).

[0124] In some embodiments, the ngRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence of any of the ngRNAs in Table 1. In some embodiments, the pegRNA comprises a sequence of any of the ngRNAs in Table 1.

[0125] In another aspect, the present disclosure provides dead single guide RNAs (dsgRNAs) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence CTCCCTCCAAGTTGTCCACG (SEQ ID NO: 764), CTGGAGCCTTCAGA (SEQ ID NO: 765), ATTTTACCCTCTGA (SEQ ID NO: 766), GCCAGGCATAATCC (SEQ ID NO: 767), TCCTGGATTATGCC (SEQ ID NO: 768), TCTTTAATGGTGCC (SEQ ID NO: 769), TTACCTCTTCTAGT (SEQ ID NO: 770), ATGCCAACTAGAAG (SEQ ID NO: 771), GCCAAATATATAATT (SEQ ID NO: 772), or CCGCGCGCGCGAAAAAGCCG (SEQ ID NO: 773). In certain embodiments, the dsgRNA comprises a spacer comprising the sequence CTCCCTCCAAGTTGTCCACG (SEQ ID NO: 764), CTGGAGCCTTCAGA (SEQ ID NO: 765), ATTTTACCCTCTGA (SEQ ID NO: 766), GCCAGGCATAATCC (SEQ ID NO: 767), TCCTGGATTATGCC (SEQ ID NO: 768), TCTTTAATGGTGCC (SEQ ID NO: 769), TTACCTCTTCTAGT (SEQ ID NO: 770), ATGCCAACTAGAAG (SEQ ID NO: 771), GCCAAATATATAATT (SEQ ID NO: 772), or CCGCGCGCGCGAAAAAGCCG (SEQ ID NO: 773). 59 / 370 B1195.70196WO00 13363760.2

[0126] In some embodiments, the dsgRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGCTTT (SEQ ID NO: 782), GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 761), or GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17). In certain embodiments, the dsgRNA comprises a backbone scaffold comprising the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGCTTT (SEQ ID NO: 782), GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 761), or GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17).

[0127] In some embodiments, the dsgRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence of any of the dsgRNAs in Table 1. In some embodiments, the dsgRNA comprises a sequence of any of the dsgRNAs in Table 1.

[0128] Additional sequences of suitable pegRNAs, ngRNAs, and dsgRNAs for prime editing CFTR within the scope of the present disclosure will be apparent to those of skill in the art. Such suitable pegRNAs, ngRNAs, and dsgRNAs sequences typically comprise a spacer sequence that is complementary to a nucleic sequence within 50 nucleotides (e.g., within 45, 40, 35, 30, 25, 20, 15, or 10 nucleotides) upstream or downstream of the target nucleotide to be edited (e.g., a target mutation in a CFTR gene).

[0129] In general, a pegRNA, ngRNA, or dsgRNA spacer is any RNA sequence having sufficient complementarity with a target polynucleotide sequence (e.g., CFTR) to hybridize with the target sequence and direct sequence-specific binding of a napDNAbp (e.g., Cas9, which may be part of a prime editor) to the target sequence. In some embodiments, the degree of complementarity between the spacer and its corresponding target sequence in CFTR, when 60 / 370 B1195.70196WO00 13363760.2optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more (or the spacer and the corresponding target sequence comprise one, two, three, four, five, six, seven, eight, nine, or ten amino acid differences). In certain embodiments, the spacer is 100% complementary to its corresponding target sequence in CFTR. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0130] The ability of a pegRNA, ngRNA, or dsgRNA to direct sequence-specific binding of a prime editor to a target sequence may also be assessed by any suitable assay. For example, a prime editor and pegRNA may be provided to a host cell having the corresponding target sequence (e.g., CFTR, or a portion thereof), such as by transfection with vectors encoding the prime editor and pegRNA or by transfection of a ribonucleoprotein (RNP) complex, followed by an assessment of preferential cleavage, nicking, or editing within the target sequence. Similarly, cleavage or editing of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, prime editor, and pegRNA to be tested and a control pegRNA different from the test pegRNA, and comparing binding or rate of cleavage or editing at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will be apparent to those skilled in the art.

[0131] In some embodiments, a pegRNA, ngRNA, or dsgRNA is about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 75, about 100, or more nucleotides in length. In some embodiments, a gRNA is about 50-150, about 60-140, about 70-130, about 80-120, or about 90-110 nucleotides in length. In some embodiments, the spacer sequence of a pegRNA, ngRNA, or dsgRNA is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides in length.

[0132] In some embodiments, a pegRNA, ngRNA, or dsgRNA comprises an optional linker sequence. For example, the gRNAs provided herein may comprise an optional linker sequence between the spacer and the backbone scaffold sequences. In certain embodiments, the optional linker sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 61 / 370 B1195.70196WO00 13363760.2nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, or at least 50 nucleotides in length. Methods of Prime Editing CFTR

[0133] Some aspects of the present disclosure provide methods of prime editing a CFTR gene. In one aspect, the present disclosure provides methods of prime editing a CFTR gene comprising contacting a nucleic acid sequence encoding the CFTR gene with a prime editor and any of the pegRNAs disclosed herein. In some embodiments, the method further comprises providing any of the ngRNAs disclosed herein. In some embodiments, the method further comprises providing any of the dsgRNAs disclosed herein.

[0134] Any of the prime editors disclosed herein, or any prime editor known in the art, can be used in the methods of the present disclosure. In some embodiments, the prime editor comprises a nucleic acid-programmable DNA-binding protein (napDNAbp) and a polymerase. In some embodiments, the prime editor comprises a napDNAbp (e.g., a Cas9 protein, such as SpCas9, or a variant thereof, such as nCas9 or dCas9) and a polymerase (e.g., a reverse transcriptase, such as an MMLV reverse transcriptase, a Tf1 reverse transcriptase, or a variant thereof). In certain embodiments, the prime editor comprises a PE6 Cas9 variant and / or a PE6 reverse transcriptase. In some embodiments, the prime editor is a PE6c prime editor. In some embodiments, the prime editor comprises PEmax architecture as provided herein.

[0135] In some embodiments, the method further comprises providing an inhibitor of the cellular mismatch repair (MMR) pathway. In some embodiments, the inhibitor of the MMR pathway comprises a dominant negative variant of MLH1 (MLH1dn). In certain embodiments, the MLH1dn comprises a truncated version of the wild type MLH1 protein (e.g., with the C-terminal amino acids 754-756 truncated as described herein).

[0136] In some embodiments, the step of contacting corrects an F508del mutation in the CFTR protein. In some embodiments, the step of contacting results in the insertion of the sequence 5′-CTT-3′ into the CFTR gene. In some embodiments, the step of contacting results in the correction of the CFTR gene to a wild-type sequence. In some embodiments, the step of contacting results in at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, or at least 50% correction of the CFTR gene to a wild-type sequence within a cell. In some 62 / 370 B1195.70196WO00 13363760.2embodiments, the step of contacting results in an increase in CFTR ion channel function to at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, or at least 50% of wild-type levels within a cell.

[0137] In some embodiments, the step of contacting results in the installation of one or more silent edits into the CFTR gene. In certain embodiments, the one or more silent edits comprise one or more PAM-disrupting edits. In certain embodiments, the one or more silent edits comprise one or more MMR pathway-inhibiting edits. Silent mutations may be introduced, for example, in the CFTR codons encoding CFTR amino acid positions I507, F508, G509, V510, S511, and / or Y512. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR I507 from ATC to ATA. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR F508 from TTT to TTC. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR G509 from GGT to GGA. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR V510 from GTT to GTG. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR V510 from GTT to GTC. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR V510 from GTT to GTA. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR S511 from TCC to AGT. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR S511 from TCC to TCA. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR S511 from TCC to TCG. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR S511 from TCC to AGC. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR S511 from TCC to TCT. In some embodiments, the one or more silent edits installed into the CFTR gene comprise changing the codon encoding CFTR Y512 from TAT to TAC.

[0138] In some embodiments, the contacting step comprises delivering one or more polynucleotides encoding the pegRNA and the prime editor (and optionally a ngRNA and / or dsgRNA) to the nucleic acid sequence encoding the CFTR gene. In some embodiments, the contacting step is performed in a cell, such as a human or non-human airway epithelial sell. 63 / 370 B1195.70196WO00 13363760.2In some embodiments, the cell is in a tissue selected from the group consisting of lung tissue, pancreatic tissue, liver tissue, kidney tissue, or intestinal tissue. In some embodiments, the contacting step is performed in vitro. In some embodiments, the contacting step is performed in vivo. In certain embodiments, the contacting step is performed in a subject. A subject may have been diagnosed with a disease, or be at risk for having a disease. In some embodiments, the method is a method for treating a disease in a subject. In some embodiments, the disease is cystic fibrosis.

[0139] In some aspects, the present disclosure contemplates use of any of the pegRNAs, ngRNAs, dsgRNAs, compositions, polynucleotides, vectors, pharmaceutical compositions, and / or cells disclosed herein in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cystic fibrosis). In some aspects, any of the pegRNAs, ngRNAs, dsgRNAs, compositions, polynucleotides, vectors, pharmaceutical compositions, and / or cells disclosed herein are for use in medicine. In some embodiments, the present disclosure provides for veterinary uses (e.g., in non-human animals) of any of the pegRNAs, ngRNAs, dsgRNAs, compositions, polynucleotides, vectors, pharmaceutical compositions, cells, and / or methods provided herein. Pharmaceutical compositions

[0140] Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the pegRNAs, ngRNAs, dsgRNAs, prime editors, compositions, systems, polynucleotides, vectors, and / or cells described herein. The term “pharmaceutical composition,” as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g., for specific delivery, increasing half-life, or other therapeutic compounds).

[0141] As used here, the term “pharmaceutically-acceptable carrier” (or “pharmaceutically acceptable excipient”) means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue, or portion of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients 64 / 370 B1195.70196WO00 13363760.2of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.). Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose;(2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, and antioxidants can also be present in the formulation. Terms such as “excipient,” “carrier,” “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” or the like are used interchangeably herein.

[0142] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject for gene editing (e.g., prime editing).

[0143] The pharmaceutical compositions described herein may be administered or packaged as a unit dose, for example. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

[0144] In some embodiments, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of 65 / 370 B1195.70196WO00 13363760.2materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierce-able by a hypodermic injection needle. The active agent in the composition is a compound of the invention. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. Polynucleotides, Vectors, Cells, and Kits

[0145] The present disclosure provides, in some aspects, polynucleotides and vectors encoding any of the pegRNAs, ngRNAs, dsgRNAs, prime editors, systems, and / or compositions described herein. In some aspects, the present disclosure provides polynucleotides and vectors encoding a pegRNA and a prime editor as disclosed herein. In some embodiments, the polynucleotides and vectors provided herein comprise DNA (e.g., plasmid DNA or viral DNA). In some embodiments, the polynucleotides and vectors provided herein comprise RNA (e.g., mRNA or viral RNA).

[0146] Cells that may contain any of the pegRNAs, ngRNAs, dsgRNAs, prime editors, systems, and / or compositions described herein are also provided by the present disclosure. The methods described herein may be used to deliver a pegRNA and prime editor (and optionally an ngRNA and / or dsgRNA) into a eukaryotic cell (e.g., a mammalian cell, such as a human cell). In some embodiments, the cell is in vitro (e.g., a cultured cell). In some embodiments, the cell is in vivo (e.g., in a subject, such as a human subject). In some embodiments, the cell is ex vivo (e.g., isolated from a subject and may be administered back to the same or a different subject).

[0147] In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some 66 / 370 B1195.70196WO00 13363760.2embodiments, a cell transiently transfected with the components of a prime editing system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a prime editing complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence.

[0148] The pegRNAs, ngRNAs, dsgRNAs, prime editors, systems, compositions, polynucleotides, and / or vectors described herein may also be assembled into kits. In some embodiments, the kit comprises polynucleotides for expression of the g pegRNAs, ngRNAs, dsgRNAs, prime editors, systems, and / or compositions described herein. In some embodiments, the kit comprises appropriate pegRNAs or nucleic acid vectors for the expression of such pegRNAs to target the Cas9 protein of a prime editor to a desired target sequence, e.g., in CFTR. In some embodiments, the pegRNAs in the kit are useful for correcting an F508del mutation in a CFTR protein.

[0149] The kits described herein may include one or more containers housing components for performing the methods described herein, and optionally instructions for use. Any of the kits described herein may further comprise components needed for performing the prime editing methods described herein. Each component of the kits, where applicable, may be provided in liquid form (e.g., in solution) or in solid form, (e.g., a dry powder). In certain cases, some of the components may be reconstitutable or otherwise processible (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water), which may or may not be provided with the kit.

[0150] In some embodiments, the kits may optionally include instructions and / or promotion for use of the components provided. As used herein, “instructions” can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and / or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use, or sale for animal administration. As used herein, “promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity 67 / 370 B1195.70196WO00 13363760.2including pharmaceutical sales, and any advertising or other promotional activity including written, oral, and electronic communication of any form, associated with the disclosure. Additionally, the kits may include other components depending on the specific application, as described herein.

[0151] The kits may contain any one or more of the components described herein in one or more containers. The components may be prepared sterilely, packaged in a syringe, and shipped refrigerated. Alternatively, they may be housed in a vial or other container for storage. A second container may have other components prepared sterilely. Alternatively, the kits may include the active agents premixed and shipped in a vial, tube, or other container.

[0152] The kits may have a variety of forms, such as a blister pouch, a shrink-wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box, or a bag. The kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc. EXAMPLES Example 1. Successive prime editing enhancements enable efficient, precise correction of CFTR F508del and functional rescue in human airway epithelial cells

[0153] Prime editing (PE) enables precise and versatile genome editing capable of installing all 12 possible substitutions as well as deletions and insertions of hundreds of base pairs without requiring double-stranded breaks. Here, the systematic, multi-tiered development of PE strategies to convert the CFTR F508del mutation, the predominant cause of cystic fibrosis (CF), to wild-type CFTR by precisely inserting the missing nucleotides was demonstrated. Initial attempts with first-generation PE systems yielded only very low editing efficiencies in human cells (<0.5%). By combining six recent advances in prime editing—epegRNAs, the PEmax architecture, MLH1dn, strategic silent edits, novel PE6 variants, and dsgRNAs—that each address different bottlenecks in PE efficiency, CFTR F508del precise correction efficiencies were increased 120-fold. Treating immortalized human bronchial epithelial cells 68 / 370 B1195.70196WO00 13363760.2homozygous for CFTR F508del with the resulting highly optimized PE system yielded an average of 51% precise correction to wild-type CFTR. In primary CF patient airway epithelial cells from multiple donors, this prime editing treatment yielded 25% average CFTR F508del correction efficiency and restored CFTR ion channel function to >50% of wild type levels, achieving functional rescue comparable to or higher than the outcome of treatment with the combination of small-molecule drugs elexacaftor+tezacaftor+ivacaftor. This prime editing strategy exhibited minimal off-target editing and a 3.4-fold higher ratio of precise correction:indel byproducts compared to previous optimized nuclease-mediated homology- directed repair (HDR) approaches. Direct, efficient F508del correction in primary CF patient airway epithelial cells, restoring CFTR function, suggests the feasibility of a durable one-time treatment for CF, and provides a blueprint for optimizing efficient PE strategies to correct other pathogenic gene variants. Initial attempts to correct CFTR F508del with PE2 and PE3

[0154] To efficiently test and optimize PE CFTR F508del correction strategies61, PE was first used to generate a clonal HEK293T cell line homozygous for this deletion in the endogenous CFTR gene (FIG.5). Next, this cell line was used with PE2 to screen pegRNAs with combinations of PBS and RTT lengths at two F508del CTT deletion-proximal protospacers with NGG PAMs (NGG1 and NGG2; see FIG.6). Correction of CFTR F508del using NGG1 pegRNAs was low (FIG.7), which was hypothesized to be due to the presence of a TTTT sequence on the non-PAM containing strand of the NGG1 protospacer. It was speculated that this TTTT may act as an RNA Polymerase III transcriptional terminator that prevents complete pegRNA PBS transcription from a U6 promoter, resulting in an incompletely transcribed pegRNA that cannot support PE (FIG.6). F508del correction was observed when NGG2 pegRNAs were used, yielding average editing efficiencies of 0.2% or less (FIG.1B).

[0155] To enhance correction of F508del, a PE3 screen of two NGG2 pegRNAs (NGG2 PBS13 RTT29 and NGG2 PBS14 RTT41) was performed against a panel of all available ngRNA protospacers with pegRNA-ngRNA inter-nick distances of approximately 125 bp (FIG.1C, FIG.1D). The best-performing PE3 NGG2 pegRNA offered up to a 2.6-fold improvement in edit installation efficiency over PE2, and maximum mean editing for any NGG2 PE3 edit was 0.5% or less.

[0156] It was hypothesized that the inefficient correction of F508del using NGG1 and NGG2 could be due to inaccessibility of the edit site to Cas effectors due to chromatin state, which 69 / 370 B1195.70196WO00 13363760.2has been reported to negatively affect all major forms of mammalian cell genome editing9,62–64. CFTR resides within a topologically associated domain whose chromatin state is tightlycontrolled by protein interactions with numerous enhancers and insulators embedded within the gene65. To distinguish chromatin accessibility from prime editor-specific editing limitations, both NGG1 and NGG2 were targeted with pegRNA-guided ABE8e- SpCas9(D10A) adenine base editor (ABE)66to test if a base editor could edit either protospacer. Efficient A•T-to-G•C editing at adenine bases was observed at NGG1 and NGG2, averaging up to 45% and 29%, respectively, (FIG.1E), demonstrating that NGG1 and NGG2 were indeed accessible by pegRNA-guided Cas-effectors. These results suggest that suboptimal execution of the PE steps that follow target site engagement was primarily responsible for inefficient CFTR F508del correction. Advances in PE technologies enhance precise correction of CFTR F508del

[0157] Several enhancements to PE were developed (FIG.2A). The first of these improvements was the development of engineered pegRNAs (epegRNAs), which protect pegRNA RTT and PBS sequences from endogenous exonuclease degradation by appending an RNA structural motif to pegRNA 3′ ends40. Inhibition or evasion of cellular DNA mismatch repair (MMR) enhances PE purity and efficiency41, and PEmax, an architecture- optimized prime editor protein, further increases editing efficiency41. The PE6 suite of laboratory-evolved and engineered reverse transcriptases and prime editor Cas9 domains also have enhanced editing capabilities34. It was sought to combine the capabilities of these technologies to improve CFTR F508del correction.

[0158] 178 epegRNAs were first screened with combinations of PBS and RTT lengths at seven protospacers to explore correction from several F508del-proximal protospacers (FIG. 8A). Four protospacers had NGG PAMs (NGG1-NGG4, including NGG1 and NGG2 from the initial PE2 and PE3 experiments), and three protospacers had NGA PAMs (NGA1- NGA3), which have been demonstrated as targetable by prime editors with SpCas9- VRQR(H840A) Cas9 nickase domains67. This first screen was completed using PE4max with epegRNAs (where PE4 denotes PE2 + MLH1dn, a dominant negative MLH1 variant that impedes MMR). Marginal rates of editing with the NGA1, NGA2, NGG1, NGG3, and NGG4 epegRNAs (<0.1% F508del correction) were observed, but up to 0.40% average correction of F508del with NGA3 was observed (FIGs.8B-8G). A large panel of 96 epegRNAs with different combinations of PBS and RTT lengths was tested (FIG.2B and FIG.8H). From the 70 / 370 B1195.70196WO00 13363760.2NGG2 PE4max panel, the epegRNA NGG2 PBS13 RTT41 strategy was identified, which yielded a mean PE4max editing rate of 0.61%, an approximately 3.8-fold improvement over the previous best PE2 pegRNA outcome. It was elected to move forward with further F508del edit optimization with the NGG2 PBS13 RTT41 epegRNA.

[0159] To further develop the editing strategy to correct F508del, the ngRNA panel from the earlier PE3 experiments was tested with the epegRNA NGG2 PBS13 RTT41 in a PE5max experiment (where PE5 denotes PE3 + MLH1dn) (FIG.9). A +104 nick that enabled a mean editing rate of 0.86% when used with the epegRNA NGG2 PBS13 RTT41 was identified, representing a two-fold improvement over the previous best PE3 editing strategy and further demonstrating the utility of epegRNAs, PEmax, and MLH1dn when paired with a ngRNA.

[0160] Once a suitable epegRNA and ngRNA pair was identified, it was sought to further enhance PE by installing benign silent edits simultaneously with the CTT insertion that corrects F508del. Silent edits were selected with two purposes: (1) to disrupt NGG2’s PAM and / or protospacer seed sequence and thereby enhance edit installation efficiency1and (2) to install additional silent edits around the PAM-disrupting edits, thereby creating a contiguous or semi-contiguous tract of mismatches that would cause the PE heteroduplex intermediate to evade MMR, enhancing editing efficiency41. In PE5max experiments with the NGG2 PBS13 RTT41 epegRNA, four PE strategies that co-installed silent edits with F508del correction (strategies SE1 to SE4) were tested. These strategies took advantage of the flexible third nucleotide position in phenylalanine, glycine, valine, and tyrosine codons and the ability of serine codons to be completely recoded (FIG.2C, FIG.10). Compared to correction of F508del alone without any additional silent mutation installation (SE0), SE2’s co-installation of two NGG2 PAM-disrupting edits and three additional silent mutations resulted in a large (five-fold) improvement in F508del correction, with a mean editing rate of 6.8% (FIG.2D). This result suggests that additional silent edits to disrupt an epegRNA protospacer’s PAM and enhance MMR evasion can offer major increases in PE efficiency, even when the co- installed silent edits are distal from the corrective edit.

[0161] The last PE enhancement that was evaluated in the F508del HEK293T cell line was the use of recently evolved PE6 prime editor protein variants34. Using the optimized NGG2 PBS13 RTT41 SE2 epegRNA with MLH1dn and the +104 ngRNA, PEmax was compared to PE6 variants PE6a-PE6g (FIG.2E). An enhanced F508del correction with PE6b and PE6c compared to PEmax was observed, with mean editing rates of 9.8%, 11%, and 7.4%, respectively, consistent with previous observations that PE6b and PE6c enhance editing 71 / 370 B1195.70196WO00 13363760.2efficiencies at a variety of targets34. Notably, PE6c offered a 1.5-fold improvement in editing activity over PEmax using the NGG2 PBS13 RTT41 SE2 epegRNA, consistent with the previous observations that PE6c can outperform PEmax at many challenging therapeutic edits that use a long RTT34. These enhancements of PE efficiencies demonstrate that, when matched with edits tailored to their respective strengths, PE6 variants can synergistically improve PE efficiencies in coordination with other PE improvements including epegRNAs, MLH1dn, and silent edits. Interdependent PE enhancements enable editing in immortalized airway epithelial cells

[0162] Having identified a suitable PE6-based strategy for the correction of CFTR F508del in HEK293T cells, the PE strategy in immortalized human bronchial epithelial cells was tested and further optimized.16HBE14o- cells homozygous was used for F508del and M470 (16HBEge-F508del) generated by the Cystic Fibrosis Foundation68. These cells show reduced CFTR protein expression and impaired chloride channel function68. The prime editor components identified in the above experiments were delivered into 16HBEge-F508del cells as RNA (in vitro transcribed mRNA for PE and MLH1dn proteins; chemically synthesized RNAs for epegRNA and ngRNA) via nucleofection. It was found that epegRNAs protected with 3′ phosphonoacetate (mP) modifications enhanced editing efficiency by 1.3-fold over epegRNAs protected with 3′ phosphorothioate modifications, consistent with past studies that have found the mP modification to enhance editing by Cas9 nucleases69(FIG.11A).

[0163] CFTR is actively transcribed in 16HBEge-F508del cells68, suggesting that the chromatin state of the gene may make it more accessible to gene editing agents than it is in HEK293T cells. It was sought to determine whether chromatin accessibility or prime editor- specific editing constraints may be the limiting factor for F508del correction in 16HBEge- F508del cells. To assess these possibilities, ABEs as well as cytosine base editors (CBEs) were targeted to NGG and NGA protospacers adjacent to F508del using standard Cas9 sgRNAs and pegRNAs. Efficient base editing was observed at all protospacers tested (87% to 98% editing for ABEs and 67% to 94% editing for CBEs (FIG.11B). However, it was observed that editing rates were lower at bases A10 and A12 of protospacer NGG2 when ABE was targeted using a pegRNA or an epegRNA compared to a sgRNA (FIG.3A). This result suggests that although Cas domains can efficiently engage CFTR in 16HBEge-F508del cells, the pegRNAs or epegRNAs used for PE may hinder the editors’ ability to bind the target site, potentially due to the reduced affinity that Cas9 displays for pegRNAs and epegRNAs 72 / 370 B1195.70196WO00 13363760.2compared to sgRNAs40resulting in a smaller fraction of prime editors existing in a guide RNA-bound, target-searching state. This observation led to the hypothesis that it may be possible to increase the accessibility of prime editors to CFTR by further modulating local chromatin context.

[0164] 5′-truncated guide RNAs with only 14-16 nucleotides of protospacer complementarity enable Cas9 to bind to a target sequence but do not support DNA cleavage70. It was hypothesized that leveraging one such catalytically “dead” guide RNA (dsgRNA) to direct prime editor protein to a site proximal to F508del would disrupt local chromatin state and increase the accessibility of the prime editor to the site of the therapeutic edit (FIG.3B); this strategy has been shown to improve prime editing efficiency in mouse cells9. Eight NGG- PAM dsgRNAs were designed with 14-nucleotide spacers that bind to sequences within 110 bases of NGG2, and their effect on F508del correction was tested when co-delivered with PE6c into 16HBEge-F508del cells. It was found that 3 of 8 tested dsgRNAs substantially improved editing efficiency, by an average of 1.9-fold. The best-performing dsgRNA, binding at position -40 relative to the nick location specified by the NGG2 epegRNA, enabled a two-fold increase in CFTR F508del correction (to a mean efficiency of 42.6%) compared to a control that used a non-targeting dsgRNA (FIG.3C). The -40 dsgRNA was included in subsequent experiments with 16HBEge-F508del cells and primary CF airway epithelial cells from patients.

[0165] Thus, over the course of efforts to maximize PE-mediated F508del correction in HEK293T and 16HBEge-F508del cell lines, four enhancements were identified—(1) epegRNAs, (2) silent edits, (3) PE6 variants, and (4) dsgRNAs—that each substantially increased editing efficiency. Since these enhancements each improve editing efficiency by a different hypothesized mechanism, it was tested how the inclusion of each enhancement, both alone and in combination, contributes to efficient editing in 16HBEge-F508del cells. It was found that all four enhancements help maximize editing efficiency in 16HBEge-F508del cells (FIG.3D). In the absence of these four key enhancements, PE5max F508del correction averaged 1.8% with 0.57% indel formation; with all four enhancements implemented, a mean F508del correction rate of 51% with 13% indel formation was observed, a 28-fold improvement in editing efficiency while also increasing edit-to-indel ratio from 3.1 to 3.9. In a separate experiment, the influence of MLH1dn on editing efficiency in the presence of these enhancements was characterized. It was found that omitting MLH1dn led to a modest reduction in F508del correction efficiency from 58% to 52% and a decrease in edit-to-indel 73 / 370 B1195.70196WO00 13363760.2ratio from 4.8 to 3.2 (FIG.3E). These findings indicate that mechanistically distinct improvements to PE systems contribute synergistically to enhanced editing efficiencies. Reevaluation of the CFTR F508del PE correction strategy

[0166] In view of the editing rates observed in the initial pegRNA optimization efforts using PE2 and PE4max (FIGs.1B and 2B), it was next assessed whether the selected epegRNA characteristics remained optimal in the presence of other enhancements. epegRNA PBS and RTT lengths were re-screened at NGG2 in CFTR F508del HEK293T cells using PE6c with MLH1dn, SE2 silent edits, the -40 dsgRNA, and the +104 ngRNA. It was found that PBS13 RTT41 was still among the most optimal PBS and RTT combinations (FIG.15A). Re- screening nicking sgRNAs also indicated that the +104 nsgRNA remained optimal (FIG. 15B). These results suggest that optimal epegRNA and ngRNA designs for the CFTR F508del corrective edit remain the same in the context of diverse prime editing enhancements, and therefore for at least some target sites, these characteristics can be optimized independently of other improvements to the prime editing system.

[0167] It was next considered whether the marginal PE2 and PE4max editing rates (FIGs.1B and 2B) may be the result of epegRNA PBS-RTT instability or cis-acting interactions between the spacer and the PBS. To circumvent these potential issues, the petRNA system in which epegRNAs are split into separate sgRNAs and circularized RTT-PBS transcripts was tested94. Using combinations of an NGG2 sgRNA and petRNAs with varied PBS and RTT lengths, F508del correction in CFTR F508del HEK293T cells was re-screened using MLH1dn, SE2 silent edits, the -40 dsgRNA, and the +104 ngRNA (FIG.15C). As previously described94, a nCas9 (here based on PEmax) and an MCP-RT (here using a PE6c-based reverse transcriptase) were used for petRNA editing. While efficient petRNA editing at DNMT1 and RUNX1 positive control targets was recapitulated (FIG.15D), CFTR F508del correction greater than 4% was not observed, indicating that the use of petRNAs did not enhance PE correction of F508del.

[0168] It was also assessed whether epegRNA screening could be accelerated using computational tools for pegRNA design. Using PE6c with MLH1dn, the -40 dsgRNA, and the +104 ngRNA, the F508del correction efficiencies of the top 24 epegRNAs predicted by PRIDICT95and the top 24 epegRNAs predicted by DeepPrime96were tested. However, neither model predicted NGG2 PBS13 RTT41 within their top 24 designs; this epegRNA 74 / 370 B1195.70196WO00 13363760.2outperformed all others generated from the suggestions of both models in the context of both SE0 and SE2 (FIGs.16A-16D). F508del correction in primary CF patient airway epithelial cells restores CFTR function

[0169] The F508del correction strategy was next tested in primary human airway epithelial cells from three CF patients with homozygous CFTR F508del alleles. Using PE6c with MLH1dn, the -40 dsgRNA, the +104 ngRNA, and the NGG2 PBS13 RTT41 SE2 epegRNA delivered by RNA electroporation, a mean F508del correction rate of 25% with 6.5% indels (FIG.4A) among all treated cells with no sorting was observed.

[0170] To determine the phenotypic impact of correcting F508del in primary CF airway epithelial cells, CFTR-mediated anion secretion in prime edited donor cells under short- circuit conditions was quantified following differentiation for three weeks at an air-liquid interface (ALI). The change in short-circuit current (Isc) was recorded following stimulation with the CFTR-activating agents forskolin and 3-isobutyl-1-methylxanthine (F&I) and inhibition with the CFTR-specific channel inhibitor CFTR(inh)-172. Donor-matched mock- treated epithelial cells (electroporated without RNA), epithelial cells treated with elexacaftor+tezacaftor+ivacaftor (ETI), and non-CF epithelial cells were included as electrophysiological controls. Compared to mock-treated CF donor epithelial cells, F&I stimulation resulted in substantially increased CFTR-dependent Isc in the prime editor-treated cells and CFTR(inh)-172 administration likewise resulted in decreased CFTR-dependent Isc in the prime editor-treated cells (FIGs.4B-4C). Compared to treatment with ETI, prime editor-treated cells exhibited a similar change in CFTR-dependent Isc when stimulated with F&I or after CFTR(inh)-172 administration (FIGs.4B-4C). These results demonstrate substantial rescue of CFTR channel activity following correction by prime editing, with CFTR function in prime editor-treated donor cells exceeding 50% of current levels in healthy (non-CF) airway epithelial cells (FIG.4C).

[0171] To assess the persistence of F508del correction during cellular differentiation, F508del correction rates in airway epithelial cells from two ALI-cultured populations was evaluated. It was found that rates of F508del correction remained consistent when compared at three days post-electroporation and after three weeks of differentiation at ALI (FIGs.8A- 8H), demonstrating the edit’s stability during cellular expansion. These findings indicate that the optimized PE strategy to correct the CFTR F508del CTT deletion not only restores expression of functional CFTR capable of much-improved anion transport in primary CF 75 / 370 B1195.70196WO00 13363760.2airway epithelial cells from patients but also that the corrective edit persists through cellular proliferation and differentiation. On-target and off-target editing analysis

[0172] Having established that enhanced prime editing systems can efficiently correct CFTR F508del and restore CFTR function in primary CF airway epithelial cells, it was sought to characterize unintended editing outcomes of the prime editing strategy—including epegRNA scaffold integration, partial silent edit incorporation, and genomic off-target editing—in detail. The highly processive Tf1 reverse transcriptase used in PE6c can generate indels by reverse transcribing the epegRNA scaffold, which can be incorporated into the target genomic DNA34. To determine if this scaffold incorporation occurs in prime-edited primary CF airway epithelial cells, HTS reads for partial and complete scaffold integration events were analyzed. The first base of the epegRNA scaffold, indexed from the 3′ end, is complementary to the first base of genomic DNA encoded beyond the RTT; it therefore cannot be distinguished between 0 and 1 bases of scaffold integration, as these lead to identical (scaffold-free) sequence outcomes. Across three edited donor cell lines, no scaffold incorporation in 98.8%-99.9% (mean: 99.2%) of reads containing F508del CTT insertion (FIG.12A) were observed. No additional scaffold incorporation in three replicates of 16HBEge-F508del cells edited with high-processivity PE6c (percentage of CTT insertion events that are scaffold-free: 97.9%-98.0%, mean 98.0%) compared to PEmax (percentage of CTT insertion events that are scaffold-free: 97.3%-98.7%, mean 98.0%) (FIG.12B) was observed. These findings are consistent with the previous observation that highly processive PE6 variants do not increase rates of scaffold incorporation when carefully matched with edits suited to their capabilities (such as using PE6c to enhance editing with the long 3′ extension of the NGG2 PBS13 RTT41 SE2 epegRNA)34. Using NUPACK71, the folding energy of the PBS and RTT of the NGG2 PBS13 RTT41 SE2 epegRNA is -7.80 kcal / mol. The choice of PE6c for this edit is consistent with the prior findings that Tf1-based PE variants (PE6b and PE6c) frequently outperform alternatives for epegRNAs with predicted secondary structure folding energies smaller in magnitude (less stable) than -23 kcal / mol34.

[0173] The CFTR F508del correction strategy developed above installs silent edits—most notably, converting a serine codon from TCC to AGT—to evade cellular mismatch repair mechanisms. Partial incorporation of these silent edits could lead to nonsynonymous changes in protein sequence. The occurrence of all possible combinations of partial silent edit 76 / 370 B1195.70196WO00 13363760.2incorporation, both with and without concomitant CTT insertion, in treated primary airway epithelial cells was searched for. It was found that partial silent edits are incorporated at a low frequency without CTT insertion, and the incorporation of any partial edits that would lead to outcomes encoding nonsynonymous protein sequences (FIGs.13A-13B) was not observed. Some of these silent edits also disrupt Cas9 protospacer and PAM sequences and thus should prevent Cas9 re-engagement after editing. It was observed the incorporation of protospacer and PAM edits without concomitant CTT insertion in primary CF airway epithelial cells with a mean frequency of 3.6% (compared to a mean CTT insertion frequency of 23%). This editing outcome effectively renders a small fraction of F508del-containing sequences un- targetable by PE, eliminating their ability to be corrected. The increase in CTT insertion frequency observed when installing silent edits therefore greatly outweighs the fraction of un- correctable sequences generated due to partial silent edit incorporation. It was thus recommended that partial silent edit incorporation be investigated in future therapeutic PE studies, especially if some silent edits encode changes to the protospacer or PAM sequences.

[0174] To characterize off-target editing frequencies of the optimized PE strategy, CIRCLE- seq72was used to identify sites within the human genome that are cleaved in vitro by Cas9 guided with the epegRNA, ngRNA, or dsgRNA used in the CFTR F508del prime editing strategy. Editing outcomes were assessed in primary CF airway epithelial cells from three separate donors at the top 32 candidate sites for each of the three guide RNAs, representing 96 total CIRCLE-seq-nominated off-target candidate sites investigated. Few differences were observed in off-target indel or substitution formation in PE-treated donor cells compared to untreated donor cells (FIG.4D and FIGs.14A-14C), consistent with the observation from multiple laboratories that PE induces few detected off-target modifications, likely due to its mechanism requiring multiple DNA hybridization events,1,3,5–17. Of the 96 total CIRCLE-seq nominated off-target sites that were assessed in primary CF airway epithelial cells, four showed a mean 1.5-fold or higher increase in indel formation in treated cells compared to untreated cells (FIG.4D), potentially suggestive of off-target editing. No off-target sites showed a mean 1.5-fold or higher increase in substitution formation in treated cells compared to untreated cells.

[0175] The indel frequencies at all four of these potential off-target sites were ≤0.1% in treated samples across all replicates, indicating that off-target modification at these sites occurs at a very low frequency. These sites were analyzed more closely to assess their potential physiological relevance (FIG.4D). One of the four sites is engaged by the 77 / 370 B1195.70196WO00 13363760.2epegRNA (epegRNA-OT01) and targets the EPCAM-DT lncRNA, whose function is unknown. The remaining three sites are engaged by the ngRNA. Two of these sites lie within intergenic regions and are unlikely to have strong physiological consequences if disrupted at a low frequency. The final site (ngRNA-OT18) is situated within an intronic region of RUNX173. Because ngRNA-OT18 is located ~60 kilobases from the nearest exon and showed a low mean indel formation rate of 0.048% in treated primary cells, this off-target site is not anticipated to have significant physiological consequences. No potential off-target sites were detected from the dsgRNA. These observations collectively suggest that the PE CFTR F508del correction strategy induces few off-target edits in the human genome under the tested conditions. Discussion

[0176] By systematically refining a PE strategy to correct CFTR F508del, large improvements in editing efficiency were achieved. The initial PE3 experiments only resulted in up to 0.42% mean F508del correction. By applying several recently described PE improvements to the CFTR F508del correction strategy, 11%, 51%, and 25% mean F508del correction in HEK293T cells, 16HBEge-F508del cells, and primary CF patient airway epithelial cells, representing 26-fold, 120-fold, and 59-fold improvements in F508del correction over the original PE3 editing attempt, respectively was achieved. Six improvements to the original PE system each contributed to the optimization of the F508del correction strategy: (1) epegRNAs, (2) PEmax, (3) MLH1dn, (4) silent edits, (5) PE6, and (6) dsgRNAs. Experiments in F508del 16HBEge-F508del cells demonstrated that, when used in combination, epegRNAs, silent edits, PE6c, and dsgRNAs can each contribute comparably to a 28-fold net improvement in editing efficiency (FIG.3D). Previous reports have noted the difficulty of PE correction of CFTR F508del8and other CF-causing mutations in CFTR8,74. It is hoped that the systematic process used to achieve efficient F508del correction starting from very low initial editing efficiency can serve as a guide for other therapeutic PE efforts.

[0177] In CF donor airway epithelial cells, 25% mean F508del correction rescued CFTR ion channel function to greater than 50% of healthy non-CF airway epithelial cells. Similar ranges of in vitro F508del mutation correction and functional CFTR rescue have been reported with Cas9 nuclease-mediated homology-directed repair (HDR) of upper-airway basal stem cells and airway epithelial cells. However, this HDR-mediated editing resulted in a nearly 1:1 edit-to-indel ratio75, whereas the PE-mediated F508del correction method offers 78 / 370 B1195.70196WO00 13363760.2a mean edit-to-indel ratio of 3.8 in primary cells. Additionally, while the HDR-mediated editing strategy used Cas9 nuclease to induce DSBs and AAV6 to provide a donor DNA template, the PE F508del correction strategy is DNA-free and takes advantage of transient RNA reagents that do not require DSBs. The DSB-independent nature of PE may offer fewer undesired and uncharacterized outcomes, and also obviate the need for donor DNA template delivery, likely simplifying delivery and reducing cellular toxicity of correction. Consistent with the inherent resistance of the mechanism of PE to off-target editing, only very low frequencies (≤0.1% at four sites) of potential off-target editing in primary CF patient airway epithelial cells following treatment with the optimized PE correction system was observed. The enhanced edit-to-indel ratio of the PE strategies developed in this study, compared to the use of Cas9 nuclease, results in unedited alleles predominantly encoding unmodified F508del CFTR protein that remains druggable by CF small-molecule correctors and potentiators, unlike the frameshifted CFTR alleles from the much higher frequency of nuclease-mediated indel byproduct formation that likely generate truncated CFTR proteins that cannot be rescued by small-molecule drugs. Methods General molecular biology

[0178] Guide RNA expression plasmids (pegRNAs, epegRNAs, ngRNAs, dsgRNAs, and sgRNAs) were cloned as previously described using isothermal assembly and synthetic gene fragments61. Guide RNA sequences are provided in Table 1. Guide RNA expression plasmids were purified using PureYield Plasmid Miniprep kits (Promega). Prime editor and MLH1dn plasmids were purified using Qiagen Plasmid Plus Midi kit (Qiagen). DNA PCR amplification was completed using Phusion U Green Multiplex PCR master mix (Thermo Fisher Scientific). Primers and gene fragments were ordered from Integrated DNA Technologies. Synthetic guide RNA generation

[0179] Synthetic pegRNAs and epegRNAs were ordered from Agilent Research Labs and contained 2′-O-methyl modifications at the first three nucleotides, 2′-O-methyl modifications at the third-to-last and second-to-last nucleotides, 3′-phosphorothioate linkages between the first three nucleotides, and 3′-phosphonoacetate linkages between the last two nucleotides. Synthetic ngRNAs were ordered from Synthego and contained 2′-O-methyl modifications at 79 / 370 B1195.70196WO00 13363760.2the first three and last three nucleotides and 3′-phosphorothioate linkages between the first three and last 2 nucleotides. Synthetic dsgRNAs were ordered from Integrated DNA Technologies and contained 2′-O-methyl modifications at the first three and last three nucleotides and phosphorothioate linkages between the three first and last three nucleotides. Guide RNA sequences are provided in Table 1. Generation of in vitro-transcribed mRNAs

[0180] Prime editor, adenine base editor, cytosine base editor, and MLH1dn-encoded mRNA were generated using in vitro transcription as previously described61. Briefly, the prime editor, base editor, or MLH1dn transcripts, containing a 5′ untranslated region (UTR), Kozak sequence, prime editor or MLH1dn open reading frame, and 3′ UTR were PCR amplified from a template plasmid containing an inactive T7 (dT7) promoter. PCR primers repair this dT7 promoter and also install a 119 nt poly(A) tail. The purified PCR dsDNA amplicon was used as an in vitro transcription template using the HiScribe T7 high-yield RNA synthesis kit (NEB). In vitro transcription followed the manufacturer's optional protocol to include CleanCap reagent AG (Trilink) and substitute N1-Methylpseudouridine-5′-triphosphate (Trilink) for uridine triphosphate. Lithium chloride precipitation was used to purify mRNA from complete in vitro transcription reactions and mRNA transcripts were reconstituted in nuclease-free water. General mammalian cell culture conditions

[0181] HEK293T (ATCC CRL-3216) cells were purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium with GlutaMax (Thermo Fisher Scientific) supplemented with 10% Fetal bovine serum (FBS; Thermo Fisher Scientific) at 37 °C with 5% CO2, as previously described61.

[0182] 16HBEge-F508del cells homozygous for CFTR F508del were a gift from the Cystic Fibrosis Foundation Therapeutics Lab and were cultured as previously described68. Briefly, 16HBEge-F508del cells were cultured in Minimum Essential Medium (MEM, Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and 1x Penicillin / Streptomycin (Thermo Fisher Scientific) at 37 °C with 5% CO2. For routine cell culture, standard tissue culture flasks were pre-treated with a thin layer of coating solution at 37 °C 5% CO2 for 2 hours, followed by coating solution removal and coated flask storage at 4 °C. Coating solution composition was LHC-8 basal medium (Thermo Fisher Scientific) 80 / 370 B1195.70196WO00 13363760.2supplemented with 1.34 μl / ml Bovine serum albumin 7.5% (Thermo Fisher Scientific), 10 μl / ml Bovine collagen solution Type 1 (Advanced BioMatrix), and 10 μl / ml Fibronectin from human plasma (Thermo Fisher Scientific).

[0183] Primary airway epithelial cells from non-CF donors were isolated from trachea or bronchi from postmortem lungs unsuitable for transplant. Primary CF airway epithelial cells were isolated from lung tissue obtained following lung transplant. Studies were approved by the University of Iowa Institutional Review Board, under United States Department of Health and Human Services registration number IRB00000099. The genotypes of the CF donors were: F508del / F508del (n=3 donors). Cells were expanded on culture plates coated with human collagen IV (Sigma) in PneumaCult-Ex Plus medium (Ex-Plus medium, STEMCELL Technologies). To establish air-liquid interface (ALI) cultures, cells were seeded onto collagen-coated, semi-permeable membranes (0.33 cm2, polycarbonate, Costar) and differentiated in PneumaCult-ALI medium (ALI medium, STEMCELL Technologies) for ≥3 weeks prior to functional assays.

[0184] All cell lines were verified to be free of mycoplasma and identity-authenticated by their suppliers. HEK293T transfection

[0185] HEK293T transfections were conducted as previously described61. Briefly, approximately 16,000 HEK293T cells were plated per well of a 96 well plate in complete culture media. After 18-24 hours, cells were transfected at 70-80% confluency with variable amounts of plasmid DNA and 0.5 µL of Lipofectamine 2000 diluted in Opti-MEM (Thermo Fisher Scientific), following manufacturer’s instructions. For a single 96 well transfection using standard prime editor systems, the following plasmids amounts were transfected: 200 ng of prime editor; 50 ng of pegRNA, epegRNA, or sgRNA; 15 ng of ngRNA (if included in experiment); 15 ng of dsgRNA (if included in experiment); and 100 ng of MLH1dn (if included in experiment). For a single 96 well transfection using the petRNA system with a split prime editor, the following plasmids amounts were transfected: 100 ng of the PEmax nCas9; 100 ng of the MCP-RT(PE6c); 25 ng of petRNA; 25 ng of petRNA-paired sgRNA; 15 ng of ngRNA; 15 ng of dsgRNA; and 100 ng of MLH1dn. Following transfection, cells were incubated at 37 °C with 5% CO2for 72 hours. 81 / 370 B1195.70196WO00 13363760.216HBEge-F508del cell and primary CF patient airway epithelial cell nucleofection

[0186] Nucleofection of 16HBEge-F508del cells and primary CF patient airway epithelial cells was completed with the SG Cell Line 4D-Nucleofector X Kit (Lonza) and a 4D- Nucleofector (Lonza). Cells from maintenance culture were dissociated using TrypLE (Thermo Fisher Scientific), and 200,000 cells were resuspended in 20 µL complete SG nucleofector solution per reaction. Prime editing RNA reagents were added the nucleofector solution cell mix as follows: 1 µg prime editor mRNA, 1 µg MLH1dn mRNA, 90 pmol pegRNA or epegRNA, 30 pmol ngRNA, and 30 pmol dsgRNA. The cell-RNA mixture was transferred into a nucleofector 16-well strip and electroporated using the program CM-137. Following nucleofection, 80 µl of pre-warmed growth media was added to each cuvette, and the 100 µL cell mix was used to seed tissue culture plates.

[0187] For 16HBEge-F508del cells, cells were transferred onto a 24 well tissue culture plate with prewarmed 16HBEge-F508del cell growth media and incubated at 37 °C in 5% CO2 for six days. On day four, cells were rinsed with PBS to eliminate dead cells and subsequently incubated with fresh growth media.

[0188] For primary CF airway epithelial cells, cells were transferred onto collagen-coated 6 well plates with pre-warmed Ex-Plus medium and incubated at 37 °C in 5% CO2. Cells were rinsed with PBS to eliminate dead cells and subsequently incubated with fresh Ex-Plus medium the following day.72 hours after nucleofection, cells were dissociated with TrypLE and seeded onto collagen-coated semi-permeable transwell membranes at density of 1.25 x 105per filter in Ex-Plus medium. Cells were maintained in Ex-Plus medium for 2-4 days and subsequently switched to ALI medium. The cells were differentiated at ALI for 3 weeks before functional analysis. A cell aliquot obtained during filter seeding was used for genomic DNA extraction and high throughput sequencing analysis. Genomic DNA preparation from cell culture

[0189] Genomic DNA (gDNA) was isolated from HEK293T and 16HBEge-F508del cell culture following a custom lysis protocol and paramagnetic bead extraction. To lyse cells, growth media was carefully removed from cell culture plates and 100 µL or 250 µL of lysis buffer (100 mM Tris-HCl pH 8.0, 200 mM NaCl, 5 mM EDTA, 0.05% SDS, 4.0 mg ml–1Proteinase K (New England Biolabs), and 12.5 mM DTT) was added to each well of a 96 well or 24 well plate, respectively. Plates with lysis buffer were incubated for 20 hours at 55 °C with shaking. To extract gDNA, one volume of lysate was thoroughly mixed with one 82 / 370 B1195.70196WO00 13363760.2volume of Ampure XP beads (Beckman Coulter Life Sciences) and incubated for 5 minutes, separated on plate magnet, washed with 70% ethanol three times with bead resuspension, and eluted in 50 µL nuclease-free water.

[0190] Genomic DNA from primary CF airway epithelial cells was extracted using QuickExtract (LGC Biosearch Technologies) lysis solution, following the manufacturer’s protocol. Preparation of HEK293T cells for fluorescence-activated cell sorting (FACS)

[0191] Briefly, for a single confluent well of HEK293T cells in a 96 well plate: 72 hours after transfection, media was carefully removed and 30 µL TrypLE Express (ThermoFisher) was used to coat well cell monolayer, which was then incubated at 37 °C with 5% CO2for 5 minutes. Following incubation, 70 µL complete HEK293T cell growth media was added, and cells were resuspended by pipetting. The cell-media mix was centrifuged at 250xg for 5 min, media supernatant was removed, and pelleted cells were resuspended in 500 µL PBS (ThermoFisher), and the cell-media mix was passed through a 35-μm cell strainer (Corning). Cells were sorted using a MA900 Cell Sorter (Sony Biotechnology). Cells were sorted into custom HEK293T cell lysis buffer (listed above), and gDNA was extracted as described for the custom lysis buffer (procedure listed above). High-throughput sequencing and data analysis

[0192] High throughput sequencing (HTS) of genomic loci was completed as previously described61. Briefly, two rounds of PCR amplification were performed using Phusion U Green Multiplex PCR master mix (Thermo Fisher Scientific). In the first round of PCR amplification (PCR1), approximately 150 ng of genomic DNA was used to template a PCR with primers containing Illumina adapter overhangs and cycled under the following conditions: 95 °C for 3 minutes; 27–30 cycles of 95 °C for 10 seconds, 58-61 °C (corresponding to the experimentally optimized Tm) for 20 seconds and 72 °C for 30 seconds, followed by 72 °C for 5 minutes. In the second round of PCR (PCR2), combinations of Illumina-barcoded forward and reverse primers were used to uniquely identify each sample and, with 1-2 µL of PCR1 as a template, were amplified under the following conditions: 95 °C for 3 minutes; 7 cycles of 95 °C for 10 seconds, 61 °C for 20 seconds, and 72 °C for 30 seconds, followed by 72 °C for 5 minutes. PCR2 reactions from the same genomic locus were pooled and gel extracted with QIAquick gel extraction kit (Qiagen) to eliminate primer 83 / 370 B1195.70196WO00 13363760.2dimers. Gel extracted amplicons were quantified using Qubit double-stranded DNA high- sensitivity assay kit (Thermo Fisher Scientific), and 4 nM libraries were run on an Illumina MiSeq 300 v2 Kit with 200–300 cycles.

[0193] Sequencing reads were demultiplexed using MiSeq Reporter (Illumina), and amplicon sequences were aligned to a reference sequence using CRISPResso292as previously described61. For prime editing experiments, the parameter QWC was set as the amplicon coordinates 10 bp 5′ upstream the nick position of the most 5′ epegRNA, pegRNA, ngRNA, or dsgRNA and 10 bp 3′ downstream the nick position of the most 3′ epegRNA, pegRNA, ngRNA, or dsgRNA. If the end of the 3′ flap generated by the RT plus 10 bp extended past one of these defined QWC bounds, the amplicon coordinate of the 3′ flap generated by the RT plus 10 bp was used in place the superseded QWC bound. Generation of a homozygous CFTR F508del HEK293T cell line using PE2

[0194] The monoclonal CFTR F508del HEK293T cell line was isolated by limiting dilution. Briefly, after identifying the most efficient PE2 strategy to install the CFTR F508del CTT deletion into the endogenous CFTR gene of HEK293T cells (FIG.5), HEK293T cells were transfected with this PE2 strategy following methods listed above. After 72 hours, transfected cells were dissociated from adherent culture, counted, and plated across 1096 well plates in 100 µL of media per well at a concentration of 0.5 cells per each well. Cells were grown for 10 days and monitored for the development of monoclonal cell populations. Identified monoclonal cultures were expanded and genotyped following HTS methods listed above. One clonal HEK293T cell line homozygous for CFTR F508del was isolated. Design of epegRNAs using computational tools

[0195] Web servers for DeepPrime (deepcrispr.info / DeepPrime / ) and PRIDICT 2.0 (pridict.it) were used to design pegRNA sequences for CTT insertion at CFTR F508del. Default settings were used for both models. The desired edited sequence used as a model input was simple CTT insertion without concomitant silent edits (i.e., SE0). The 24 pegRNA sequences with the highest predicted editing efficiency were synthesized as epegRNAs and tested experimentally. SE2 epegRNAs were designed by incorporating the SE2 silent edits into the SE0 pegRNA sequences generated by each model. 84 / 370 B1195.70196WO00 13363760.2Ussing chamber assay

[0196] Three weeks following nucleofection, primary airway epithelial cell cultures were treated for 24 hours with 10 µM forskolin (Cayman) and 100 µM 3-isobutyl-1- methylxanthine (IBMX, Sigma) prior to bioelectric studies. Donor-matched naïve CF cultures treated for 24 hours with ETI (2 μM of Elexacaftor (VX-445, Cayman), 18 μM of Tezacaftor (VX-661, Cayman), and 1 μM of Ivacaftor (VX-770, Cayman) in the presence of 10 mg / ml normal human serum (Sigma) as described previously54were used as a control for CFTR modulator treatment. Non-CF cultures treated with 10 µM forskolin and 100 µM IBMX were used as non-CF positive control.

[0197] Epithelial cell cultures were mounted in Ussing chambers (Physiologic Instruments, Inc., San Diego, CA) in symmetrical Cl- buffered Ringers solutions consisting of (mM): 135 NaCl, 5 HEPES, 0.6 KH2PO4, 2.4 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, 5 dextrose, pH titrated to 7.40 at 37° C with NaOH. The command voltage was set to 0 mV, and short-circuit current (Isc) was monitored. The following drugs were used during the assay: apical amiloride (Sigma Aldrich, 100 μM from 100 mM DMSO stock) to inhibit ENaC, apical 4, 4’-dilsothiocyano-2, 2’-stilbenedifulonic acid (DIDS, Sigma Aldrich, 100 μM from 100 mM DMSO stock) to inhibit non-CFTR Cl–channels, apical forskolin (Cayman Chemical, 10 μM from 10 mM DMSO stock) and IBMX (100 μM from 100 mM ethanol stock) to stimulate CFTR channel activity, and CFTRinh-172 (Sigma Aldrich, 10 µM from 10 mM DMSO stock) to block CFTR. Prediction of pegRNA secondary structure

[0198] NUPACK71(old.nupack.org) was used to predict the secondary structure and folding energies of pegRNAs. The pegRNA RTT and PBS (for epegRNA NGG2 PBS13 RTT41 SE2: UGGCACCAUUAAAGAAAAUAUCAUCUUUGGUGUGAGUUACGAUGAAUAUAGA UA (SEQ ID NO: 204)) sequence was queried using default parameters (type: RNA; temperature: 37 ˚C; maximum complex size: 1 strand). Quantification of pegRNA scaffold insertion

[0199] Quantification of pegRNA scaffold insertion was completed using a custom Python script described previously1. The script searches for occurrences of sequences of the pegRNA scaffold adjacent to on-target edits within high-throughput sequencing data, beginning with a single base at the 3′ end of the scaffold (proximal to the pegRNA reverse transcriptase 85 / 370 B1195.70196WO00 13363760.2template, referenced in the analysis as base 1). The script iterates through the entire scaffold sequence; in its ith iteration, insertion events representing integration of scaffold sequences 1- i are quantified. Scaffold insertion was quantified using genomic DNA from three prime edited primary CF airway epithelial cell cultures as well as prime edited 16HBEge-F508del cells. Analysis of partial edit incorporation

[0200] Partial edit incorporation was quantified using CRISPResso2 in HDR mode, as described above (see “High-throughput sequencing and data analysis”). For each sample, a separate CRISPResso2 run was executed for every possible combination of partial SE2 edit incorporation, both with and without concomitant CTT insertion. Partial edit quantification was performed using genomic DNA from three prime edited primary CF airway epithelial cell cultures as well as three unedited controls. Off-target analysis

[0201] Off-target site nomination was performed using CIRCLE-seq as previously described93. The top 32 CIRCLE-seq nominated off-target sites, as defined by nuclease read count, for each of the three guide RNAs used to edit primary CF airway epithelial cells (epegRNA NGG2 PBS13 RTT41 SE2, ngRNA +104, dsgRNA -40) were selected for deep sequencing analysis. Primers were designed to amplify each of the 96 nominated off-target sites within a 240-280 bp amplicon using NCBI Primer-BLAST. Off-target sites were amplified from purified genomic DNA and prepared for high-throughput sequencing as described above (see “High-throughput sequencing and data analysis”). Off target amplicon sequencing was performed using genomic DNA from three prime edited primary CF airway epithelial cell cultures as well as three unedited controls.

[0202] Indels and substitutions at nominated off-target sites were quantified from high- throughput sequencing data using CRISPResso292. Indels were quantified within a window from 10 bp 5′ upstream of the end of the epegRNA homology template to 10 bp 3′ downstream of the Cas9 nick position; this was achieved by setting the parameter WC to ‘18’ and the parameter W to ‘31’. Substitutions were quantified within a window from 0 bp 5′ upstream of the end of the epegRNA homology template to 0 bp 3′ downstream of the Cas9 nick position; this was achieved by setting the parameter WC to ‘18’ and the parameter W to ‘21’. For both indel and substitution quantifications, the discard_indel_reads parameter set as 86 / 370 B1195.70196WO00 13363760.2‘TRUE’ and the q parameter set as ‘30’. Off-target sites were further investigated if the mean indel or substitution rate at the site for prime edited samples was 1.5-or-more-fold greater than mean indel or substitution rate for the same site in untreated samples. Example 2: Co-installation of Translationally Silent Edits in CFTR

[0203] Co-installation of translationally silent edits (i.e., edits that change a target gene’s nucleotide sequence but not its protein sequence) via prime editing was found to improve editing efficiency for the cystic fibrosis-causing mutation CFTR.F508∆.108 silent edit strategies for CFTR.F508∆ correction were investigated, and numerous strategies with increased efficiency were found. These silent edit strategies were also investigated in the context of other prime editing parameters (notably, the primer binding site and reverse transcriptase template lengths of the epegRNA as well as the protospacer sequence length of the epegRNA), and it was found that they improve editing efficiency even when these parameters are varied. Co-installation of translationally silent edits was found to improve editing efficiency for CFTR.F508∆ correction (FIG.17). A screen of 72 different silent edit strategies (“Screen A”), with PBS13 / RTT41 and a 19 bp protospacer, was conducted (FIG. 18). The top-performing pegRNA (OP253) showed 1.56x-fold higher editing than SE2. It was found that improved silent edit strategies can recode codons both before and after the position of the therapeutic edit (F at position 508) (FIG.19). Silent edits can also improve editing efficiency in the context of different (e)pegRNA parameters like PBS, RTT, and protospacer length (FIGs.20A-20B). Another screen of 36 different silent edit strategies (some with varied PBS and RTT lengths, all 19 bp protospacer sequence) was also conducted (“Screen B”) (FIG.21). The top silent edits from Screens A and B with different pegRNA PBS / RTT lengths were also screened (“Screen C”) (FIG.22).

[0204] The most optimal epegRNA from Example 1 was CFTR-F508del-NGG2-PAM20- OGS-SE2-PBS13-RTT41: (Spacer in bold, scaffold-F in italics, RTT in underline, PBS in bold italics, linker in bold underline, tevopreQ1 motif in italics underline) TCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTA GTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTAAAGAAAA TATCATCTTTGGTGTGAGTTACGATGAATATAGATACGCGGTTCTATCTAGTTACG CGTTAAACCAACTAGAA (SEQ ID NO: 744). 87 / 370 B1195.70196WO00 13363760.2

[0205] The most optimal epegRNA following the screening described in Example 2 was CFTR-F508del-NGG2-PAM19-SCF-OPO253-PBS13-RTT45: (Spacer in bold, scaffold-F in italics, RTT in underline, PBS in bold italics, linker in bold underline, tevopreQ1 motif in italics underline) CTGTATCTATATTCATCATGTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGCCTGGCACCATTAAAGA AAATATCATCTTTGGTGTGTCATACGATGAATATAGATACGCGGTTCTATCTAGTTA CGCGTTAAACCAACTAGAA (SEQ ID NO: 251). 88 / 370 B1195.70196WO00 13363760.2DIfohNgeQ:-tl gRpA E OlS Nufn lg eee / N 2 A R 5 3 2 5 4 2 52A G A AA TTTGG C A GD GA TTTGG C D G N AdR NlA A TT o)g′TGCACGCACGI)A AA TT G GC C ACI)AAA C AG GCAAACQ7T GAGGC G AAACQ7TG G e Rfpe-rgf3 T a-′TTAAG ACTE1:TATAAGC ACTE1:TATA TCTTTACAAGGS(OT TTACAAGGS(OT T oepcs5(TC TC T T GC TC G G A G A G T A G A C N G G A G A T A G A C N G G A R Nr)′GTTTAQ:GTT:TAQ GTT ge- Rec3 TC CEO TC CEO TCT CTATSNCTATSNTA peA-rgea-p′s5(TATA( DI )TATA(C DI )TAT N o p A g T A C T 9 g T A C T 9 g T A argdeormti0E 0E 0 P mdeS S E S AtNe2 2 2 G G G RgretG G G gatisN N N oitiR;R;R;d 1 n 1 1 S92 1 1 S23 15 o C n PT S3 B T B PT T B PT T 0 3 3 0 lel lO lle eRd9e82 Rd92eRd W6 p TF 0K T80K T80 912.CytC5E F F H T C5E F F H T C5 F 07.06 e 573 r 916 13 u 3 gib b b B 1 F 1 F 1 F 1 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;1R;R1S8 1;3 11 114 T S4 S4 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 1 b b F 1 F 1 FGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)GCACGCACG)GCACGCACG)AGC TAG GCAAACQ71 TAG GCAAACQ71 TAG G AAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT ATTACA S(T A TC T T TC T T TC T T AGG O T G A GC T A G A C N G G A G A GC TC G G A T A G A C N G G A G A GC T A G A C N G G A GTTA:TA:T:TCTCQ GT EO TCTCQ GT EO TCTA CQ GTT EO TCT CTAT T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTA TDI )9 T gAT T A 0E 0 S E 0 S E 0 S E S 2 2 2 2 G G G G G G G G N N N N R;2R;2R;2R;1 1 1 2 S92 S23 5 18 B PT T B PT S3 T B PT S3 T B PT T 0 3 3 3 0 leleleleO Rd9 T82 Rd9 82 Rd92 Rd W6 F 0K T 5E F 0K T8 5E F 0KE T8 F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 1 1 1 b F F F 1 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;2R;R1S1 2;4 14 319 T S4 S2 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 1 b b F 1 F 1 FGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)GCACGCACG)GCACGCACG)AGC TAG GCAAACQ71 TAG GCAAACQ71 TAG G AAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT ATTACA S(T A TC T T TC T T TC T T AGG O T G A GC T A G A C N G G A G A GC TC G G A T A G A C N G G A G A GC T A G A C N G G A GTTA:TA:T:TCTCQ GT EO TCTCQ GT EO TCTA CQ GTT EO TCT CTAT T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTA TDI )9 T gAT T A 0E 0 S E 0 S E 0 S E S 2 2 2 2 G G G G G G G G N N N N R;3R;3R;3R;1 1 1 3 S23 S53 8 11 B PT T B PT S3 T B PT S4 T B PT T 0 3 3 3 0 leleleleO Rd9 T82 Rd9 82 Rd92 Rd W6 F 0K T 5E F 0K T8 5E F 0KE T8 F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 1 1 1 b F F F 1 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;3R;R1S4 4;4 19 412 T S2 S3 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 1 b b F 1 F 1 FGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)GCACGCACG)GCACGCACG)AGC TAG GCAAACQ71 TAG GCAAACQ71 TAG G AAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT ATTACA S(T A TC T T TC T T TC T T AGG O T G A GC T A G A C N G G A G A GC TC G G A T A G A C N G G A G A GC T A G A C N G G A GTTA:TA:T:TCTCQ GT EO TCTCQ GT EO TCTA CQ GTT EO TCT CTAT T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTA TDI )9 T gAT T A 0E 0 S E 0 S E 0 S E S 2 2 2 2 G G G G G G G G N N N N R;4R;4R;4R;1 1 1 4 S53 S83 1 14 B PT T B PT S4 T B PT S4 T B PT T 0 3 3 3 0 leleleleO Rd9 T82 Rd9 82 Rd92 Rd W6 F 0K T 5E F 0K T8 5E F 0KE T8 F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 1 1 1 b F F F 1 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTGCGDIGAAAA ) AAAATTT GT AAAA TTGCGDIG ) AAAATTT G TTGC C CG GC CAC C C AC C C AC GG CAAACQ7T A E1 TAGAGG CAAAG CQ7TG A E1 TAGAGGC CAAAG CQ7TG A E1 TAGAGGC CAAA GC ACT:TTATGC ACT:TTATGC ACT:TTA G AC ATA CAGGS(OTCTTATA CAGGS T A G A(OTCTTATAAGGS C N G G A G A G T A G A(OTCTTTACTAAG A G T A G A C N G G A G A G C C N G G A G A GC T A G Q: : : :EO GTTA S TCTCQO GTTA TATES TCTCQO GTTA TATES TCTCQO TATE (NC TA( NC T( NC T S T 9 g T A C( NTDI )9 T gA T A C TDI )9 T gA A T A CDI )TA A TDI )9 0E 0 0 S E S E S 2 2 2 G G G G G G N N N 62 5 1 2 7 1 11 0 e3 3 3 0 ll lO Rd92e9e9 Rd2 Rd2 W T8 F 0K T8 5E F 0KE T8 F 0K 6 E 912.T C F H T C5 F H T C5 F H T 07.06 57 93 16 13 c B31 1c1cF F 1 FGAAAATT GT A D GAAAATT GT GAAAATT GT GAA AAATTTGCG TGC C CACGI)AAAATTTGCGD AAATTTGCGD AA GC C CACGI)AGC C CACGI)AGC TAGA AGG GCAAA ACCQ7T E1:TAGA AGG AA GCAA CQ7T E1:TAGA AGG AA GCAA CQ7T E1:TAG TTATT CA TS(TTAT CA CTS(TTAT CA CTS(TTA TC TAT AGG OTCTTAT AGG OTCTTAT AGG OTCT G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A GTTTAQ:GTTTAQ:GTTAQ:GTT TC C O TC C O TCTC O TCT CTATES TATES TATES TA TATA( NC DI )TATA( NC D)TATA( NC D)TAT g T A C T 9 g T A C TI9 g T A C TI9 g T A 0E 0 0 0 S E S E S E S 2 2 2 2 G G G G G G G G N N N N 50 4 1 01 68 55 0 3 3 0 lelel3lO Rd9 T82 d9e9eW K R82 K Rd82 d K R8 6 F 0E T0E T0E T0 912.C5 F F H T C5 F F H T C5 F F H T C5 F 07.06 57 93 16 cc c13 B31 1 1 1cF F F 1 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT TTGCGDIGAAAA ) AAAATTT GT AAAA TTGCGDIG ) AAAATTT G TTGC C CG GC C AC C C AC C C AC GG CAAACQ7T A E1 TAGAGGC CAAAG CQ7TG A E1 TAGAGGC CAAAG CQ71 TG A TAGAGGC CAAA GC ACT:TTATGC ACT:TTATGC ACTE:TA G AC ATA CAGGS(OTCTTATAAGGS G T A G A(OTCTTATAAGGS C N G G A G A G T A G A(OTTCTTTACTAAG A G T A G A C N G G A G A C C C N G G A G A GC T A G Q:GTTA E Q:GTTAQ:GTTA:O S TCTC O TATE TCTC O TATE TCTCQO TATE (NC T S( NC T S A C( NC T SNTDI )9 T gA A T A C TDI )9 T gA A T TDI )9 T gA A T A C(TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N 44 7 2 14- 0 e3 3 3 0 ll lO Rd92e9e9 Rd2 Rd2 W T8 F 0K T8 5E F 0KE T8 F 0K 6 E 912.T C F H T C5 F H T C5 F H T 07.06 57 93 16 13 c B31 1c1cF F 1 FGAAAATT GT A D GAAAATT GT GAAAATT GT GAA AAATTTGCG TGC CIAAATTTGCGDIAAATTTGCGD AA TAGA AGGCACG)AGC C CACG)AGC C CACGI)AGC GCAAA ACCQ7T GAGG TE1:TA AGCAAA A CQ7T E1:TAGA AGG AA GCAA CQ7T E1:TAG TTATTACA S(TTAT CA CTS(TTAT CA CTS(TTA TC T TCAGG OTCTTATCAGG OTCTTAT AGG OTCT G G A G A G T A G A C N G G A G A G T A G A C N G G A G A GC T A G A C N G G A GTT TCTA CQ:GTTTAQ:GTTTAQ:GTTT CTATEO S TATA( NTC C CTATEO SNTC C O TATES TCTA DI )TATA(C DI )TATA( NC DI )TAT g T A C T 9 g T A C T 9 g T A C T 9 g T A 0E 0E 0E 0 S S S E S 2 2 2 2 G G G G G G G G N N N N 11 2 1 - E 2 P E 6 P 21 0 3 0 lel3l3lO Rd9e9e9eT82 W K Rd82 d2 d K R8 R8 6 F 0E T0E T0KE T0 912.C5 F F H T C5 F F H T C5 F F H T C5 F 07.06 57 93 16 cc c13 B31 1 1 1cF F F 1 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTGCGDIGAAAA ) AAAATTT GT AAAA TTGCGDIG ) AAAATTT G TTGC C CG GC CAC C C AC C C AC GG CAAACQ7T A E1 TAGAGG CAAAG CQ7TG A E1 TAGAGGC CAAAG CQ7TG A E1 TAGAGGC CAAA GC ACT:TTATGC ACT:TTATGC ACT:TTA G AC ATA CAGGS(OTCTTATA CAGGS T A G A(OTCTTATAAGGS C N G G A G A G T A G A(OTCTTTACTAAG A G T A G A C N G G A G A G C C N G G A G A GC T A G Q: : : :EO GTTA S TCTCQO GTTA TATES TCTCQO GTTA TATES TCTCQO TATE (NC TA( NC T( NC T S T 9 g T A C( NTDI )9 T gA T A C TDI )9 T gA A T A CDI )TA A TDI )9 0E 0 0 S E S E S 2 2 2 G G G G G G N N N 52 7 1 1 5 1 01 0 e3 3 3 0 ll lO Rd92e9e9 Rd2 Rd2 W T8 F 0K T8 5E F 0KE T8 F 0K 6 E 912.T C F H T C5 F H T C5 F H T 07.06 57 93 16 13 c B31 1c1cF F 1 FGAAAATT GT A D GAAAATT GT GAAAATT GT GAA AAATTTGCG TGC C CACGI)AAAATTTGCGD AAATTTGCGD AA GC C CACGI)AGC C CACGI)AGC TAGA AGG GCAAA ACCQ7T E1:TAGA AGG AA GCAA CQ7T E1:TAGA AGG AA GCAA CQ7T E1:TAG TTATT CA TS(TTAT CA CTS(TTAT CA CTS(TTA TC TAT AGG OTCTTAT AGG OTCTTAT AGG OTCT G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A GTTTAQ:GTTTAQ:GTTAQ:GTT TC C O TC C O TCTC O TCT CTATES TATES TATE TA TATA( NC D)TATA( NC D)TATAS( NC D)TAT g T A C TI9 g T A C TI9 g T A C TI9 g T A 0E 0 0 0 S E S E S E S 2 2 2 2 G G G G G G G G N N N N 401 68 55 44 0 3 3 0 lelel3lO Rd9 T82 d9e9eW K R82 K Rd82 d K R8 6 F 0E T0E T0E T0 912.C5 F F H T C5 F F H T C5 F F H T C5 F 07.06 57 93 16 cc c13 B31 1 1 1cF F F 1 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTGCGDIGAAAA ) AAAATTT GT AAAA TTGCGDIG ) AAAATTT G TTGC C CG GC CAC C C AC C C AC GG CAAACQ7T A E1 TAGAGG CAAAG CQ7TG A E1 TAGAGGC CAAAG CQ7TG A E1 TAGAGGC CAAA GC ACT:TTATGC ACT:TTATGC ACT:TTA G AC ATA CAGGS(OTCTTATA CAGGS T A G A(OTCTTATAAGGS C N G G A G A G T A G A(OTCTTTACTAAG A G T A G A C N G G A G A G C C N G G A G A GC T A G Q: : : :EO GTTA S TCTCQO GTTA TATES TCTCQO GTTA TATES TCTCQO TATE (NC TA( NC T( NC T S T 9 g T A C( NTDI )9 T gA T A C TDI )9 T gA A T A CDI )TA A TDI )9 0E 0 0 S E S E S 2 2 2 G G G G G G N N N 2 1 7141 -1- 0 e3 3 3 0 ll lO Rd92e9e9 Rd2 Rd2 W T8 F 0K T8 5E F 0KE T8 F 0K 6 E 912.T C F H T C5 F H T C5 F H T 07.06 57 93 16 13 c B31 1c1cF F 1 FGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)GCACGCACG)GCACGCACG)AGC TAG GCAAACQ71 TAG GCAAACQ71 TAG G AAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT ATTACA S(T A TC T T TC T T TC T T AGG O T G A GC T A G A C N G G A G A GC TC G G A T A G A C N G G A G A GC T A G A C N G G A AA C:TA:T:CAA AT QO GT TCTCQO GTTAQ GTTT TE TC CEO TC CA A ANCTA SNCTATSNTA gTATTE)T G A AS(DI01 T gATA T A C(TDI )9 T gATA T A C(C TDI )9 T gAT T A e8eE 8E BtiBti0 AdeAdeE 0 S E S 2 1 2 2 G G G G G G G G N N N N 1 2R;0R;G G 10 01 G S 1 G 3 S3 N N B PT T B PT T 0 le39le3le3 0 leO Rd T82 Rd9 82 Rd9 82 Rd W6 F 0K 5E TF 0K T 5E F 0KE T8 F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 16 ee133 b B 1 1 1 2 b F F F 2 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;0R;R1S2 0;3 13 014 T S3 S3 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 2 b b F 2 F 2 FGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)GCACGCACG)GCACGCACG)AGC TAG GCAAACQ71 TAG GCAAACQ71 TAG G AAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT ATTACA S(T A TC T T TC T T TC T T AGG O T G A GC T A G A C N G G A G A GC TC G G A T A G A C N G G A G A GC T A G A C N G G A GTTA:TA:T:TCTCQ GT EO TCTCQ GT EO TCTA CQ GTT EO TCT CTAT T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTA TDI )9 T gAT T A 0E 0 S E 0 S E 0 S E S 2 2 2 2 G G G G G G G G N N N N R;0R;0R;0R;1 1 1 0 S53 S63 7 18 B PT T B PT S3 T B PT S3 T B PT T 0 3 3 3 0 leleleleO Rd9 T82 Rd9 82 Rd92 Rd W6 F 0K T 5E F 0K T8 5E F 0KE T8 F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 2 2 2 b F F F 2 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;0R;R1S1 0;4 12 013 T S4 S4 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 2 b b F 2 F 2 FGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)GCACGCACG)GCACGCACG)AGC TAG GCAAACQ71 TAG GCAAACQ71 TAG G AAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT ATTACA S(T A TC T T TC T T TC T T AGG O T G A GC T A G A C N G G A G A GC TC G G A T A G A C N G G A G A GC T A G A C N G G A GTTA:TA:T:TCTCQ GT EO TCTCQ GT EO TCTA CQ GTT EO TCT CTAT T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTA TDI )9 T gAT T A 0E 0 S E 0 S E 0 S E S 2 2 2 2 G G G G G G G G N N N N R;1R;1R;1R;1 1 1 1 S03 S13 2 13 B PT T B PT S3 T B PT S3 T B PT T 0 3 3 3 0 leleleleO Rd9 T82 Rd9 82 Rd92 Rd W6 F 0K T 5E F 0K T8 5E F 0KE T8 F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 2 2 2 b F F F 2 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;1R;R1S4 1;3 15 116 T S3 S3 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 2 b b F 2 F 2 FTD GAAAATT GTD GAAAATT GTD GAAAATT GT GAA GIAAAATTTGCGIAAATTTGCGIAAATTTGCGDIAA G)GCACGCACG)AGCACGCACG)AGC CGCACG)AGC CQ71 TAG GCAAACQ7T G G AAACQ7T GAG AAACQ7T G TE:T AG ACTE1:TA AGC ACTE1:TA AGC AC E1:TA GS(OTTATTACAAG S(TTAT CA S(TTAT CA TS(TTA TC T T G A G A GC G OTCTTAT T A G A C N G G A G A GCAGG OTCTTAT AGG OTCT A C N G T A G A C N G G A G A GC T A G A C N G G A GTTA:TTA:TTA:TT TCTCQO G TATE TCTCQO G TCQO G T TATE TCTATE TC CT T S gA A T A C( NC T S TDI )9 T gA A T A C( NC T SNCTAT TDI )9 T gA A T A C(TDI )9 T gA T A 0E 0E 0E 0 S S S E S 2 2 2 2 G G G G G G G G N N N N R;1R;1R;1R;1S73 18 11 112 T S3 S4 S4 B P T B PT T B PT T B PT T 0 3 0 lele3l3lO Rd92 T8 Rd92eRd92eRd W6 F 0K T8 5E F 0KE T8 F 0KE T8 F 0 912.C F H T C5 F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 2 2 2 b F F F 2 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;1R;R1S3 2;4 10 211 T S3 S3 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 2 b b F 2 F 2 FGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)GCACGCACG)GCACGCACG)AGC TAG GCAAACQ71 TAG GCAAACQ71 TAG G AAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT ATTACA S(T A TC T T TC T T TC T T AGG O T G A GC T A G A C N G G A G A GC TC G G A T A G A C N G G A G A GC T A G A C N G G A GTTA:TA:T:TCTCQ GT EO TCTCQ GT EO TCTA CQ GTT EO TCT CTAT T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTA TDI )9 T gAT T A 0E 0 S E 0 S E 0 S E S 2 2 2 2 G G G G G G G G N N N N R;2R;2R;2R;1 1 1 2 S23 S33 4 15 B PT T B PT S3 T B PT S3 T B PT T 0 3 3 3 0 leleleleO Rd9 T82 Rd9 82 Rd92 Rd W6 F 0K T 5E F 0K T8 5E F 0KE T8 F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 2 2 2 b F F F 2 FAATT GTD GAAAATT GT GAAAATT GT GAAAATT G ATTTGCG AAATTTGCGD AAATTTGCGD AAAT TGC AC AI)A C AI)A C AI)A CT AGGC C AAG GC C CG GC C CG GC AC GCAA CQ7T E1:TAGA AGG AA GCAA CQ7T E1:TAGA AGG AA Q7T GCAA CE1:TAGA AGGC CAAA T CA CTS(TTAT CA CTS(TTAT CA CTS TTATGCAAC TAT AGG OTCTTAT AGG OTCTTAT AGG G A(OTCT A AG GC T A G A C N G G A G A GC T T G A T A G A C N G G A G A GC T A C N G G A G A GC T A G AQ:GTTAQ:GTTAQ:GTTAQ:C O TCTC O TCTC O TCTC O TES TATES TATES TATE A C( NC TA T 9 g T A C( NC T TI9 g T A C( NC T S TI9 g T A C( NDI )TAD)TA AD)TA A TDI )9 0E 0 S E 0 S E S 2 2 2 G G G G G G N N N R;2R;R;1S6 2 3 17 218 B PT S3 T B PT S3 T B PT T 0 3 0 9l3 3 3 O 2e9le9le9 Rd2 d2 d2 W KE T8 F 0K RT8 R C5E F 0K T8 F H T C5E F 0K 6 E 912.H T F H T C5 F H T 07.06 57 93 16 13 b B31 2 b b F 2 F 2 FD GAAAATT GTD GAAAATT GTD GAAAATT GTD GAA I AAAATTTGCGIAAAATTTGCGIAAATTTGCGIAA Q)7 GCACGCACG)GCACGCACG)AGCACGCACG)AGC E1 T : TAG G T AGCAAA ACCQ7 TE1 T : TAG G AAACQ7T G G AAACQ7T G T AGC ACTE1:TA AGC ACTE1:TA S(OT ATTACAAGGS(OT ATTACA S(TTAT CA S(TTA TC T T GC TC T T AGG OTCTTAT AGG OTCT C N G G A G A T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A GTTAQ:GTTA:GTTA:GTT TCTC O TCTCQO CTCQO CT CTATE T TAS gA T A C( NCTATE T T TATE TTA AS TDI )9 T gA T A C( NC T S TDI )9 T gA A T A C( NC T TDI )9 T gA T A 0E 0 0 0 S E S E S E S 2 2 2 2 G G G G G G G G N N N N R;2R;R R1 2;2;3 S14 12 13 10 T S4T S4 S3 B P T B P T B PT T B PT T 0 3 3 0 lelele3lO Rd9 T82 Rd92 Rd92eRd W6 F 0K T8 5E F 0KE T8 F 0KE T8 F 0 912.C F H T C5 F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 2 2 2 b F F F 2 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;3R;R1S1 3;3 12 313 T S3 S3 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 2 b b F 2 F 2 FTD GAAAATT GTD GAAAATT GTD GAAAATT GT GAA GIAAAATTTGCGIAAATTTGCGIAAATTTGCGDIAA G)GCACGCACG)AGCACGCACG)AGC CGCACG)AGC CQ71 TAG GCAAACQ7T G G AAACQ7T GAG AAACQ7T G TE:T AG ACTE1:TA AGC ACTE1:TA AGC AC E1:TA GS(OTTATTACAAG S(TTAT CA S(TTAT CA TS(TTA TC T T G A G A GC G OTCTTAT T A G A C N G G A G A GCAGG OTCTTAT AGG OTCT A C N G T A G A C N G G A G A GC T A G A C N G G A GTTA:TTA:TTA:TT TCTCQO G TATE TCTCQO G TCQO G T TATE TCTATE TC CT T S gA A T A C( NC T S TDI )9 T gA A T A C( NC T SNCTAT TDI )9 T gA A T A C(TDI )9 T gA T A 0E 0E 0E 0 S S S E S 2 2 2 2 G G G G G G G G N N N N R;3R;3R;3R;1S43 15 16 317 T S3 S3 S3 B P T B PT T B PT T B PT T 0 3 0 lele3l3lO Rd92 T8 Rd92eRd92eRd W6 F 0K T8 5E F 0KE T8 F 0KE T8 F 0 912.C F H T C5 F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 2 2 2 b F F F 2 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;3R;R1S8 3;3 11 312 T S4 S4 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 2 b b F 2 F 2 FGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)GCACGCACG)GCACGCACG)AGC TAG GCAAACQ71 TAG GCAAACQ71 TAG G AAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT ATTACA S(T A TC T T TC T T TC T T AGG O T G A GC T A G A C N G G A G A GC TC G G A T A G A C N G G A G A GC T A G A C N G G A GTTA:TA:T:TCTCQ GT EO TCTCQ GT EO TCTA CQ GTT EO TCT CTAT T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTA TDI )9 T gAT T A 0E 0 S E 0 S E 0 S E S 2 2 2 2 G G G G G G G G N N N N R;3R;4R;4R;1 1 1 4 S34 S03 1 12 B PT T B PT S3 T B PT S3 T B PT T 0 3 3 3 0 leleleleO Rd9 T82 Rd9 82 Rd92 Rd W6 F 0K T 5E F 0K T8 5E F 0KE T8 F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 2 2 2 b F F F 2 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;4R;R1S3 4;3 14 415 T S3 S3 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 2 b b F 2 F 2 FGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)GCACGCACG)GCACGCACG)AGC TAG GCAAACQ71 TAG GCAAACQ71 TAG G AAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT ATTACA S(T A TC T T TC T T TC T T AGG O T G A GC T A G A C N G G A G A GC TC G G A T A G A C N G G A G A GC T A G A C N G G A GTTA:TA:T:TCTCQ GT EO TCTCQ GT EO TCTA CQ GTT EO TCT CTAT T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTA TDI )9 T gAT T A 0E 0 S E 0 S E 0 S E S 2 2 2 2 G G G G G G G G N N N N R;4R;4R;4R;1 1 1 4 S63 S73 8 11 B PT T B PT S3 T B PT S4 T B PT T 0 3 3 3 0 leleleleO Rd9 T82 Rd9 82 Rd92 Rd W6 F 0K T 5E F 0K T8 5E F 0KE T8 F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 2 2 2 b F F F 2 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;4R;R1S2 4;4 13 510 T S4 S3 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 2 b b F 2 F 2 FGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)GCACGCACG)GCACGCACG)AGC TAG GCAAACQ71 TAG GCAAACQ71 TAG G AAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT ATTACA S(T A TC T T TC T T TC T T AGG O T G A GC T A G A C N G G A G A GC TC G G A T A G A C N G G A G A GC T A G A C N G G A GTTA:TA:T:TCTCQ GT EO TCTCQ GT EO TCTA CQ GTT EO TCT CTAT T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTA TDI )9 T gAT T A 0E 0 S E 0 S E 0 S E S 2 2 2 2 G G G G G G G G N N N N R;5R;5R;5R;1 1 1 5 S13 S23 3 14 B PT T B PT S3 T B PT S3 T B PT T 0 3 3 3 0 leleleleO Rd9 T82 Rd9 82 Rd92 Rd W6 F 0K T 5E F 0K T8 5E F 0KE T8 F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 2 2 2 b F F F 2 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;5R;R1S5 5;3 16 517 T S3 S3 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 2 b b F 2 F 2 FGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)GCACGCACG)GCACGCACG)AGC TAG GCAAACQ71 TAG GCAAACQ71 TAG G AAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT ATTACA S(T A TC T T TC T T TC T T AGG O T G A GC T A G A C N G G A G A GC TC G G A T A G A C N G G A G A GC T A G A C N G G A GTTA:TA:T:TCTCQ GT EO TCTCQ GT EO TCTA CQ GTT EO TCT CTAT T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTA TDI )9 T gAT T A 0E 0 S E 0 S E 0 S E S 2 2 2 2 G G G G G G G G N N N N R;5R;5R;5R;1 1 1 5 S83 S14 2 13 B PT T B PT S4 T B PT S4 T B PT T 0 3 3 3 0 leleleleO Rd9 T82 Rd9 82 Rd92 Rd W6 F 0K T 5E F 0K T8 5E F 0KE T8 F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 2 2 2 b F F F 2 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;6R;R1S0 6;3 11 612 T S3 S3 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 2 b b F 2 F 2 FGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)GCACGCACG)GCACGCACG)AGC TAG GCAAACQ71 TAG GCAAACQ71 TAG G AAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT ATTACA S(T A TC T T TC T T TC T T AGG O T G A GC T A G A C N G G A G A GC TC G G A T A G A C N G G A G A GC T A G A C N G G A GTTA:TA:T:TCTCQ GT EO TCTCQ GT EO TCTA CQ GTT EO TCT CTAT T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTA TDI )9 T gAT T A 0E 0 S E 0 S E 0 S E S 2 2 2 2 G G G G G G G G N N N N R;6R;6R;6R;1 1 1 6 S33 S43 5 16 B PT T B PT S3 T B PT S3 T B PT T 0 3 3 3 0 leleleleO Rd9 T82 Rd9 82 Rd92 Rd W6 F 0K T 5E F 0K T8 5E F 0KE T8 F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 2 2 2 b F F F 2 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;6R;R1S7 6;3 18 611 T S3 S4 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 2 b b F 2 F 2 FGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)GCACGCACG)GCACGCACG)AGC TAG GCAAACQ71 TAG GCAAACQ71 TAG G AAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT ATTACA S(T A TC T T TC T T TC T T AGG O T G A GC T A G A C N G G A G A GC TC G G A T A G A C N G G A G A GC T A G A C N G G A GTTA:TA:T:TCTCQ GT EO TCTCQ GT EO TCTA CQ GTT EO TCT CTAT T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTA TDI )9 T gAT T A 0E 0 S E 0 S E 0 S E S 2 2 2 2 G G G G G G G G N N N N R;6R;6R;7R;1 1 1 7 S24 S34 0 11 B PT T B PT S3 T B PT S3 T B PT T 0 3 3 3 0 leleleleO Rd9 T82 Rd9 82 Rd92 Rd W6 F 0K T 5E F 0K T8 5E F 0KE T8 F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 2 2 2 b F F F 2 FATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AACQ7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA E1:TA AGCAA CE1:TA AGCA CE1:TA A CA CA CTS TTAT CA CTS TTAT CAACTS TTATGCAAC AT AGG(OTCTTAT AGG(OTCTTAT AGG T A G A(O T A AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC TC T T C N G G A G A GC T A G Q:GTTAQ:GTTA:GTTA:EO S TCTC O TATE CTCQO CTCQO S TTATE TTATE (NC T TI9 g T A C( NC T S TI9 g T A C( NC T S TI9 g T A C( ND)TA AD)TA AD)TA A TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;7R;R1S2 7;3 13 714 T S3 S3 B P T B PT T B PT T 0 3 3 3 0 le9leleO Rd2 d92 d92 W T8 F 0K RT8 R 5E F 0K T8K 69 F H T C5E F 0E 12.T C F H T C5 F H T 07.06 57 93 16 13 b B31 2 b b F 2 F 2 FGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)GCACGCACG)GCACGCACG)AGC TAG GCAAACQ71 TAG GCAAACQ71 TAG G AAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT ATTACA S(T A TC T T TC T T TC T T AGG O T G A GC T A G A C N G G A G A GC TC G G A T A G A C N G G A G A GC T A G A C N G G A GTTA:TA:T:TCTCQ GT EO TCTCQ GT EO TCTA CQ GTT EO TCT CTAT T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTAT TDI )9 T gATAS T A C( NCTA TDI )9 T gAT T A 0E 0 S E 0 S E 0 S E S 2 2 2 2 G G G G G G G G N N N N R;7R;7R;7R;1 1 1 7 S53 S63 7 18 B PT T B PT S3 T B PT S3 T B PT T 0 3 3 3 0 leleleleO Rd9 T82 Rd9 82 Rd92 Rd W6 F 0K T 5E F 0K T8 5E F 0KE T8 F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 16 133 b b b B 1 2 2 2 b F F F 2 FAATT GTD GAAAATT GT GAAAATT GT GAAAATT G ATTTGCG AAATTTGCGD AAATTTGCGD AAAT TGC AC AI)A C AI)A C AI)A CT AGGC C AAG GC C CG GC C CG GC AC GCAA CQ7T E1:TAGA AGG AA GCAA CQ7T E1:TAGA AGG AA Q7T GCAA CE1:TAGA AGGC CAAA T CA CTS(TTAT CA CTS(TTAT CA CTS TTATGCAAC TAT AGG OTCTTAT AGG OTCTTAT AGG G A(OTCT A AG GC T A G A C N G G A G A GC T T G A T A G A C N G G A G A GC T A C N G G A G A GC T A G AQ:GTTAQ:GTTAQ:GTTAQ:C O TCTC O TCTC O TCTC O TES TATES TATES TATE A C( NC TA T 9 g T A C( NC T TI9 g T A C( NC T S TI9 g T A C( NDI )TAD)TA AD)TA A TDI )9 0E 0 S E 0 S E S 2 2 2 G G G G G G N N N R;7R;R;1S1 7 4 12 713 B PT S4 T B PT S4 T B PT T 0 3 0 9l3 3 3 O 2e9le9le9 Rd2 d2 d2 W KE T8 F 0K RT8 R C5E F 0K T8 F H T C5E F 0K 6 E 912.H T F H T C5 F H T 07.06 57 93 16 13 b B31 2 b b F 2 F 2 FGAAAATT GTD GAAAATT GT GAAAATT GT GAA AAAATTTGCG AAAATTTGCGD AAA TTTGCGD AA AGC C TAGATGGCACGI)GC C CACGI)GCAC CACGI)AGC GCAAA ACCQ8A GAGG TE1:TA TGCAAA A CQ8A E1:TAGATGG AA GCAA CQ8A E1:TAG TTATTACA S(TTAT CA CTS(TTAT CA CTS(TTA TC T TCAGG OTCTTAT AGG OTCTTAT AGG OTCT G G A G A G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A GTTTAQ:GTTTAQ:GTT:TAQ GTT TC CEO TC C O TC C O TCT CTATS TATA( NCTATES TATES TA DI )TATA( NC DI )TATA( NC D)TAT g T A C T 9 g T A C T 9 g T A C TI9 g T A 0E 1E 2 3 S S E S E S 2 2 2 2 G G G G G G G G N N N N 0E 1E 2E 3 S S S E S 0 3 0 lel3l3lO Rd9e9e9eT82 W K Rd82 d2 d K R8 R8 6 F 0E T0E T0KE T0 912.C5 F F H T C5 F F H T C5 F F H T C5 F 07.06 57 93 16 13 B3 d d d 1 2 2 2 d F F F 2 FATTT GT GAA CTTG ACGDI)AAAAA ATTT GT GAA CTTGCGDI)AAAAA ATTT GT GAA TTGCGDI)AAAAA ATTT G TTGC C CG GC CAC C C AC C C AC GG CAAACQ8A A E1:TAGTGG CAAAG CQ8AG A E1 TAGTGGC CAAAG CQ8AG A E1 TAGTGGC CAAA GC ACT TTATGC ACT:TTATGC ACT:TTATG AC ATA CAGGS(OTCTTATA CAGGS A G A(OTCTTATA C N G G A G A GCAGGS T A G A(OTCTTACTAAG A G T A G A C N G G A G A G T C N G G A G A GC T A G Q:GTTA: : :EO TCTCQ GTT EO TCTA CQO GTTA TCTCQO S( NCTAT TAS( NCTATE TAS( NCTATE TAS g T A C( NTDI )9 T gA T A C TDI )9 T gA T A C TDI )9 TA TDI )9 4E 2 S E 2 S E S 2 2 2 G G G G G G N N N xa4 maE S E 6 P E P 0 e3 3 3 0 ll lO Rd92e9e9 Rd2 Rd2 W T8 F 0K T8 5E F 0KE T8 F 0K 6 E 912.T C F H T C5 F H T C5 F H T 07.06 57 93 16 13 d2eB31 2eF F 2 FGAAAATT GTD GAAAATT GT GAAAATT GT GAA AAAATTTGCG AAAATTTGCGD AAA TTTGCGD AA AGC C TAGATGGCACGI)GC CGCACGI)GCAC CACGI)AGC GCAAA ACCQ8A GAG TE1:TA TGCAAA ACCQ8A E1:TAGATGG GCAAA A CQ8A E1:TAG TTATTACAA S(TTAT CA TS(TTAT CA CTS(TTA TC T TC GG OTCTTATCAGG OTCTTATCAGG OTCT G G A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A GTT TCTA CQ:TA:TA:T O GTT Q GTT Q GTT CTATES TNTC C TATEO SNTC C TATEO SNTCTA gATA T(C DI )TATA(C DI )TATA(C DI )TAT A C T 9 g T A C T 9 g T A C T 9 g T A 2E 2 2 2 S E S E S E S 2 2 2 2 G G G G G G G G N N N N b6c6 deE E 6 6 P P E P E P 0 3 0 lel3l3lO Rd9e9e9eT82 W K Rd82 d2 d K R8 R8 6 F 0E T0E T0KE T0 912.C5 F F H T C5 F F H T C5 F F H T C5 F 07.06 57 93 16 ee e13 B31 2 2 2eF F F 2 FATTT GT TTGCGD GAAAATT GT AAAATTTGCGD GAAAATT GT AAA TTTGCGD C CACGI)GC C CACGI)GCAC CACGI)GG AACQ8A GAGG AACQ8A GAGG AACQ8 GCA CAACTE1 S(:TA TTATTGCA CAACTE1 S(:TA TTATTGCA CAACTE1 S:ATCAGG OTCTTAT AGG OTCTTA AGG T A G A C N G G A G A GC T A G T A G A C N G G A C(O G A G T A G A C N Q:T: :EO GTTAQ GTTTAQ S( NTC C O CTATE TC C O TE T SNCTA SNTDI )9 T gA A T A C(TDI )9 T gATA T A C(TDI )9 e8 2E 2 E BtiS E S Ade2 2 2 G G G G G G N N N f A 6 g6 N E E R P Pgs0 3 3 el0 lelgeO Rd92e9 Rd2 Ed B8 W T8K 8K 0 6 F 0E TF 0E H5 912.T C5 F H T C5 F H T 61F- 07.06 57 93 16 ee13 B31 2 2aF F 3 FGAAAATT GT A D GAAAATT GT GAAAATT G AAATTTGCG TGCACIAAATTTGCGD AAATTTGC T GGCAC A GQ)A 9TGCAC GGCACGIQ)A 9TGCACGCAC AG A AC G AAAC G G AAA TTAAGC ACTE1:TA AGC ACTE1:TA AGC AC TCTTTACTAAGGS G A GC(OTTA TCTTTACTA S T A G A C N G G A G A GCAGG(OTTA TCTTTACTAAG G G A T A G A C N G G A G A GC T A G TACT:TA T:TA T:GTTA O TCTA O TCTA O TC CTTCQ G EN)TCTCQ G EN)TCTCQEN)T AAT T AS(DI11 CT T AAT T AS(DI11 CT T AAT T AS(DI11 e8eE 8 BtiEt2 AdeBiAdeE S 2 2 2 G G G G G G N N N A A N N R R gege. ppet.n e 0 l elel0 gEed gEed gEed O B8 B8 B8 W6 H0 H0 H0 9 65 1F- 65 1F- 65 1F- 102.7.06 57 93 16 13 a B31 3a3cF F 3 FGAAAATT GT ) A D GAAAATT GT GAAAATT GT GAA AAATTTGCG 91 TGC C CACGI)AAAATTTGCGD GC C CACGI)AAAATTTGCGD AA GC C CACGI)AGC : TAGA AGG AA GCAA CQ9T E1:TAGA AGG AA GCAA CQ9T E1:TAGA AGG CAAACQ9T E1:TAG OTTA TCTTTACTA CT AGGS(OTTA TCTTTACTA CT AGGS(OTTA TCTTG TACTAACT AGGS G A G A G T A G A(OTTA TCT N G G A G A GC T A G A C N G G A G A GC T A G A C N G C C N G G A TACT:TACT:TACT:TAC GTTA O GTTA O TTA O TT TCTTCQ TEN)TCTTCQ G TEN)TCTCQ G EN)TCT C T AA T AS(DI11 C T AA T AS(DI11 CT T AAT T AS(DI11 CT T AA T 2E 2 2 2 S E S E S E S 2 2 2 2 G G G G G G G G N N N N 10 0 1 01 54 14 e 0 l e e e 0 gEed glEed glegleO B80 B8 Ed 0 B8 Ed 0 B8 W6 H65 H H H0 9 1F- 65 1F- 65 1F- 65 1F- 102.7.06 57 93 16 cc c13 B31 3 3 3cF F F 3 FATTT GT CTTGCGDIGAAAA ) AAAATTT GT TTGCGDIGAAAA ) AAAATTT GT AAAA TTGCGDIG ) AAAATTT G TTGC CACG C C AC C C AC C C AC GG A Q9TG AGGCA GQ9TG AGGCA GQ9TG AGGC CA AC 1 TAGA CA AC 1 TAGA CA AC 1 TAGA CAAA GC ACTE:TTATGC ACTE:TA G ACTE:TA G AC ATAAGGS(OTCTTATAAGGS G G A G A G T A G A(OTTCTTTACTAAGGS C N G G A G A G T A G A(OT TTACAAG A GC T A G A C N C C TC T T C N G G A G A GC T A G : TA T:TA T:TA T:QO GTCTA O TCTA O TCTA O EN)TCTCQ G EN)TCTCQ G EN)TCTCQEN)S(DI11 CT T AAT T AS(DI11 CT T AAT T AS(DI11 CT T AAT T AS(DI11 2E 2 S E 2 S E S 2 2 2 G G G G G G N N N 4 0 4 2 3 -4- ele 0 l el0 gEed gegeO B8 Ed Ed W 0 B80 B8 6 H65 H 1F- 65 H0 9 1F- 65 1 1F- 02.7.06 57 93 16 13 c B31 3c3cF F 3 FA T AA AAGTGG A T AA AAGTGG A T AA AGT G G A TT T A TT T AATT GT AAAAT GC D G T GC D G T GC D TGCACTT GCACG GITAG GCAAACQ)AAAA 91 TGCACTT GCACG GI)AAAA TT G AG GCAAACQ91 TGCACGCACGI)AG GCAAACQ91 TTAAG ACTE:TTAAG ACTE:TTAAG ACTE:TCTTTACTAAGGS(OTTCTTTACTAAGGS C N G G A G A G T A G A(O TTCTTACAAGGS C N G G A G A G T A G A(O A G A GC T T G G T A G A C C C N TA T:TA T:A T:GTC O C O T C O TCTA TCQ GTTAQ GTTAQ CTATEN)TCTC CTATEN)TCTTCTEN)T A T AS(DI11 T A T AS(DI11 C T AA T AS(DI11 2E 0E 0 S S E S 2 2 2 G G G G G G N N N 6 - 0 / - - / - - / / 1 - / - - / + e e 00 glgleglEed Eed Eed O B8 8 8 W 0 B B 6 H 0 0 9 65 H 1F- 65 H 1F- 65 1 1F- 02.7.06 57 93 16 13 c B31 3 d3 d F F 3 FGAAAATT GT A D GAAAATT GTD GAAAATT GT GAAA AAATTTGCG TGCACIAAATTTGCGIAAATTTGCGDIAAA T GGCAC A GQ)A 9 TGCAC GGCAC A GQ)A 9 TGCACGCACGQ)A 9TGCA AG CA AC G A AC G G AAAC G TTAAG ACTE1:TA AGC ACTE1:TA AGC ACTE1:TA A TCTTTACTAAGGS G A GC(O TTA TCTTTACTAAGGS(O TTA TCTTTACTAAGGS(OTTA TCTTT G G A T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G TA T:TA T:TA T:A T GTC CTA O TCA O TCA O TTCA TCQ GCTTCQ GCT Q G T T EN)T EN)T TCN)TCTC CTAT T A 1 CT T AAT T A 1 CT T AATE T A 1 CT T AAT T AS(DI1S(DI1S(DI1 T A 2E 0 S E 0 S E 2 S E S 2 2 2 2 G G G G G G G G N N N N - / - / - / - / + + / - - / + / - / - / - / - / -+ / + e 00 gleglelelEedegegeO B8 Ed Ed Ed B8 B8 B8 W6 H0 H0 H0 H0 9 65 1F- 65 1F- 65 1F- 65 1F- 102.7.06 57 93 16 13 B3 d d d 1 3 3 3 d F F F 3 FATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TGCGD GAAAA AA TTTTG C AI)A CI)A TI)A A T C CG GC CACG C C AC C C A GG AA Q9 T AGG AA Q9 TG AGGCAAGQ9TG AGGCA GCA C CAACTE1:TAG TTAA CA C TGC ACTE1:TAG TTAA CA C TGC ACTE1:TAG TTAA CA TGC A AT AGGS(O T T A AAGGS A(O T A AAGGS C N G G A G A G T A G A(O T A AA GC C T T TC T T TC T T A T A G A C N G G A G A GC T A G C C N G G A G A GC T A : A T:T:T:QO T GTCA O TATCA O TAC O EN)TCTTCQ GCT Q GTTAQ CTATEN)T TC CTATEN)TCTC CT TEN)S(DI11 T A T AS(DI11 T A T AS(DI11 T AA T AS(DI11 2E 0 S E 0 S E S 2 2 2 G G G G G G N N N - / + / + / ++ + / / - / - - / / - - / + ele 0 l el0 gEed gegeO B8 Ed Ed W 0 B80 B8 6 H65 H 1F- 65 H0 9 1F- 65 1 1F- 02.7.06 57 93 16 13 B3 d 1 3 d3 d F F 3 FA T AA AAGTGG A T AA AAGTGG A T AA AAGT G G A TT T G A TT T A TT GT AAAAT GC D T GC D G T GC D TGCACTT GCACG GITAGAGCAAACQ)AAAA 91 TGCACTT GCACG GIAG GCAAACQ)AAAA 91 TGCACTT G GCACGI)AG GCAAACQ91 TTATG ACTE:TTAAG ACTE:TTAAG ACTE:TCTTACTAAGGS(O TTCTTTACTAAGGS G G A G A G T A G A(OTTCTTTACAAGGS C N G G A G A G T A G A(O G G A G A GC T A G A C N C TC C N TA T:T:T:GTCTA O TATCTA O TATCTA O TCTCQ G Q G Q CTATEN)TCTC CTATEN)TCTC CTATEN)T A T AS(DI11 T A T AS(DI11 T A T AS(DI11 0E 2 2 S E S E S 2 2 2 G G G G G G N N N - / + / + / +- / - / / ++ + / / - + / - e 00 gleglelEed EegeO B8 d H0 B8 Ed 0 B8 W 0 69 65 H 1F- 65 H 1F- 65 1 1F- 02.7.06 57 93 16 13 B3 d 1 3 d3 d F F 3 FGAAAATT GT A D GAAAATT GT GAAAATT GT GAAAA AAATTTGCG TGC CIAAATTTGCGDIAAATTTGCGD AAAT TAGAGGCACG)AGC CGCACG)AGC C CACGI)AGC C AAACQ9T GAG AAACQ9T GAGG AAACQ9 T GAG TTAAGC ACTE1:TA AGC AC E1:TA AGC A E1:TA AG TCTTTACTA CAGGS(OTTA TCTTTACTA TS CAGG(OTTA TCTTTACTA CTS CAGG(O TTA TCTTTA G G A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A G A TA T:A T:A T:A T TCA O TTCA O TTCA O TTC G T TNG T G T TA CTCQ)TCTCQN)TCTCQNG ) TCTCQ CT T AATE T AS(DI11 CT T AATE T AS(DI11 CT T AATE T AS(DI11 CT T AATE T AS(0E 2 2 2 S E S E S E S 2 2 2 2 G G G G G G G G N N N N + / + - / ++ / / / - + / +- / ++ / / ++ / / ++ / + ele 0 l ele 0 gEed gEed gEed glEed O B8 B8 B8 8 W 0 0 B 6 H H 0 0 9 65 1F- 65 H 1F- 65 H 1F- 65 1 1F- 02.7.06 57 93 16 133 d d d B 1 3 3 3 d F F F 3 FTTGGT AA C D G AATTTGGT AA C D G AATTTGGT TTACGI)AAA CACTTACGI)AAA CACTTAC CG GC CAAAG C ACCQ9 TG GAGGCAAG TE1:TA CQ9 TG GAGGCAAG C TAAGCAACTE1:TAT AGCAACT TA CAGGS(O TTCTTTACTAAGGS(O T A TCTTTACTAAGG G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A : TA T:TA T:O N GTCTA O TCTA O ) TC Q G Q TTCEN)TCTCEN)DI11 C T AAT T AS(DI11 CT T AAT T AS(DI11 2E 2 2 2 S E S E S E S 2 2 2 2 G G G G G G G G N N N N ddet etA aaertkceN rtR n ogeu m E Ppel l00 ytai l ll ytaiytaiytaiO ranyma eh isranyalehranyaleranyaleW6 r FeitawrtillmirFeitawrti sllmirFeith awrti sllmirFeit wrhti sll912.p C piapeecp C piapeecp C piapeecp Capiapeec07.06 57 93 16 aa a13 B31 4 4 4 d F F F 4 FDIGAAAA Q)AAAATTTGGT AA CTTAC CGDIG ) AAA 9 TGCA GC G 7TGC E1:TAGAG GCAAA ACCQE1:TAG S(O TTA TCTTTACTA T CAGGS(OTTA TCT C N G G A G A G T A G A C N G G A GTT:TCTA CQO GTTT TE TC CTA CTA TATAS( ND)TAT G T A C TI9 G T A lelRd a / T8l e0laRd T8l0laFtsFtsna / n C5 FniC5 FniA A 4N N 2 2 0R 0R G G 1 ggsG G + n4- d N N A A N 8 9 N R R 1T 1T ggsT T n d R R l 00 yra tai l3 3 O nyleyra tainyle92 92 W mirFei at h p Cawrtpii sap llm eecirFei at h p Cawrti sllKE K 6 E 91 piapeecH T H T 02.7.06 57 93 163 d 1 1 1 B31 4 d4 F F F FdedeATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AA Q7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA CE1:TA AGCAA CE1:TA A CA CE1:TA A CA CA CTS TTAT CA CTS TTATGCAACT TTATGCAAC ATCAGG(OTCTTAT AGG N G G A G A GC(OTCTTAT AGGS T A G A C N G G A G A GC T A G A(OTCTTAT AG A G T A G A C C N G G A G A GC T A G Q:GTTA:GTTA:GTTA:EO TCTCQO T TCQO TCQO S( NCTATE CTATE TCTATE T T SNC T SNC T SNTDI )9 GA A T A C(TDI )T 9 GA A T A C(TDI )T 9 GA A T A C(TDI )9 lelleleRd T80laRd t T8l0laRd T8l0laF 5sFtsFtsC FniC5 FniC5 Fni2 2 2 G G G G G G N N N 02 12 2 T 2 T TT TT R R R 0 3 3 0 9 9 3 O 2 2 92 W K K 6 E E KE 912.H T H T H T 07.06 57 93 16 F 1 1 13 1 B31 d F F ededeGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)T TGCACGCACG)GCACGCACG)AGC AG GCAAACQ71 AG GCAAACQ71 TAG GCAAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT TTACAAGGS(OT AAG ACTE1:TAT TTACAA S(T A TC T TC TC T TC TC T TC GG OTCT G G A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A GTTA:TA:TA:T TCTCQO GTT QO GTT Q GTT TE TC CTE TC CEO TC CTA SNCTA S CTATS CTA TATA(TATA( NT TA( NT T G T A C TDI )9 G T A C TDI )9 GA T A C TDI )9 GA T A lellelle lleRd Rd Rd RdlT80laT80laT8laT8laF 5tsF 5tsF 05tsF 0tsC FniC FniC FniC5 Fni2 2 2 2 G G G G G G G G N N N N 32 4 5 6 T 2T 2 2 T T TT TT R R R R 0 39 3 3 3 0 O 2 92 92 92 W KE KE KE K 6 E 912.H T H T H T H T 07.06 57 93 16 1 133 F 1F 1F 1 B 1 dd dF ee edeATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AA Q7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA CE1:TA AGCAA CE1:TA A CA CE1:TA A CA CA CTS TTAT CA CTS TTATGCAACT TTATGCAAC ATCAGG(OTCTTAT AGG N G G A G A GC(OTCTTAT AGGS T A G A C N G G A G A GC T A G A(OTCTTAT AG A G T A G A C C N G G A G A GC T A G Q:GTTA:GTTA:GTTA:EO TCTCQO T TCQO TCQO S( NCTATE CTATE TCTATE T T SNC T SNC T SNTDI )9 GA A T A C(TDI )T 9 GA A T A C(TDI )T 9 GA A T A C(TDI )9 lelleleRd T80laRd t T8l0laRd T8l0laF 5sFtsFtsC FniC5 FniC5 Fni2 2 2 G G G G G G N N N 72 82 9 T 2 T TT TT R R R 0 3 3 0 9 9 3 O 2 2 92 W K K 6 E E KE 912.H T H T H T 07.06 57 93 16 F 1 1 13 1 B31 d F F ededeGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)T TGCACGCACG)GCACGCACG)AGC AG GCAAACQ71 AG GCAAACQ71 TAG GCAAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT TTACAAGGS(OT AAG ACTE1:TAT TTACAA S(T A TC T TC TC T TC TC T TC GG OTCT G G A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A GTTA:TA:TA:T TCTCQO GTT QO GTT Q GTT TE TC CTE TC CEO TC CTA SNCTA S CTATS CTA TATA(TATA( NT TA( NT T G T A C TDI )9 G T A C TDI )9 GA T A C TDI )9 GA T A lellelle lleRd Rd Rd RdlT80laT80laT8laT8laF 5tsF 5tsF 05tsF 0tsC FniC FniC FniC5 Fni2 2 2 2 G G G G G G G G N N N N 03 1 4 6 T 3T 3 3 T T TT TT R R R R 0 39 3 3 3 0 O 2 92 92 92 W KE KE KE K 6 E 912.H T H T H T H T 07.06 57 93 16 1 133 F 1F 1F 1 B 1 dd dF ee edeATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT GT CTTG ACGDIGAAAA ) AAAATTT G TTGC C CG GC C CG GC C CG C C AC GG AA Q7T GAGG AA Q7T GAGG AA Q7TG GAGGCAA GCAA CE1:TA AGCAA CE1:TA A CA CE1:TA A CA CA CTS TTAT CA CTS TTATGCAACT TTATGCAAC ATCAGG(OTCTTAT AGG G G A G A GC(OTCTTAT AGGS T A G A C N G G A G A GC T A G A(OTCTTAT AG A G T A G A C N C N G G A G A GC T A G Q:GTTAQ:GTTA:AA C:EO TCTC O TCTCQO AAT O S( NCTATE T T S CTATE C CAAA Q TI9 G T A C( NT SNTTEN)D)A A TDI )T 9 GA A T A C(TDI )9 A gTA T G A AS(DI01 lelRdl edlT8 F 0laR tsT80lat0 C5 FniF C5sFniE S 2 2 1 G G G G G G N N N 7R;3 8 1 T 3 10 T TT S2 R R B PT T 0 3 0 9 39le3 O 2 2 K Rd92 W6 E KE T8 F 0K 9 H T H T C5E 1 F H T 02.7.06 57 93 16 F 1 3 13 1 B31 d F F ededeGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)T TGCACGCACG)GCACGCACG)AGC AG GCAAACQ71 AG GCAAACQ71 TAG GCAAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT TTACAAGGS(OT AAG ACTE1:TAT TTACAAG S(T A TC T T TC T T TC T T G OTCT G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A AA C:A C:A C:A CAA AT QO A CAA AT QO A CAAT QO A CAA CA A CA A AA A TTEN)TTEN)CA EN)CA ATAS0 ATA 0 ATATT 0 ATA g T G A A(DI1 g T G A AS(DI1 g T G A AS(DI1 g T G 0E 0E 0E 0 S S S E S 1 1 1 1 G G G G G G G G N N N N R;1R;1R;R;1S32 1S6 1 2 1 1 S92 1S2 PT T PT T PT 3 B B B T B PT T 0 e39e39e3 0 ll l9leO Rd T82 W K Rd82 d2 d K R8 R8 6 F 0E T0E T0KE T0 912.C5 F F H T C5 F F H T C5 F F H T C5 F 07.06 57 93 16 3 133 F 3F 3F 3 B 1 dd dF ee edeATTT GT CTTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT G TTGC CACGI)AGC C CACGI)AGC C CACGI)AGC C CAC GG AACQ7T GAGG AACQ7T GAGG AACQ7T GAGG AA GCAA E1:TA AGCAA E1:TA AGCAA E1:TA AGCA CA CTS(TTAT CA CTS(TTAT CA CTS(TTAT CAAC AT AGG OTCTTAT AGG OTCTTAT AGG OTCTTAT AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G : AA C:AA C:AA C:QO CAAT O AT O AT TEN)CAAA Q CA EN)CAAA Q CA EN)CAA O A QN)AS(DI01 A gTATT T G A AS(DI01 A gTATT T G A AS(DI01 A gTATTE T G A AS(DI01 0E 0 S E 0 S E S 1 1 1 G G G G G G N N N R;1R;R;1S5 2 3 1S0 213 T 2T S2 B P T B P T B PT T 00 le39le39le39 O Rd2 Rd2 d2 W T8 F 0K T8 5E F 0K R E T8 F 0K 6 E 912.T C F H T C5 F H T C5 F H T 07.06 57 93 16 F 3 3 13 3 B31 d F F ededeGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)T TGCACGCACG)GCACGCACG)AGC AG GCAAACQ71 AG GCAAACQ71 TAG GCAAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT TTACAAGGS(OT AAG ACTE1:TAT TTACAAG S(T A TC T T TC T T TC T T G OTCT G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A AA C:A C:A C:A CAA AT QO A CAA AT QO A CAAT QO A CAA CA A CA A AA A TTEN)TTEN)CA EN)CA ATAS0 ATA 0 ATATT 0 ATA g T G A A(DI1 g T G A AS(DI1 g T G A AS(DI1 g T G 0E 0E 0E 0 S S S E S 1 1 1 1 G G G G G G G G N N N N R;2R;2R;R;1S62 1S9 2 2 1 2 S23 1S5 PT T PT T PT 3 B B B T B PT T 0 e39e39e3 0 ll l9leO Rd T82 W K Rd82 d2 d K R8 R8 6 F 0E T0E T0KE T0 912.C5 F F H T C5 F F H T C5 F F H T C5 F 07.06 57 93 16 3 133 F 3F 3F 3 B 1 dd dF ee edeATTT GT CTTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT G TTGC CACGI)AGC C CACGI)AGC C CACGI)AGC C CAC GG AACQ7T GAGG AACQ7T GAGG AACQ7T GAGG AA GCAA E1:TA AGCAA E1:TA AGCAA E1:TA AGCA CA CTS(TTAT CA CTS(TTAT CA CTS(TTAT CAAC AT AGG OTCTTAT AGG OTCTTAT AGG OTCTTAT AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G : AA C:AA C:AA C:QO CAAT O AT O AT TEN)CAAA Q CA EN)CAAA Q CA EN)CAA O A QN)AS(DI01 A gTATT T G A AS(DI01 A gTATT T G A AS(DI01 A gTATTE T G A AS(DI01 0E 0 S E 0 S E S 1 1 1 G G G G G G N N N R;3R;R;1S0 3 2 1S3 316 T 2T S2 B P T B P T B PT T 00 le39le39le39 O Rd2 Rd2 d2 W T8 F 0K T8 5E F 0K R E T8 F 0K 6 E 912.T C F H T C5 F H T C5 F H T 07.06 57 93 16 F 3 3 13 3 B31 d F F ededeGAAAATT GTD GAAAATT GTD GAAAATT GTD GAA AAAATTTGCGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)T TGCACGCACG)GCACGCACG)AGC AG GCAAACQ71 AG GCAAACQ71 TAG GCAAACQ7T G TTAAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT TTACAAGGS(OT AAG ACTE1:TAT TTACAAG S(T A TC T T TC T T TC T T G OTCT G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A AA C:A C:A C:A CAA AT QO A CAA AT QO A CAAT QO A CAA CA A CA A AA A TTEN)TTEN)CA EN)CA ATAS0 ATA 0 ATATT 0 ATA g T G A A(DI1 g T G A AS(DI1 g T G A AS(DI1 g T G 0E 0E 0E 0 S S S E S 1 1 1 1 G G G G G G G G N N N N R;3R;3R;R;1S92 1S2 3 3 1 4 S53 1S0 PT T PT T PT 2 B B B T B PT T 0 e39e39e3 0 ll l9leO Rd T82 W K Rd82 d2 d K R8 R8 6 F 0E T0E T0KE T0 912.C5 F F H T C5 F F H T C5 F F H T C5 F 07.06 57 93 16 3 133 F 3F 3F 3 B 1 dd dF ee edeATTT GT CTTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT G TTGC CACGI)AGC C CACGI)AGC C CACGI)AGC C CAC GG AACQ7T GAGG AACQ7T GAGG AACQ7T GAGG AA GCAA E1:TA AGCAA E1:TA AGCAA E1:TA AGCA CA CTS(TTAT CA CTS(TTAT CA CTS(TTAT CAAC AT AGG OTCTTAT AGG OTCTTAT AGG OTCTTAT AG A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G : AA C:AA C:AA C:QO CAAT O AT O AT TEN)CAAA Q CA EN)CAAA Q CA EN)CAA O A QN)AS(DI01 A gTATT T G A AS(DI01 A gTATT T G A AS(DI01 A gTATTE T G A AS(DI01 0E 0 S E 0 S E S 1 1 1 G G G G G G N N N R;4R;R;1S3 4 2 1S6 419 T 2T S2 B P T B P T B PT T 00 le39le39le39 O Rd2 Rd2 d2 W T8 F 0K T8 5E F 0K R E T8 F 0K 6 E 912.T C F H T C5 F H T C5 F H T 07.06 57 93 16 F 3 3 13 3 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GAA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAAA TGCACGCACG)T A TGCACGCACG)TGCACGCACG)GC AG G GCAAA A CQ7 E1:TAGAG GCAAACQ7 E1:TAGAGCAAACQ7 E1 T : TAG TTA CT T TTACAAGGS(OT A ACT T TTACAAGGS(OT A G ACT T TTACAAGGS(T A TC T TC TC T T TC T T OTCT G G A G A G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A AA C:A C:TCA:TC CAA CAAT O A AT O ACATQO ACA A Q CAA TTEN)CA A Q T EN)TCCTES T (N)TCC ATA 0 ATATT 0 AGGA 2 AGG g T G A AS(DI1 g T G A AS(DI1 g T G C ADI1 g T G 0E 0 0 0 S E S E S E S 1 1 1 1 G G A A G G G G N N N N R;4R;4R;0R;1S23 1S53 1S8 2 2 1S82 B PT T B PT T B PT T B PT T 00 le3l3l3lO Rd9e9e9eT82 W K Rd82 K Rd82 d K R8 6 F 05E TF 0E T0E T0 912.F H T C5 F F C F H T C5 F H T C5 F 07.06 57 93 3 b b 16 133 F 3F 4F 4 B 1 dd dF ee edeATTT GT CTTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT G TTGC CACGI)AGC C CACGI)AGC C CACGI)AGC C CAC GG AACQ7T GAGG AACQ7T GAGG AACQ7T GAGG AA GCA C ACTE1:TA T TTAATGCA C ACTE1:TATAAGCAACTE1:TATAAGCAAC A A S CAGG(OTCTTATAAGGS(OTTCTTTACTAAGGS(OTTCTTTACTAAG A G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G Q:ATC AAQ:ATC AAQ:ATC AAQ:EO TC TEO TC TEO TC TEO S( N)TCCT G S A( N)TCCT G S A( N)TCCT G S A( N)ADI21 A gG T G C ADI21 A gG T G C ADI21 A gG T G C ADI21 0E 0 0 S E S E S 1 1 1 A A A G G G N N N R;3R;1 4R;5 S82 1S82 1S82 B PT T B PT T B PT T 0 3 3 3 0 lel lO Rd9eT82 Rd92e9 Rd2 W6 F 0KE T80KE T80K 912.T C5 F F H T C5 F F H T C5E F H T 07.06 57 93 b b 163 4F 4 b4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GAA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAAA TGCACGCACG)T TGCACGCACG)TGCACGCACG)GC AG GCAAACQ71 AG GCAAACQ71 AG GCAAACQ71 TAG TTAA TTG ACTE:TT ACAAGGS(OT AAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT TTACAAGGS(OT A TC T TC TC T T GC TC T T TCT G G A G A G T A G A C N G G A G A T A G A C N G G A G A GC T A G A C N G G A ATCA:TCA:TCA:TC TCA TCCTQO A A TESN)TC TCCTQO A A TESN)TC TCCTQO A A TESN)TC TCC AGGA(2 AGGA(2 GGA(GG g T G C ADI1 g T G C ADI1 A g T G C ADI21 A g T G 0E 0 0 0 S E S E S E S 1 1 1 1 A A A A G G G G N N N N R;6R;R;R;1 0 2 3 S82 18 18 18 T S3T S3 S3 B P T B P T B PT T B PT T 0 3 3 0 lelel3lO Rd92 d9ed9eW T8K R 2 T8K R82 K Rd8 6 F 0E F 0E TF 0E TF 0 912.C5 F H T C5 F H T C5 F H T C5 F 07.06 57 93 b4 b b b 16 133 F 4F 4F 4 B 1 dd dF ee edeATTT GT CTTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT G TTGC CACGI)AGC C CACGI)AGC C CACGI)AGC C CAC GG AACQ7T GAGG AACQ7T GAGG AACQ7T GAGG AA GCA C ACTE1:TA T TTAATGCA C ACTE1:TATAAGCAACTE1:TATAAGCAAC A A S CAGG(OTCTTATAAGGS(OTTCTTTACTAAGGS(OTTCTTTACTAAG A G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G Q:ATC AAQ:ATC AAQ:ATC AAQ:EO TC TEO TC TEO TC TEO S( N)TCCT G S A( N)TCCT G S A( N)TCCT G S A( N)ADI21 A gG T G C ADI21 A gG T G C ADI21 A gG T G C ADI21 0E 0 0 S E S E S 1 1 1 A A A G G G N N N R;4R;1 5R;6 S83 1S83 1S83 B PT T B PT T B PT T 0 3 3 3 0 lel lO Rd9eT82 Rd92e9 Rd2 W6 F 0KE T80KE T80K 912.T C5 F F H T C5 F F H T C5E F H T 07.06 57 93 b b 163 4F 4 b4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GAA ) AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAAA 71 TGCACGCACG):TAG G T CAAACQ71 TGCACGCACG)AG GCAAACQ71 TGCACGCACG)GC AG GCAAACQ7T G AAG ACTE:TTAAG ACTE:TTAAG ACTE1:TAT OT TTACAAGGS(OT TTACAAGGS(OT TTACAA S(T A TC T T TC T T TC T T GG OTCT N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A TATCT:AQO TA T:TCAQO TACTQ:O TAC GCTTCE GCT E GTTAE GTT T SN)T TCSNTCTCSNTCT CT g AAT(T A ADI3 T 1 C g AAT()T T A ADI31 C g AAT()T T A ADI31 C g AA T 0E 0E 0E 0 S S S E S 2 2 2 2 A A A A G G G G N N N N R;0R;2R;3R;1S62 1S62 1S6 4 2 1S62 B PT T B PT T B PT T B PT T 00 le3l3l3lO Rd92eRd92e9eRd2 Rd W T8K 8K 8K 8 6 F 0E TF 0E TF 0E TF 0 912.C5 F H T C5 F H T C5 F H T C5 F 07.06 57 93 c4c c c16 133 F 4F 4F 4 B 1 dd dF ee edeATTT GT CTTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT G TTGC CACGI)AGC C CACGI)AGC C CACGI)AGC C CAC GG AACQ7T GAGG AACQ7T GAGG AACQ7T GAGG AA GCA C ACTE1:TA T TTAATGCA C ACTE1:TATAAGCAACTE1:TATAAGCAAC A A S CAGG(OTCTTATAAGGS(OTTCTTTACTAAGGS(OTTCTTTACTAAG A G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G Q:ACT:ACT:ACT:EO T Q GTTAEO T Q GTTAEO T Q GTTAEO S( N)TCTTCTS( N)TCTTCTS( N)TCTTCTS( N)ADI31 C g AA T A ADI31 C g AA T A ADI31 C g AA T A ADI31 0E 0 0 S E S E S 2 2 2 A A A G G G N N N R;5R;1 6R;0 S62 1S62 1S63 B PT T B PT T B PT T 0 3 3 3 0 lel lO Rd9eT82 Rd92e9 Rd2 W6 F 0KE T80KE T80K 912.T C5 F F H T C5 F F H T C5E F H T 07.06 57 93 cc163 4F 4c4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GAA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAAA TGCACGCACG)T TGCACGCACG)TGCACGCACG)GC AG GCAAACQ71 AG GCAAACQ71 AG GCAAACQ71 TAG TTAA TTG ACTE:TT ACAAGGS(OT AAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT TTACAAGGS(OT A TC T TC TC T T GC TC T T TCT G G A G A G T A G A C N G G A G A T A G A C N G G A G A GC T A G A C N G G A TACT:ACT:ACT:AC GT AQO TT AQO TT AQO TT CTTCE GCTTCE GCT E G T T SN)T SN)T TCSN)TCT CT g AAT(T A ADI31 CT g AAT(T A ADI3 T 1 C g AAT(T T A ADI31 C g AA T 0E 0 0 0 S E S E S E S 2 2 2 2 A A A A G G G G N N N N R;3R;R;R;1 4 5 6 S63 16 16 16 T S3T S3 S3 B P T B P T B PT T B PT T 0 3 3 0 lelel3lO Rd92 d9ed9eW T8K R 2 T8K R82 K Rd8 6 F 0E F 0E TF 0E TF 0 912.C5 F H T C5 F H T C5 F H T C5 F 07.06 57 93 c4c c c16 133 F 4F 4F 4 B 1 dd dF ee edeATTT GT CTTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT G TTGC CACGI)AGC C CACGI)AGC C CACGI)AGC C CAC GG AACQ7T GAGG AACQ7T GAGG AACQ7T GAGG AA GCA C ACTE1:TA T TTAATGCA C ACTE1:TATAAGCAACTE1:TATAAGCAAC A A S CAGG(OTCTTATAAGGS(OTTCTTTACTAAGGS(OTTCTTTACTAAG A G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G Q:E AA C:AA C:AA C:O AC O AC O AC S( N)CT TAAA Q CT EN)TAAA Q CT EN)TAA O A QN)ADI31 T gCGCA T G A AS(DI41 T gCGCA T G A AS(DI41 T gCGCAE T G A AS(DI41 0E 0 0 S E S E S 3 3 3 A A A G G G N N N R;0R;1 2R;3 S51 1S51 1S51 B PT T B PT T B PT T 0 3 3 3 0 lel lO Rd9eT82 Rd92e9 Rd2 W6 F 0KE T80KE T80K 912.T C5 F F H T C5 F F H T C5E F H T 07.06 57 93 d d 163 4F 4 d4 1 B31 d F F ededeD GAAAATTGGTD GAAAATT GTD GAAAATT GTD GAA I AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAAA Q)7 E1 TGCACGCACG)AG GCAAACQ71 TGCACGCACG)AG GCAAACQ71 TGCACGCACG)GC AG G AAACQ7T G S(:TT OT AAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT AT CA S(T A TC T T TC T T TCTTA AGG O T G A G A GC T TC C N G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A AA C:C:C:CTAC T QO AA CTAC QO AA CTAC O AA CTA AAA AA AA Q A GC EN)TA EN)TA EN)TA TC A 4 TCGCA 4 TCGCA 4 CG g T G A AS(DI1 g T G A AS(DI1 g T G A AS(DI1 T g T G 0E 0E 0 0 S S E S E S 3 3 3 3 A A A A G G G G N N N N R;4R;5R;R;1S51 1S5 6 1 1S5 0 1 1S52 B PT T B PT T B PT T B PT T 00 le3l3l3lO Rd92eRd92e9eRd2 Rd W T8K 8K 8K 8 6 F 0E TF 0E TF 0E TF 0 912.C5 F H T C5 F H T C5 F H T C5 F 07.06 57 93 d4 d d d 16 133 F 4F 4F 4 B 1 dd dF ee edeATTT GT CTTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT G TTGC CACGI)AGC C CACGI)AGC C CACGI)AGC C CAC GG AACQ7T GAGG AACQ7T GAGG AACQ7T GAGG AA GCA C ACTE1:TA T TTAATGCA C ACTE1:TATAAGCAACTE1:TATAAGCAAC A A S CAGG(OTCTTATAAGGS(OTTCTTTACTAAGGS(OTTCTTTACTAAG A G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G : C:C:C:QO AA CTAC QO AA CTAC QO AA CTAC O AAA AAA AA Q AEN)T EN)T EN)TA EN)AS(DI41 T gCGCA T G A AS(DI41 T gCGCA T G A AS(DI41 T gCGCA T G A AS(DI41 0E 0 0 S E S E S 3 3 3 A A A G G G N N N R;2R;1 3R;4 S52 1S52 1S52 B PT T B PT T B PT T 0 3 3 3 0 lel lO Rd9eT82 Rd92e9 Rd2 W6 F 0KE T80KE T80K 912.T C5 F F H T C5 F F H T C5E F H T 07.06 57 93 d d 163 4F 4 d4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GAA ) AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAAA 71 TGCACGCACG):TAG G T CAAACQ71 TGCACGCACG)AG GCAAACQ71 TGCACGCACG)GC AG GCAAACQ7T G AAG ACTE:TTAAG ACTE:TTAAG ACTE1:TAT OT TTACAAGGS(OT TTACAAGGS(OT TTACAA S(T A TC T TC TC T TC TC T TC GG OTCT N G G A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A AA C:A C:A C:A CTAC T A QO A CTAC QO A CAAT QO A CAA A A GCAEN)TAAA G EN)CAAA EN)CAA TCS4 TC CA 4 ATATT 0 ATA g T G A A(DI1 g T G A AS(DI1 g T G A AS(DI1 g T G 0E 0E 0E 0 S S S E S 3 3 1 1 A A G G G G G G N N N N R;5R;6R;1R;1S52 1S52 1S0 1 2 1S32 B PT T B PT T B PT T B PT T 00 le3l3l3lO Rd92eRd92e9eRd2 Rd W T8K 8K 8K 8 6 F 0E TF 0E TF 0E TF 0 912.C5 F H T C5 F H T C5 F H T C5 F 07.06 57 93 d4 de e16 133 F 4F 4F 4 B 1 dd dF ee edeATTT GT CTTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT G TTGC CACGI)AGC C CACGI)AGC C CACGI)AGC C CAC GG AACQ7T GAGG AACQ7T GAGG AACQ7T GAGG AA GCA C ACTE1:TA T TTAATGCA C ACTE1:TATAAGCAACTE1:TATAAGCAAC A A S CAGG(OTCTTATAAGGS(OTTCTTTACTAAGGS(OTTCTTTACTAAG A G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G : AAAC:AAAC:AA C:QO CA T QO CA T QO CAAT O CAAA AA AA Q TEN)EN)CA EN)CA EN)AS(DI01 A gTATT T G A AS(DI01 A gTATT T G A AS(DI01 A gTATT T G A AS(DI01 0E 0 0 S E S E S 1 1 1 G G G G G G N N N R;1R;1 1R;1 S62 1S92 1S23 B PT T B PT T B PT T 0 3 3 3 0 lel lO Rd9eT82 Rd92e9 Rd2 W6 F 0KE T80KE T80K 912.T C5 F F H T C5 F F H T C5E F H T 07.06 57 93 ee163 4F 4e4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GAA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAAA TGCACGCACG)T A TGCACGCACG)TGCACGCACG)GC AG G GCAAA A CQ7 E1:TAGAG GCAAACQ7 E1:TAGAGCAAACQ7 E1 T : TAG TTA CT T TTACAAGGS(OT A ACT T TTACAAGGS(OT A G ACT T TTACAAGGS(T A TC T TC TC T T TC T T OTCT G G A G A G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A AA C:AA C:AA C:A CAAT O CAAT O AAT O AAA CAAA Q CAAA Q C CAAA Q C A TTEN)TTEN)TTEN)CA ATAS D0 ATAS0 ATA 0 ATA g T G A A( I1 g T G A A(DI1 g T G A AS(DI1 g T G 0E 0 0 0 S E S E S E S 1 1 1 1 G G G G G G G G N N N N R;1R;R;R;1 2 2 2 S53 10 13 16 T S2T S2 S2 B P T B P T B PT T B PT T 0 3 3 0 lelel3lO Rd92 d9ed9eW T8K R 2 T8K R82 K Rd8 6 F 0E F 0E TF 0E TF 0 912.C5 F H T C5 F H T C5 F H T C5 F 07.06 57 93 e4e e e16 133 F 4F 4F 4 B 1 dd dF ee edeATTT GT CTTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT G TTGC CACGI)AGC C CACGI)AGC C CACGI)AGC C CAC GG AACQ7T GAGG AACQ7T GAGG AACQ7T GAGG AA GCA C ACTE1:TA T TTAATGCA C ACTE1:TATAAGCAACTE1:TATAAGCAAC A A S CAGG(OTCTTATAAGGS(OTTCTTTACTAAGGS(OTTCTTTACTAAG A G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G : AAAC:AAAC:AA C:QO CA T QO CA T QO CAAT O CAAA AA AA Q TEN)EN)CA EN)CA EN)AS(DI01 A gTATT T G A AS(DI01 A gTATT T G A AS(DI01 A gTATT T G A AS(DI01 0E 0 0 S E S E S 1 1 1 G G G G G G N N N R;2R;1 2R;2 S92 1S23 1S53 B PT T B PT T B PT T 0 3 3 3 0 lel lO Rd9eT82 Rd92e9 Rd2 W6 F 0KE T80KE T80K 912.T C5 F F H T C5 F F H T C5E F H T 07.06 57 93 ee163 4F 4e4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GAA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAAA TGCACGCACG)T A TGCACGCACG)TGCACGCACG)GC AG G GCAAA A CQ7 E1:TAGAG GCAAACQ7 E1:TAGAGCAAACQ7 E1 T : TAG TTA CT T TTACAAGGS(OT A ACT T TTACAAGGS(OT A G ACT T TTACAAGGS(T A TC T TC TC T T TC T T OTCT G G A G A G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A AA C:AA C:AA C:A CAAT O CAAT O AAT O AAA CAAA Q CAAA Q C CAAA Q C A TTEN)TTEN)TTEN)CA ATAS D0 ATAS0 ATA 0 ATA g T G A A( I1 g T G A A(DI1 g T G A AS(DI1 g T G 0E 0 0 0 S E S E S E S 1 1 1 1 G G G G G G G G N N N N R;3R;R;R;1 3 3 3 S02 13 19 12 T S2T S2 S3 B P T B P T B PT T B PT T 0 3 3 0 lelel3lO Rd92 d9ed9eW T8K R 2 T8K R82 K Rd8 6 F 0E F 0E TF 0E TF 0 912.C5 F H T C5 F H T C5 F H T C5 F 07.06 57 93 e4e e e16 133 F 4F 4F 4 B 1 dd dF ee edeATTT GT CTTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT G TTGC CACGI)AGC C CACGI)AGC C CACGI)AGC C CAC GG AACQ7T GAGG AACQ7T GAGG AACQ7T GAGG AA GCA C ACTE1:TA T TTAATGCA C ACTE1:TATAAGCAACTE1:TATAAGCAAC A A S CAGG(OTCTTATAAGGS(OTTCTTTACTAAGGS(OTTCTTTACTAAG A G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G : AAAC:AAAC:AA C:QO CA T QO CA T QO CAAT O CAAA AA AA Q TEN)EN)CA EN)CA EN)AS(DI01 A gTATT T G A AS(DI01 A gTATT T G A AS(DI01 A gTATT T G A AS(DI01 0E 0 0 S E S E S 1 1 1 G G G G G G N N N R;3R;1 4R;4 S53 1S02 1S32 B PT T B PT T B PT T 0 3 3 3 0 lel lO Rd9eT82 Rd92e9 Rd2 W6 F 0KE T80KE T80K 912.T C5 F F H T C5 F F H T C5E F H T 07.06 57 93 ee163 4F 4e4 1 B31 d F F ededeD GAAAATTGGTD GAAAATT GTD GAAAATT GTD GAA I AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAAA Q)7 E1 TGCACGCACG)AG GCAAACQ71 TGCACGCACG)AG GCAAACQ71 TGCACGCACG)GC AG G AAACQ7T G S(:TT OT AAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT AGC ACTE1:TAT TTACAAGGS(OT AT CA S(T A TC T T TC T T TCTTA AGG O T G A G A GC T TC C N G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A AA C:A C: :C A A A AA C AA A T A QO CA T QO CAAT O CAA C AA A TTEN)CA A EN)CAAA QEN)CAA ATA 0 ATATT 0 ATATT 0 TA g T G A AS(DI1 g T G A AS(DI1 g T G A AS(DI1 A g T G 0E 0E 0 0 S S E S E S 1 1 1 1 G G G G G G G G N N N N R;4R;4R;R;1S62 1S9 4 2 1S2 4 3 1S53 B PT T B PT T B PT T B PT T 00 le3l3l3lO Rd92eRd92e9eRd2 Rd W T8K 8K 8K 8 6 F 0E TF 0E TF 0E TF 0 912.C5 F H T C5 F H T C5 F H T C5 F 07.06 57 93 e4e e e16 133 F 4F 4F 4 B 1 dd dF ee edeATTT GT CTTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT G TTGC CACGI)AGC C CACGI)AGC C CACGI)AGC C CAC GG AACQ7T GAGG AACQ7T GAGG AACQ7T GAGG AA GCA C ACTE1:TA T TTAATGCA C ACTE1:TATAAGCAACTE1:TATAAGCAAC A A S CAGG(OTCTTATAAGGS(OTTCTTTACTAAGGS(OTTCTTTACTAAG A G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G : T:T:T:O TCACQO TCACQO TC CQ Q GC TE)GGTE GC GE GCAGEO N AT SNATGTSNATGTSNA 1 gT TG TA T()C 1 gT TG TA T()C 1 gT TG T T()S(DI0 CDI5 CDI5 C A CDI51 0E 0 0 S E S E S 3 3 3 G G G G G G N N N R;0R;1 1R;2 S04 1S04 1S04 B PT T B PT T B PT T 0 3 3 3 0 lel lO Rd9eT82 Rd92e9 Rd2 W6 F 0KE T80KE T80K 912.T C5 F F H T C5 F F H T C5E F H T 07.06 57 93 ff163 4F 4f4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GAA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAAA TGCACGCACG)T TGCACGCACG)TGCACGCACG)GC AG GCAAACQ71 AG GCAAACQ71 AG GCAAACQ71 TAG TTAA TTG ACTE:TT ACAAGGS(OT AAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT TTACAAGGS(OT A TC T TC TC T T GC TC T T TCT G G A G A G T A G A C N G G A G A T A G A C N G G A G A GC T A G A C N G G A T TC:T TC:T TC:T T GC CA ATGGQO CA TE G SN)C ATGGQO CA TE G SN)C ATGGQO CA TE G SN)C ATG C gT TG TA T(CDI51 C gT TG TA T(CDI51 C gT TG TA T(CDI51 C gT TG T 0E 0 0 0 S E S E S E S 3 3 3 3 G G G G G G G G N N N N R;3R;R;R;1 4 6 0 S04 10 10 11 T S4T S4 S5 B P T B P T B PT T B PT T 0 3 3 0 lelel3lO Rd92 d9ed9eW T8K R 2 T8K R82 K Rd8 6 F 0E F 0E TF 0E TF 0 912.C5 F H T C5 F H T C5 F H T C5 F 07.06 57 93 f4f f f16 133 F 4F 4F 4 B 1 dd dF ee edeAATT GTD GAAAATT GTD GAAAATT GT GAAAATT G ATTTGCGIAAATTTGCG AAATTTGCGD AAATTTGC AC AGGCAC A GQ)A 7TGCACGCACGIQ)A 7TGCACGCACGIQ)A 7TGCACGCAC A AC G G AAAC G G AAC G G AA TGC T C ACTE1:TA T TTAATGC C ACTE1:TA TTAAGCAACTE1:TATAAGCAAC A A CAGGS(OTCTTATA S CAGG(OTCTTTACTA S CAGG(OTTCTTTACTAAG G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A G A GC T A G C GQ:T O T GCAC GQ:T:T:O T GCAC GQO T GCAC GQO TESNC ATGTESNC E C E ATGTSNATGTS A()C 1 gT TG TA T()C 1 gT TG TA T()C 1 gT T T T( N)TDI5 CDI5 CDI5 C GA CDI51 0E 0 S E 0 S E S 3 3 3 G G G G G G N N N R;1R;R;1 21 3 S15 S15 1S1 B PT T B PT 5 T B PT T 0 39 3 0 l 2ele3le3 O K Rd9 82 Rd9 82 Rd92 W6 E TF 0KE T0K T80K 912.H T C5 F F H T C5E F F H T C5E F H T 07.06 573 f 9163 4fF 4f4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GAA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAAA TGCACGCACG)T TGCACGCACG)TGCACGCACG)GC AG GCAAACQ71 AG GCAAACQ71 AG GCAAACQ71 TAG TTAA TTG ACTE:TT ACAAGGS(OT AAG ACTE:TT TTACAAGGS(OT AAG ACTE:TT TTACAAGGS(OT A TC T TC TC T T GC TC T T TCT G G A G A G T A G A C N G G A G A T A G A C N G G A G A GC T A G A C N G G A TCTC:T TC:TG:TG GCA ATGGQO CA TE G SN)C ATGGQO TT TE T ACQO TT SN)AGCTTE T A SN)AGC CTGA(5 CTGA(5 TTT(TT g T T T CDI1 g T T T CDI1 C g C C T CDI61 C g C C 0E 0 0 0 S E S E S E S 3 3 4 4 G G G G G G G G N N N N R;4R;R;R;1 6 0 2 S15 11 11 11 T S5T S5 S5 B P T B P T B PT T B PT T 0 3 3 0 lelel3lO Rd92 d9ed9eW T8K R 2 T8K R82 K Rd8 6 F 0E F 0E TF 0E TF 0 912.C5 F H T C5 F H T C5 F H T C5 F 07.06 57 93 f4fg g 16 133 F 4F 4F 4 B 1 dd dF ee edeATTT GT CTTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT G TTGC CACGI)AGC C CACGI)AGC C CACGI)AGC C CAC GG AACQ7T GAGG AACQ7T GAGG AACQ7T GAGG AA GCA C ACTE1:TA T TTAATGCA C ACTE1:TATAAGCAACTE1:TATAAGCAAC A A S CAGG(OTCTTATAAGGS(OTTCTTTACTAAGGS(OTTCTTTACTAAG A G T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G Q:G:G: :EO TT TTACTQEO TT TTACTQEO TTG TTACTQEO S( N)AGTCTTS AGCTS AGCTS C 1 g C C T( N)TT C 1 g C C T( N)TT C 1 g C C T( N)DI6 C TDI6 C TDI6 C T CDI61 0E 0 0 S E S E S 4 4 4 G G G G G G N N N R;3R;1 4R;5 S15 1S15 1S15 B PT T B PT T B PT T 0 3 3 3 0 lel lO Rd9eT82 Rd92e9 Rd2 W6 F 0KE T80KE T80K 912.T C5 F F H T C5 F F H T C5E F H T 07.06 57 93 g g 163 4F 4 g4 1 B31 d F F ededeA T AA AAGTGG T A T AA AAGTGG A T AA AAGTGG DIG A TT D G A TT T G A TT T AA T GCG AA T GCGD T GC D Q)A 7TGCA ACTTACI)A ACTTACI)AAAACTTACGI)GGCA GQ7TGCAGGCA GQ7 TGCAGGC GQ7 E1 S(:TAG OTTAA CA AC TCTTG TACTAACTE1:TAG AGGS(OTTAA CA AC TCTTG TAC ACTE1:TAG TAAGGS(O TTAA CAAAC 1 TCTTG TAC ACTE:TAAGGS C N G G A G A GC T A G A C N G G A G A GC T A C(O G A C N G G A G A G T A G A C N TTGCQ:TTGC:TTGC:TTAT O TTATQO TTA QO GC E GC E TE A TS TS GCTS CTT g C CT T( N)A CDI61 CTT g C CT T( N)A CDI61 CTT g C CT T( N)CDI61 0E 0 0 S E S E S 4 4 4 G G G G G G N N N R;6R;R;1 01 2 S15 S16 11 T T PT S6 B P B T B PT T 0 3 0 lele3l3 O Rd9 T82 Rd9e82 Rd92 W6 F 0KE T0KE T80K 912.C5 F F H T C5 F F H T C5E F H T 07.06 57 93 g g 163 4F 4 g4 1 B31 d F F ededeGAAAATT GT A D GAAAATT GTD GAAAATT GT AAATTTGCG TGCACGCACGI)AAAATTTGCG GC CGCACGI)AAAATTTGCGD GC CGCACGI)TAG GCAAACQ7 T GAG AAACQ7 T GAG AAACQ7 TTAAG ACTE1:TA AGC ACTE1:TA AGC ACTE1:TCTTTACTAAGGS(O TTA TCTTTACTAAGGS(O TTA TCTTTACTAAGGS(O G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N TTGC:TTGC:TTGC:TTA QO TTA QO TTA QO AG CTCT TTES g C CT T( N)AGCTTES CDI61 CTT g C CT T( N)AGCTTES CDI61 CTT g C CT T( N)CDI61 0E 0 0 S E S E S 4 4 4 G G G G G G N N N R;3R;R;1 4 5 S16 1S1 11 PT 6 S6 B T B PT T B PT T 0 e39e3 0 ll9le39 O Rd T82 W K Rd82 d2 K R8K 6 F 0E TF 0E T0E 912.C5 F H T C5 F F H T C5 F H T 07.06 57 93 g g g 163 4 4 13 F 4 B 1 d F d F eedeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)T TGCACGCACG)TGCACGCACG)G AG GCAAACQ71 AG GCAAACQ71 AG GCAAACQ71 TA TTAA TC TG ACTE:TTAAG ACTE:TTAAG ACTE:TT TTACTA CAGGS(O TTCTTTACTA CAGGS(OTTCTTTACTA CAGGS(OTTC G G A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G TTGC:TTA:TTA:T TTATQEO G A CTS TCTCQEO GTCTCQO GTC G TAT TATE T CTT g C CT T( N)C CDI61 T gATAS T A C( NTDI )C 9 T gATAS T A C( NTDI )C 9 T gA T 0E 0 0 0 S E S E S E S 4 2 2 2 G G G G G G G G N N N N R;6R;0R;0R;1S16 1S03 1 0 S13 1S23 B PT T B PT T B PT T B PT T 0 le3l3 3 0 O Rd92eld9eld9ed W T8K RT82 K R 2 T8K RT8 6 F 05E F 05E F 0E F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 g4 h h h 16 133 F 4F 4F 4 B 1 dd dF ee edeAAA AATTT GT TTGCGDIGAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT TTG C CGCACG)AGC CGCACGI)AGC C CACGI)AGC C CA GAG AAACQ7T GAG AAACQ7T GAGG AACQ7T GAGG A AAGC ACTE1:TA AGC A E1:TA AGCAA E1:TA AGCAA TTTACTA CAGGS(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTAA A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A G A GC T A TA:TA:T: :TCQEO GT TCTCQ GT EO TCTA CQ GTT EO TCTA CQEO AT TASNTATSNTATSNTATS C(C T 9 gATA T A C(C TA T 9 g T A C(C TA T 9 g T A C( NADI )TDI )TADI )TA TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;0R;0R;1 0 S33 1S43 15 T T PT S3 B P B T B PT T 0 3 0 9l2e3le3l3 O K Rd9 82 Rd92eRd92 W6 E TF 0K T8 5E F 0K T8 5E F 0K 9 F H T C5E 12.H T C F H T C F H T 07.06 57 93 h 163 4 h F 4 h4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)T TGCACGCACG)TGCACGCACG)G AG GCAAACQ71 AG GCAAACQ71 AG GCAAACQ71 TA TTAA TC TG ACTE:TTAAG ACTE:TTAAG ACTE:TT TTACTA CAGGS(OTTCTTTACTAAGGS A GC(OTTCTTTACTAAGGS(OTTC G G A G A G T A G A C N G G A G T A G A C N G G A G A GC T A G A C N G G GTTA:TTA:TTA:T TCTCQO G TCQO G TCQO G TATE TCTATE TC TE TC C T S g T A C( NC T SNCTAT SNCT TA A TDI )9 T gA A T A C(TDI )9 T gA A T A C(TDI )9 T gA T 0E 0 0 0 S E S E S E S 2 2 2 2 G G G G G G G G N N N N R;0R;0R;0R;1S63 1S73 1 0 S83 1S14 B PT T B PT T B PT T B PT T 0 le3l3 3 0 O Rd92eld9eld9ed W T8K RT82 K R 2 T8K RT8 6 F 05E F 05E F 0E F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 h4 h h h 16 133 F 4F 4F 4 B 1 dd dF ee edeAAA AATTT GT TTGCGDIGAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT TTG C CGCACG)AGC CGCACGI)AGC C CACGI)AGC C CA GAG AAACQ7T GAG AAACQ7T GAGG AACQ7T GAGG A AAGC ACTE1:TA AGC A E1:TA AGCAA E1:TA AGCAA TTTACTA CAGGS(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTAA A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A G A GC T A TA:TA:T: :TCQEO GT TCTCQ GT EO TCTA CQ GTT EO TCTA CQEO AT TASNTATSNTATSNTATS C(C T 9 gATA T A C(C TA T 9 g T A C(C TA T 9 g T A C( NADI )TDI )TADI )TA TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;0R;0R;1 1 S24 1S34 10 T T PT S3 B P B T B PT T 0 3 0 9l2e3le3l3 O K Rd9 82 Rd92eRd92 W6 E TF 0K T8 5E F 0K T8 5E F 0K 9 F H T C5E 12.H T C F H T C F H T 07.06 57 93 h 163 4 h F 4 h4 1 B31 d F F ededeGTD GAAAATT GTD GAAAATT GTD GAAAATT GTD GA C CGIAAAATTTGCGIAAAATTTGCGIAAATTTGCGIA AG)GC CQ7 ACGCACG)GCACGCACG)AGCACGCACG)AG 1 TAG GCAAACQ71 T G G AAACQ7T G G AAACQ7T CTE:T AG ACTE:TA AGC ACTE1:TA AGC ACTE1:TA TTA TC TT CTA OTTA TCTTTACT AGG OTTA TCTTTACT AGG OTT GGS(O T A AGGS(A S(A S(TC G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G A G A GC T A G A C N G G GTTAQ:GTTA:GTTA:GT TCTC O TCTCQO TCTCQO C CTATE ATAS( NCTATE ATAS( NCTATE ATAS T (NCT T g T A C TDI )9 T g T A C TDI )9 T g T A C TDI )9 T gA T 0E 0E 0 0 S S E S E S 2 2 2 2 G G G G G G G G N N N N R;1R;1R;R;1S13 1S2 1 3 1S3 1 3 1S43 B PT T B PT T B PT T B PT T 00 le3l3l3lO Rd92eRd92e9eRd2 Rd W T8K 8K 8K 8 6 F 0E TF 0E TF 0E TF 0 912.C5 F H T C5 F H T C5 F H T C5 F 07.06 57 93 h4 h h h 16 133 F 4F 4F 4 B 1 dd dF ee edeAAA AATTT GT TTGCGDIGAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT TTG C CGCACG)AGC CGCACGI)AGC C CACGI)AGC C CA GAG AAACQ7T GAG AAACQ7T GAGG AACQ7T GAGG A AAGC ACTE1:TA AGC A E1:TA AGCAA E1:TA AGCAA TTTACTA CAGGS(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTAA A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A G A GC T A TA:TA:T: :TCQEO GT TCTCQ GT EO TCTA CQ GTT EO TCTA CQEO AT TASNTATSNTATSNTATS C(C T 9 gATA T A C(C TA T 9 g T A C(C TA T 9 g T A C( NADI )TDI )TADI )TA TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;1R;1R;1 1 S53 1S63 17 T T PT S3 B P B T B PT T 0 3 0 9l2e3le3l3 O K Rd9 82 Rd92eRd92 W6 E TF 0K T8 5E F 0K T8 5E F 0K 9 F H T C5E 12.H T C F H T C F H T 07.06 57 93 h 163 4 h F 4 h4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)T TGCACGCACG)TGCACGCACG)G AG GCAAACQ71 AG GCAAACQ71 AG GCAAACQ71 TA TTAA TC TG ACTE:TTAAG ACTE:TTAAG ACTE:TT TTACTA CAGGS(OTTCTTTACTAAGGS A GC(OTTCTTTACTAAGGS(OTTC G G A G A G T A G A C N G G A G T A G A C N G G A G A GC T A G A C N G G GTTA:TTA:TTA:T TCTCQO G TCQO G TCQO G TATE TCTATE TC TE TC C T S g T A C( NC T SNCTAT SNCT TA A TDI )9 T gA A T A C(TDI )9 T gA A T A C(TDI )9 T gA T 0E 0 0 0 S E S E S E S 2 2 2 2 G G G G G G G G N N N N R;1R;1R;1R;1S83 1S14 1 1 S24 1S34 B PT T B PT T B PT T B PT T 0 le3l3 3 0 O Rd92eld9eld9ed W T8K RT82 K R 2 T8K RT8 6 F 05E F 05E F 0E F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 h4 h h h 16 133 F 4F 4F 4 B 1 dd dF ee edeAAA AATTT GT TTGCGDIGAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT TTG C CGCACG)AGC CGCACGI)AGC C CACGI)AGC C CA GAG AAACQ7T GAG AAACQ7T GAGG AACQ7T GAGG A AAGC ACTE1:TA AGC A E1:TA AGCAA E1:TA AGCAA TTTACTA CAGGS(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTAA A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A G A GC T A TA:TA:T: :TCQEO GT TCTCQ GT EO TCTA CQ GTT EO TCTA CQEO AT TASNTATSNTATSNTATS C(C T 9 gATA T A C(C TA T 9 g T A C(C TA T 9 g T A C( NADI )TDI )TADI )TA TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;2R;2R;1 2 S03 1S13 12 T T PT S3 B P B T B PT T 0 3 0 9l2e3le3l3 O K Rd9 82 Rd92eRd92 W6 E TF 0K T8 5E F 0K T8 5E F 0K 9 F H T C5E 12.H T C F H T C F H T 07.06 57 93 h 163 4 h F 4 h4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)T TGCACGCACG)TGCACGCACG)G AG GCAAACQ71 AG GCAAACQ71 AG GCAAACQ71 TA TTAA TC TG ACTE:TTAAG ACTE:TTAAG ACTE:TT TTACTA CAGGS(OTTCTTTACTAAGGS A GC(OTTCTTTACTAAGGS(OTTC G G A G A G T A G A C N G G A G T A G A C N G G A G A GC T A G A C N G G GTTA:TTA:TTA:T TCTCQO G TCQO G TCQO G TATE TCTATE TC TE TC C T S g T A C( NC T SNCTAT SNCT TA A TDI )9 T gA A T A C(TDI )9 T gA A T A C(TDI )9 T gA T 0E 0 0 0 S E S E S E S 2 2 2 2 G G G G G G G G N N N N R;2R;2R;2R;1S33 1S43 1 2 S53 1S63 B PT T B PT T B PT T B PT T 0 le3l3 3 0 O Rd92eld9eld9ed W T8K RT82 K R 2 T8K RT8 6 F 05E F 05E F 0E F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 h4 h h h 16 133 F 4F 4F 4 B 1 dd dF ee edeAAA AATTT GT TTGCGDIGAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT TTG C CGCACG)AGC CGCACGI)AGC C CACGI)AGC C CA GAG AAACQ7T GAG AAACQ7T GAGG AACQ7T GAGG A AAGC ACTE1:TA AGC A E1:TA AGCAA E1:TA AGCAA TTTACTA CAGGS(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTAA A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A G A GC T A TA:TA:T: :TCQEO GT TCTCQ GT EO TCTA CQ GTT EO TCTA CQEO AT TASNTATSNTATSNTATS C(C T 9 gATA T A C(C TA T 9 g T A C(C TA T 9 g T A C( NADI )TDI )TADI )TA TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;2R;2R;1 2 S73 1S83 11 T T PT S4 B P B T B PT T 0 3 0 9l2e3le3l3 O K Rd9 82 Rd92eRd92 W6 E TF 0K T8 5E F 0K T8 5E F 0K 9 F H T C5E 12.H T C F H T C F H T 07.06 57 93 h 163 4 h F 4 h4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)T TGCACGCACG)TGCACGCACG)G AG GCAAACQ71 AG GCAAACQ71 AG GCAAACQ71 TA TTAA TC TG ACTE:TTAAG ACTE:TTAAG ACTE:TT TTACTA CAGGS(OTTCTTTACTAAGGS A GC(OTTCTTTACTAAGGS(OTTC G G A G A G T A G A C N G G A G T A G A C N G G A G A GC T A G A C N G G GTTA:TTA:TTA:T TCTCQO G TCQO G TCQO G TATE TCTATE TC TE TC C T S g T A C( NC T SNCTAT SNCT TA A TDI )9 T gA A T A C(TDI )9 T gA A T A C(TDI )9 T gA T 0E 0 0 0 S E S E S E S 2 2 2 2 G G G G G G G G N N N N R;2R;2R;3R;1S24 1S34 1 3 S03 1S13 B PT T B PT T B PT T B PT T 0 le3l3 3 0 O Rd92eld9eld9ed W T8K RT82 K R 2 T8K RT8 6 F 05E F 05E F 0E F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 h4 h h h 16 133 F 4F 4F 4 B 1 dd dF ee edeAAA AATTT GT TTGCGDIGAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT TTG C CGCACG)AGC CGCACGI)AGC C CACGI)AGC C CA GAG AAACQ7T GAG AAACQ7T GAGG AACQ7T GAGG A AAGC ACTE1:TA AGC A E1:TA AGCAA E1:TA AGCAA TTTACTA CAGGS(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTAA A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A G A GC T A TA:TA:T: :TCQEO GT TCTCQ GT EO TCTA CQ GTT EO TCTA CQEO AT TASNTATSNTATSNTATS C(C T 9 gATA T A C(C TA T 9 g T A C(C TA T 9 g T A C( NADI )TDI )TADI )TA TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;3R;3R;1 3 S23 1S33 14 T T PT S3 B P B T B PT T 0 3 0 9l2e3le3l3 O K Rd9 82 Rd92eRd92 W6 E TF 0K T8 5E F 0K T8 5E F 0K 9 F H T C5E 12.H T C F H T C F H T 07.06 57 93 h 163 4 h F 4 h4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)T TGCACGCACG)TGCACGCACG)G AG GCAAACQ71 AG GCAAACQ71 AG GCAAACQ71 TA TTAA TC TG ACTE:TTAAG ACTE:TTAAG ACTE:TT TTACTA CAGGS(OTTCTTTACTAAGGS A GC(OTTCTTTACTAAGGS(OTTC G G A G A G T A G A C N G G A G T A G A C N G G A G A GC T A G A C N G G GTTA:TTA:TTA:T TCTCQO G TCQO G TCQO G TATE TCTATE TC TE TC C T S g T A C( NC T SNCTAT SNCT TA A TDI )9 T gA A T A C(TDI )9 T gA A T A C(TDI )9 T gA T 0E 0 0 0 S E S E S E S 2 2 2 2 G G G G G G G G N N N N R;3R;3R;3R;1S53 1S63 1 3 S73 1S83 B PT T B PT T B PT T B PT T 0 le3l3 3 0 O Rd92eld9eld9ed W T8K RT82 K R 2 T8K RT8 6 F 05E F 05E F 0E F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 h4 h h h 16 133 F 4F 4F 4 B 1 dd dF ee edeAAA AATTT GT TTGCGDIGAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT TTG C CGCACG)AGC CGCACGI)AGC C CACGI)AGC C CA GAG AAACQ7T GAG AAACQ7T GAGG AACQ7T GAGG A AAGC ACTE1:TA AGC A E1:TA AGCAA E1:TA AGCAA TTTACTA CAGGS(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTAA A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A G A GC T A TA:TA:T: :TCQEO GT TCTCQ GT EO TCTA CQ GTT EO TCTA CQEO AT TASNTATSNTATSNTATS C(C T 9 gATA T A C(C TA T 9 g T A C(C TA T 9 g T A C( NADI )TDI )TADI )TA TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;3R;3R;1 3 S14 1S24 13 T T PT S4 B P B T B PT T 0 3 0 9l2e3le3l3 O K Rd9 82 Rd92eRd92 W6 E TF 0K T8 5E F 0K T8 5E F 0K 9 F H T C5E 12.H T C F H T C F H T 07.06 57 93 h 163 4 h F 4 h4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)T TGCACGCACG)TGCACGCACG)G AG GCAAACQ71 AG GCAAACQ71 AG GCAAACQ71 TA TTAA TC TG ACTE:TTAAG ACTE:TTAAG ACTE:TT TTACTA CAGGS(OTTCTTTACTAAGGS A GC(OTTCTTTACTAAGGS(OTTC G G A G A G T A G A C N G G A G T A G A C N G G A G A GC T A G A C N G G GTTA:TTA:TTA:T TCTCQO G TCQO G TCQO G TATE TCTATE TC TE TC C T S g T A C( NC T SNCTAT SNCT TA A TDI )9 T gA A T A C(TDI )9 T gA A T A C(TDI )9 T gA T 0E 0 0 0 S E S E S E S 2 2 2 2 G G G G G G G G N N N N R;4R;4R;4R;1S03 1S13 1 4 S23 1S33 B PT T B PT T B PT T B PT T 0 le3l3 3 0 O Rd92eld9eld9ed W T8K RT82 K R 2 T8K RT8 6 F 05E F 05E F 0E F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 h4 h h h 16 133 F 4F 4F 4 B 1 dd dF ee edeAAA AATTT GT TTGCGDIGAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT TTG C CGCACG)AGC CGCACGI)AGC C CACGI)AGC C CA GAG AAACQ7T GAG AAACQ7T GAGG AACQ7T GAGG A AAGC ACTE1:TA AGC A E1:TA AGCAA E1:TA AGCAA TTTACTA CAGGS(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTAA A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A G A GC T A TA:TA:T: :TCQEO GT TCTCQ GT EO TCTA CQ GTT EO TCTA CQEO AT TASNTATSNTATSNTATS C(C T 9 gATA T A C(C TA T 9 g T A C(C TA T 9 g T A C( NADI )TDI )TADI )TA TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;4R;4R;1 4 S43 1S53 16 T T PT S3 B P B T B PT T 0 3 0 9l2e3le3l3 O K Rd9 82 Rd92eRd92 W6 E TF 0K T8 5E F 0K T8 5E F 0K 9 F H T C5E 12.H T C F H T C F H T 07.06 57 93 h 163 4 h F 4 h4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)T TGCACGCACG)TGCACGCACG)G AG GCAAACQ71 AG GCAAACQ71 AG GCAAACQ71 TA TTAA TC TG ACTE:TTAAG ACTE:TTAAG ACTE:TT TTACTA CAGGS(OTTCTTTACTAAGGS A GC(OTTCTTTACTAAGGS(OTTC G G A G A G T A G A C N G G A G T A G A C N G G A G A GC T A G A C N G G GTTA:TTA:TTA:T TCTCQO G TCQO G TCQO G TATE TCTATE TC TE TC C T S g T A C( NC T SNCTAT SNCT TA A TDI )9 T gA A T A C(TDI )9 T gA A T A C(TDI )9 T gA T 0E 0 0 0 S E S E S E S 2 2 2 2 G G G G G G G G N N N N R;4R;4R;4R;1S73 1S83 1 4 S14 1S24 B PT T B PT T B PT T B PT T 0 le3l3 3 0 O Rd92eld9eld9ed W T8K RT82 K R 2 T8K RT8 6 F 05E F 05E F 0E F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 h4 h h h 16 133 F 4F 4F 4 B 1 dd dF ee edeAAA AATTT GT TTGCGDIGAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT TTG C CGCACG)AGC CGCACGI)AGC C CACGI)AGC C CA GAG AAACQ7T GAG AAACQ7T GAGG AACQ7T GAGG A AAGC ACTE1:TA AGC A E1:TA AGCAA E1:TA AGCAA TTTACTA CAGGS(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTAA A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A G A GC T A TA:TA:T: :TCQEO GT TCTCQ GT EO TCTA CQ GTT EO TCTA CQEO AT TASNTATSNTATSNTATS C(C T 9 gATA T A C(C TA T 9 g T A C(C TA T 9 g T A C( NADI )TDI )TADI )TA TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;4R;5R;1 5 S34 1S03 11 T T PT S3 B P B T B PT T 0 3 0 9l2e3le3l3 O K Rd9 82 Rd92eRd92 W6 E TF 0K T8 5E F 0K T8 5E F 0K 9 F H T C5E 12.H T C F H T C F H T 07.06 57 93 h 163 4 h F 4 h4 1 B31 d F F ededeGAAAATTGGTD GAAAATT GTD GAAAATT GTD GA AAAATTT CGIAAAATTTGCGIAAAATTTGCGIAA TGCACGCACG)T TGCACGCACG)TGCACGCACG)G AG GCAAACQ71 AG GCAAACQ71 AG GCAAACQ71 TA TTAA TC TG ACTE:TTAAG ACTE:TTAAG ACTE:TT TTACTA CAGGS(OTTCTTTACTAAGGS A GC(OTTCTTTACTAAGGS(OTTC G G A G A G T A G A C N G G A G T A G A C N G G A G A GC T A G A C N G G GTTA:TTA:TTA:T TCTCQO G TCQO G TCQO G TATE TCTATE TC TE TC C T S g T A C( NC T SNCTAT SNCT TA A TDI )9 T gA A T A C(TDI )9 T gA A T A C(TDI )9 T gA T 0E 0 0 0 S E S E S E S 2 2 2 2 G G G G G G G G N N N N R;5R;5R;5R;1S23 1S33 1 5 S43 1S53 B PT T B PT T B PT T B PT T 0 le3l3 3 0 O Rd92eld9eld9ed W T8K RT82 K R 2 T8K RT8 6 F 05E F 05E F 0E F 0 912.C F H T C F H T C5 F H T C5 F 07.06 57 93 h4 h h h 16 133 F 4F 4F 4 B 1 dd dF ee edeAAA AATTT GT TTGCGDIGAAAA AAATTT GT TTGCGD GAAAA AAATTT GT TTGCGD GAAAA AAATTT TTG C CGCACG)AGC CGCACGI)AGC C CACGI)AGC C CA GAG AAACQ7T GAG AAACQ7T GAGG AACQ7T GAGG A AAGC ACTE1:TA AGC A E1:TA AGCAA E1:TA AGCAA TTTACTA CAGGS(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTA CTS CAGG(OTTA TCTTTACTAA A G A G T A G A C N G G A G A G T A G A C N G G A G A G T A G A C N G G A G A GC T A TA:TA:T: :TCQEO GT TCTCQ GT EO TCTA CQ GTT EO TCTA CQEO AT TASNTATSNTATSNTATS C(C T 9 gATA T A C(C TA T 9 g T A C(C TA T 9 g T A C( NADI )TDI )TADI )TA TDI )9 0E 0E 0 S S E S 2 2 2 G G G G G G N N N R;5R;5R;1 5 S63 1S73 18 T T PT S3 B P B T B PT T 0 3 0 9l2e3le3l3 O K Rd9 82 Rd92eRd92 W6 E TF 0K T8 5E F 0K T8 5E F 0K 9 F H T C5E 12.H T C F H T C F H T 0...

Claims

CLAIMS What is claimed is:

1. A method of prime editing a cystic fibrosis transmembrane conductance regulator (CFTR) gene comprising contacting a nucleic acid sequence encoding the CFTR gene with a prime editor and a prime editing guide RNA (pegRNA), wherein the pegRNA comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence TCTGTATCTATATTCATCAT (SEQ ID NO: 1), ACCATTAAAGAAAATATCAT (SEQ ID NO: 2), ATTATGCCTGGCACCATTAA (SEQ ID NO: 3), CTGTATCTATATTCATCATA (SEQ ID NO: 4), TTCATCATAGGAAACACCAA (SEQ ID NO: 5), CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), or CTGTATCTATATTCATCAT (SEQ ID NO: 8).

2. The method of claim 1, wherein the pegRNA comprises a spacer comprising the sequence TCTGTATCTATATTCATCAT (SEQ ID NO: 1), ACCATTAAAGAAAATATCAT (SEQ ID NO: 2), ATTATGCCTGGCACCATTAA (SEQ ID NO: 3), CTGTATCTATATTCATCATA (SEQ ID NO: 4), TTCATCATAGGAAACACCAA (SEQ ID NO: 5), CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), or CTGTATCTATATTCATCAT (SEQ ID NO: 8).

3. The method of claim 1 or 2, wherein the pegRNA comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence TCTGTATCTATATTCATCAT (SEQ ID NO: 1) or CTGTATCTATATTCATCAT (SEQ ID NO: 8).

4. The method of any one of claims 1-3, wherein the pegRNA comprises a spacer comprising the sequence TCTGTATCTATATTCATCAT (SEQ ID NO: 1) or CTGTATCTATATTCATCAT (SEQ ID NO: 8). 323 / 370 B1195.70196WO00 13363760.

25. The method of any one of claims 1-4, wherein the pegRNA spacer further comprises a G at its 5' end.

6. The method of any one of claims 1-5, wherein the pegRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17), GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 18), or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTAT CAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 20).

7. The method of any one of claims 1-6, wherein the pegRNA comprises a backbone scaffold comprising the sequence: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17), GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 18), or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTAT CAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 20).

8. The method of any one of claims 1-7, wherein the pegRNA comprises a reverse transcription template (RTT) of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

9. The method of any one of claims 1-8, wherein the pegRNA comprises a reverse transcription template (RTT) comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence AGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 21), TAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 22), 324 / 370 B1195.70196WO00 13363760.2CATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 23), CACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 24), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 25), GCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 26), CATAGGAAACACCAAAGATG (SEQ ID NO: 27), AAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 28), AAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 29), TTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 30), ATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 31), CCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 32), ACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 33), CTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 34), CCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 35), TCATTGGTGTTTCCTATG (SEQ ID NO: 36), ATCATTGGTGTTTCCTATG (SEQ ID NO: 37), TATCATTGGTGTTTCCTATG (SEQ ID NO: 38), ATATCATTGGTGTTTCCTATG (SEQ ID NO: 39), AATATCATTGGTGTTTCCTATG (SEQ ID NO: 40), AAATATCATTGGTGTTTCCTATG (SEQ ID NO: 41), AAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 42), GAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 43), AGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 44), AAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 45), AAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 46), TAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 47), TTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 48), ATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 49), ACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 50), GCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 51), GGCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 52), TGGCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 53), CATCATAGGAAACACCAAAGATG (SEQ ID NO: 54), ATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 55), TATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 56), ATCTATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 57), 325 / 370 B1195.70196WO00 13363760.2TGTATCTATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 58), GGAAACACCAAAGATGATATTTTCTTTA (SEQ ID NO: 59), ATTCATCATAGGAAACACCAAAGATGATATTTTCTTTA (SEQ ID NO: 60), AAAATATCATCTTTGGTGTTTCCTAT (SEQ ID NO: 61), ACCATTAAAGAAAATATCATCTTTGGTGTTTCCTAT (SEQ ID NO: 62), AAAATATCATCTTTG (SEQ ID NO: 63), ACCATTAAAGAAAATATCATCTTTG (SEQ ID NO: 64), GGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGC (SEQ ID NO: 65), TATTCATCATAGGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGC (SEQ ID NO: 66), GGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGCATAATCCAGGA (SEQ ID NO: 67), ATTCATCATAGGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGCATAATCC AGGA (SEQ ID NO: 68), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGTTACG (SEQ ID NO: 69), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGTTACG (SEQ ID NO: 70), TGGCACCATTAAAGAAAATATCATCTTCGGTGTGAGTTATG (SEQ ID NO: 71), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGTTATG (SEQ ID NO: 72), TGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 73), TGGCACCATTAAAGAAAATATCATCTTTGGCGTAAGCTACG (SEQ ID NO: 76), TGGCACCATTAAAGAAAATATCATATTTGGTGTTAGCTACG (SEQ ID NO: 77), TGGCACCATTAAAGAAAATATCATATTTGGCGTAAGCTACG (SEQ ID NO: 78), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCAGCTACG (SEQ ID NO: 79), TGGCACCATTAAAGAAAATATCATCTTTGGTGTAAGCTACG (SEQ ID NO: 80), TGGCACCATTAAAGAAAATATCATCTTTGGCGTGAGCTACG (SEQ ID NO: 81), TGGCACCATTAAAGAAAATATCATATTTGGTGTGAGCTACG (SEQ ID NO: 82), TGGCACCATTAAAGAAAATATCATATTTGGTGTCAGCTACG (SEQ ID NO: 83), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCAGCTACG (SEQ ID NO: 84), TGGCACCATTAAAGAAAATATCATATTTGGTGTAAGCTACG (SEQ ID NO: 85), TGGCACCATTAAAGAAAATATCATATTTGGCGTCAGCTACG (SEQ ID NO: 86), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTAGCTACG (SEQ ID NO: 87), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCATACG (SEQ ID NO: 88), TGGCACCATTAAAGAAAATATCATATTTGGCGTGAGCTACG (SEQ ID NO: 89), TGGCACCATTAAAGAAAATATCATATTCGGTGTGAGCTACG (SEQ ID NO: 90), 326 / 370 B1195.70196WO00 13363760.2TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCATACG (SEQ ID NO: 91), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCATACG (SEQ ID NO: 92), TGGCACCATTAAAGAAAATATCATCTTTGGCGTATCATACG (SEQ ID NO: 93), TGGCACCATTAAAGAAAATATCATCTTTGGCGTAAGTTACG (SEQ ID NO: 94), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCGTACG (SEQ ID NO: 95), TGGCACCATTAAAGAAAATATCATCTTCGGCGTCAGCTACG (SEQ ID NO: 96), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCGTACG (SEQ ID NO: 97), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTAGCTACG (SEQ ID NO: 98), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCGTACG (SEQ ID NO: 99), TGGCACCATTAAAGAAAATATCATCTTTGGGGTATCATACG (SEQ ID NO: 100), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCGTACG (SEQ ID NO: 101), TGGCACCATTAAAGAAAATATCATCTTTGGCGTATCGTACG (SEQ ID NO: 102), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCGTACG (SEQ ID NO: 103), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGTTACG (SEQ ID NO: 70), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCGTACG (SEQ ID NO: 104), TGGCACCATTAAAGAAAATATCATCTTCGGTGTATCATACG (SEQ ID NO: 105), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCGTACG (SEQ ID NO: 106), TGGCACCATTAAAGAAAATATCATCTTCGGCGTAAGCTACG (SEQ ID NO: 107), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGCTACG (SEQ ID NO: 108), TGGCACCATTAAAGAAAATATCATCTTTGGCGTTTCGTACG (SEQ ID NO: 109), TGGCACCATTAAAGAAAATATCATCTTTGGTGTAAGTTACG (SEQ ID NO: 110), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCATACG (SEQ ID NO: 111), TGGCACCATTAAAGAAAATATCATATTCGGTGTAAGCTACG (SEQ ID NO: 112), TGGCACCATTAAAGAAAATATCATCTTCGGCGTTAGCTACG (SEQ ID NO: 113), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCATACG (SEQ ID NO: 114), TGGCACCATTAAAGAAAATATCATCTTCGGTGTATCGTACG (SEQ ID NO: 115), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 116), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCAGTTACG (SEQ ID NO: 117), TGGCACCATTAAAGAAAATATCATATTTGGTGTATCATACG (SEQ ID NO: 118), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCATACG (SEQ ID NO: 119), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCGTACG (SEQ ID NO: 120), TGGCACCATTAAAGAAAATATCATATTCGGTGTATCGTACG (SEQ ID NO: 121), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCTTACG (SEQ ID NO: 122), 327 / 370 B1195.70196WO00 13363760.2TGGCACCATTAAAGAAAATATCATATTTGGTGTATCGTACG (SEQ ID NO: 123), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGCTACG (SEQ ID NO: 124), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCTTACG (SEQ ID NO: 125), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCTTACG (SEQ ID NO: 126), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCTTACG (SEQ ID NO: 127), TGGCACCATTAAAGAAAATATCATATTCGGCGTCAGCTACG (SEQ ID NO: 128), TGGCACCATTAAAGAAAATATCATCTTTGGGGTATCGTACG (SEQ ID NO: 129), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCATACG (SEQ ID NO: 130), TGGCACCATTAAAGAAAATATCATCTTCGGCGTGAGCTACG (SEQ ID NO: 131), TGGCACCATTAAAGAAAATATCATCTTTGGCGTTAGCTACG (SEQ ID NO: 132), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCTCATACG (SEQ ID NO: 133), TGGCACCATTAAAGAAAATATCATCTTTGGAGTTTCGTACG (SEQ ID NO: 134), TGGCACCATTAAAGAAAATATCATATTCGGTGTTAGCTACG (SEQ ID NO: 135), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCTCGTACG (SEQ ID NO: 136), TGGCACCATTAAAGAAAATATCATATTTGGTGTAAGTTACG (SEQ ID NO: 137), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCTTACG (SEQ ID NO: 138), TGGCACCATTAAAGAAAATATCATCTTCGGTGTGTCATACG (SEQ ID NO: 139), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCTCGTACG (SEQ ID NO: 140), TGGCACCATTAAAGAAAATATCATCTTCGGCGTATCGTACG (SEQ ID NO: 141), TGGCACCATTAAAGAAAATATCATATTTGGTGTTAGTTACG (SEQ ID NO: 142), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCTTACG (SEQ ID NO: 143), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCTTACG (SEQ ID NO: 144), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCTTACG (SEQ ID NO: 145), TGGCACCATTAAAGAAAATATCATCTTTGGGGTGAGCTACG (SEQ ID NO: 146), AAGAAAATATCATATTTGGAGTAAGTTACG (SEQ ID NO: 147), AAGAAAATATCATATTTGGAGTAAGCTACG (SEQ ID NO: 148), AAGAAAATATCATATTTGGAGTGAGTTACG (SEQ ID NO: 149), AAAGAAAATATCATATTTGGAGTAAGTTACG (SEQ ID NO: 150), AAGAAAATATCATATTTGGTGTTTCGTACG (SEQ ID NO: 151), AAGAAAATATCATATTTGGTGTTAGCTACG (SEQ ID NO: 152), AAAGAAAATATCATATTTGGAGTAAGCTACG (SEQ ID NO: 153), AAGAAAATATCATATTTGGAGTAAGTTATG (SEQ ID NO: 154), AAAGAAAATATCATATTTGGAGTGAGTTACG (SEQ ID NO: 155), 328 / 370 B1195.70196WO00 13363760.2AAGAAAATATCATATTTGGTGTAAGCTACG (SEQ ID NO: 156), AAGAAAATATCATATTTGGTGTTTCATACG (SEQ ID NO: 157), AAGAAAATATCATATTTGGAGTCTCATACG (SEQ ID NO: 158), AAGAAAATATCATATTTGGAGTATCATACG (SEQ ID NO: 159), AAGAAAATATCATATTCGGAGTTTCATACG (SEQ ID NO: 160), AAGAAAATATCATATTTGGAGTATCGTACG (SEQ ID NO: 161), AAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 162), AAGAAAATATCATATTTGGAGTTAGTTACG (SEQ ID NO: 163), AAGAAAATATCATATTTGGTGTAAGTTACG (SEQ ID NO: 164), AAGAAAATATCATATTCGGTGTTAGCTACG (SEQ ID NO: 165), AAGAAAATATCATATTTGGAGTGAGCTACG (SEQ ID NO: 166), AAGAAAATATCATATTTGGAGTAAGCTATG (SEQ ID NO: 167), AAGAAAATATCATATTCGGAGTAAGTTACG (SEQ ID NO: 168), AAGAAAATATCATATTTGGCGTTAGCTACG (SEQ ID NO: 169), AAGAAAATATCATATTTGGTGTTAGTTACG (SEQ ID NO: 170), AAGAAAATATCATATTTGGGGTAAGTTACG (SEQ ID NO: 171), AAGAAAATATCATATTCGGAGTCTCATACG (SEQ ID NO: 172), AAGAAAATATCATATTTGGAGTGAGCTATG (SEQ ID NO: 173), AAGAAAATATCATATTTGGAGTTAGCTACG (SEQ ID NO: 174), AAGAAAATATCATATTTGGGGTAAGTTATG (SEQ ID NO: 175), AAGAAAATATCATATTCGGAGTGAGTTACG (SEQ ID NO: 176), AAGAAAATATCATATTTGGAGTCAGCTACG (SEQ ID NO: 177), AAGAAAATATCATATTTGGTGTCTCGTACG (SEQ ID NO: 178), AAGAAAATATCATATTTGGAGTCTCGTACG (SEQ ID NO: 179), AAGAAAATATCATATTTGGAGTGTCATACG (SEQ ID NO: 180), AAGAAAATATCATATTTGGAGTTTCATACG (SEQ ID NO: 181), AAGAAAATATCATATTTGGTGTCAGCTACG (SEQ ID NO: 182), AAGAAAATATCATATTTGGTGTGAGCTACG (SEQ ID NO: 183), AAGAAAATATCATATTCGGTGTAAGCTACG (SEQ ID NO: 184), AAGAAAATATCATATTCGGTGTTTCGTACG (SEQ ID NO: 185), AAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 186), TAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 187), TTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 188), 329 / 370 B1195.70196WO00 13363760.2ATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 189), ACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 190), GCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 191), GGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 192), GCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 193), AAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 194), TAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 195), TTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 196), ATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 197), ACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 198), GCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 199), GGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 200), TGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 201), GCCTGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 202), or TGCCTGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 203).

10. The method of any one of claims 1-9, wherein the pegRNA comprises an RTT comprising the sequence: AGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 21), TAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 22), CATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 23), CACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 24), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 25), GCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 26), CATAGGAAACACCAAAGATG (SEQ ID NO: 27), AAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 28), AAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 29), TTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 30), ATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 31), CCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 32), ACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 33), CTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 34), 330 / 370 B1195.70196WO00 13363760.2CCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 35), TCATTGGTGTTTCCTATG (SEQ ID NO: 36), ATCATTGGTGTTTCCTATG (SEQ ID NO: 37), TATCATTGGTGTTTCCTATG (SEQ ID NO: 38), ATATCATTGGTGTTTCCTATG (SEQ ID NO: 39), AATATCATTGGTGTTTCCTATG (SEQ ID NO: 40), AAATATCATTGGTGTTTCCTATG (SEQ ID NO: 41), AAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 42), GAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 43), AGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 44), AAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 45), AAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 46), TAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 47), TTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 48), ATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 49), ACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 50), GCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 51), GGCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 52), TGGCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 53), CATCATAGGAAACACCAAAGATG (SEQ ID NO: 54), ATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 55), TATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 56), ATCTATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 57), TGTATCTATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 58), GGAAACACCAAAGATGATATTTTCTTTA (SEQ ID NO: 59), ATTCATCATAGGAAACACCAAAGATGATATTTTCTTTA (SEQ ID NO: 60), AAAATATCATCTTTGGTGTTTCCTAT (SEQ ID NO: 61), ACCATTAAAGAAAATATCATCTTTGGTGTTTCCTAT (SEQ ID NO: 62), AAAATATCATCTTTG (SEQ ID NO: 63), ACCATTAAAGAAAATATCATCTTTG (SEQ ID NO: 64), GGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGC (SEQ ID NO: 65), TATTCATCATAGGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGC (SEQ ID NO: 66), GGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGCATAATCCAGGA (SEQ ID NO: 67), ATTCATCATAGGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGCATAATCC 331 / 370 B1195.70196WO00 13363760.2AGGA (SEQ ID NO: 68), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGTTACG (SEQ ID NO: 69), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGTTACG (SEQ ID NO: 70), TGGCACCATTAAAGAAAATATCATCTTCGGTGTGAGTTATG (SEQ ID NO: 71), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGTTATG (SEQ ID NO: 72), TGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 73), TGGCACCATTAAAGAAAATATCATCTTTGGCGTAAGCTACG (SEQ ID NO: 76), TGGCACCATTAAAGAAAATATCATATTTGGTGTTAGCTACG (SEQ ID NO: 77), TGGCACCATTAAAGAAAATATCATATTTGGCGTAAGCTACG (SEQ ID NO: 78), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCAGCTACG (SEQ ID NO: 79), TGGCACCATTAAAGAAAATATCATCTTTGGTGTAAGCTACG (SEQ ID NO: 80), TGGCACCATTAAAGAAAATATCATCTTTGGCGTGAGCTACG (SEQ ID NO: 81), TGGCACCATTAAAGAAAATATCATATTTGGTGTGAGCTACG (SEQ ID NO: 82), TGGCACCATTAAAGAAAATATCATATTTGGTGTCAGCTACG (SEQ ID NO: 83), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCAGCTACG (SEQ ID NO: 84), TGGCACCATTAAAGAAAATATCATATTTGGTGTAAGCTACG (SEQ ID NO: 85), TGGCACCATTAAAGAAAATATCATATTTGGCGTCAGCTACG (SEQ ID NO: 86), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTAGCTACG (SEQ ID NO: 87), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCATACG (SEQ ID NO: 88), TGGCACCATTAAAGAAAATATCATATTTGGCGTGAGCTACG (SEQ ID NO: 89), TGGCACCATTAAAGAAAATATCATATTCGGTGTGAGCTACG (SEQ ID NO: 90), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCATACG (SEQ ID NO: 91), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCATACG (SEQ ID NO: 92), TGGCACCATTAAAGAAAATATCATCTTTGGCGTATCATACG (SEQ ID NO: 93), TGGCACCATTAAAGAAAATATCATCTTTGGCGTAAGTTACG (SEQ ID NO: 94), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCGTACG (SEQ ID NO: 95), TGGCACCATTAAAGAAAATATCATCTTCGGCGTCAGCTACG (SEQ ID NO: 96), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCGTACG (SEQ ID NO: 97), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTAGCTACG (SEQ ID NO: 98), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCGTACG (SEQ ID NO: 99), TGGCACCATTAAAGAAAATATCATCTTTGGGGTATCATACG (SEQ ID NO: 100), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCGTACG (SEQ ID NO: 101), TGGCACCATTAAAGAAAATATCATCTTTGGCGTATCGTACG (SEQ ID NO: 102), 332 / 370 B1195.70196WO00 13363760.2TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCGTACG (SEQ ID NO: 103), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGTTACG (SEQ ID NO: 70), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCGTACG (SEQ ID NO: 104), TGGCACCATTAAAGAAAATATCATCTTCGGTGTATCATACG (SEQ ID NO: 105), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCGTACG (SEQ ID NO: 106), TGGCACCATTAAAGAAAATATCATCTTCGGCGTAAGCTACG (SEQ ID NO: 107), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGCTACG (SEQ ID NO: 108), TGGCACCATTAAAGAAAATATCATCTTTGGCGTTTCGTACG (SEQ ID NO: 109), TGGCACCATTAAAGAAAATATCATCTTTGGTGTAAGTTACG (SEQ ID NO: 110), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCATACG (SEQ ID NO: 111), TGGCACCATTAAAGAAAATATCATATTCGGTGTAAGCTACG (SEQ ID NO: 112), TGGCACCATTAAAGAAAATATCATCTTCGGCGTTAGCTACG (SEQ ID NO: 113), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCATACG (SEQ ID NO: 114), TGGCACCATTAAAGAAAATATCATCTTCGGTGTATCGTACG (SEQ ID NO: 115), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 116), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCAGTTACG (SEQ ID NO: 117), TGGCACCATTAAAGAAAATATCATATTTGGTGTATCATACG (SEQ ID NO: 118), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCATACG (SEQ ID NO: 119), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCGTACG (SEQ ID NO: 120), TGGCACCATTAAAGAAAATATCATATTCGGTGTATCGTACG (SEQ ID NO: 121), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCTTACG (SEQ ID NO: 122), TGGCACCATTAAAGAAAATATCATATTTGGTGTATCGTACG (SEQ ID NO: 123), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGCTACG (SEQ ID NO: 124), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCTTACG (SEQ ID NO: 125), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCTTACG (SEQ ID NO: 126), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCTTACG (SEQ ID NO: 127), TGGCACCATTAAAGAAAATATCATATTCGGCGTCAGCTACG (SEQ ID NO: 128), TGGCACCATTAAAGAAAATATCATCTTTGGGGTATCGTACG (SEQ ID NO: 129), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCATACG (SEQ ID NO: 130), TGGCACCATTAAAGAAAATATCATCTTCGGCGTGAGCTACG (SEQ ID NO: 131), TGGCACCATTAAAGAAAATATCATCTTTGGCGTTAGCTACG (SEQ ID NO: 132), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCTCATACG (SEQ ID NO: 133), TGGCACCATTAAAGAAAATATCATCTTTGGAGTTTCGTACG (SEQ ID NO: 134), 333 / 370 B1195.70196WO00 13363760.2TGGCACCATTAAAGAAAATATCATATTCGGTGTTAGCTACG (SEQ ID NO: 135), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCTCGTACG (SEQ ID NO: 136), TGGCACCATTAAAGAAAATATCATATTTGGTGTAAGTTACG (SEQ ID NO: 137), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCTTACG (SEQ ID NO: 138), TGGCACCATTAAAGAAAATATCATCTTCGGTGTGTCATACG (SEQ ID NO: 139), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCTCGTACG (SEQ ID NO: 140), TGGCACCATTAAAGAAAATATCATCTTCGGCGTATCGTACG (SEQ ID NO: 141), TGGCACCATTAAAGAAAATATCATATTTGGTGTTAGTTACG (SEQ ID NO: 142), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCTTACG (SEQ ID NO: 143), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCTTACG (SEQ ID NO: 144), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCTTACG (SEQ ID NO: 145), TGGCACCATTAAAGAAAATATCATCTTTGGGGTGAGCTACG (SEQ ID NO: 146), AAGAAAATATCATATTTGGAGTAAGTTACG (SEQ ID NO: 147), AAGAAAATATCATATTTGGAGTAAGCTACG (SEQ ID NO: 148), AAGAAAATATCATATTTGGAGTGAGTTACG (SEQ ID NO: 149), AAAGAAAATATCATATTTGGAGTAAGTTACG (SEQ ID NO: 150), AAGAAAATATCATATTTGGTGTTTCGTACG (SEQ ID NO: 151), AAGAAAATATCATATTTGGTGTTAGCTACG (SEQ ID NO: 152), AAAGAAAATATCATATTTGGAGTAAGCTACG (SEQ ID NO: 153), AAGAAAATATCATATTTGGAGTAAGTTATG (SEQ ID NO: 154), AAAGAAAATATCATATTTGGAGTGAGTTACG (SEQ ID NO: 155), AAGAAAATATCATATTTGGTGTAAGCTACG (SEQ ID NO: 156), AAGAAAATATCATATTTGGTGTTTCATACG (SEQ ID NO: 157), AAGAAAATATCATATTTGGAGTCTCATACG (SEQ ID NO: 158), AAGAAAATATCATATTTGGAGTATCATACG (SEQ ID NO: 159), AAGAAAATATCATATTCGGAGTTTCATACG (SEQ ID NO: 160), AAGAAAATATCATATTTGGAGTATCGTACG (SEQ ID NO: 161), AAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 162), AAGAAAATATCATATTTGGAGTTAGTTACG (SEQ ID NO: 163), AAGAAAATATCATATTTGGTGTAAGTTACG (SEQ ID NO: 164), AAGAAAATATCATATTCGGTGTTAGCTACG (SEQ ID NO: 165), AAGAAAATATCATATTTGGAGTGAGCTACG (SEQ ID NO: 166), AAGAAAATATCATATTTGGAGTAAGCTATG (SEQ ID NO: 167), 334 / 370 B1195.70196WO00 13363760.2AAGAAAATATCATATTCGGAGTAAGTTACG (SEQ ID NO: 168), AAGAAAATATCATATTTGGCGTTAGCTACG (SEQ ID NO: 169), AAGAAAATATCATATTTGGTGTTAGTTACG (SEQ ID NO: 170), AAGAAAATATCATATTTGGGGTAAGTTACG (SEQ ID NO: 171), AAGAAAATATCATATTCGGAGTCTCATACG (SEQ ID NO: 172), AAGAAAATATCATATTTGGAGTGAGCTATG (SEQ ID NO: 173), AAGAAAATATCATATTTGGAGTTAGCTACG (SEQ ID NO: 174), AAGAAAATATCATATTTGGGGTAAGTTATG (SEQ ID NO: 175), AAGAAAATATCATATTCGGAGTGAGTTACG (SEQ ID NO: 176), AAGAAAATATCATATTTGGAGTCAGCTACG (SEQ ID NO: 177), AAGAAAATATCATATTTGGTGTCTCGTACG (SEQ ID NO: 178), AAGAAAATATCATATTTGGAGTCTCGTACG (SEQ ID NO: 179), AAGAAAATATCATATTTGGAGTGTCATACG (SEQ ID NO: 180), AAGAAAATATCATATTTGGAGTTTCATACG (SEQ ID NO: 181), AAGAAAATATCATATTTGGTGTCAGCTACG (SEQ ID NO: 182), AAGAAAATATCATATTTGGTGTGAGCTACG (SEQ ID NO: 183), AAGAAAATATCATATTCGGTGTAAGCTACG (SEQ ID NO: 184), AAGAAAATATCATATTCGGTGTTTCGTACG (SEQ ID NO: 185), AAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 186), TAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 187), TTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 188), ATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 189), ACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 190), GCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 191), GGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 192), GCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 193), AAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 194), TAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 195), TTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 196), ATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 197), ACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 198), GCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 199), GGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 200), 335 / 370 B1195.70196WO00 13363760.2TGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 201), GCCTGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 202), or TGCCTGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 203).

11. The method of any one of claims 1-10, wherein the pegRNA comprises an RTT comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence: TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 25) or TGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 73).

12. The method of any one of claims 1-11, wherein the pegRNA comprises an RTT comprising the sequence: TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 25) or TGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 73).

13. The method of any one of claims 1-12, wherein the pegRNA comprises a primer binding site (PBS) comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence: ATGAATATAGA (SEQ ID NO: 205), ATGAATATAGAT (SEQ ID NO: 206), ATGAATATAGATA (SEQ ID NO: 207), ATGAATATAGATAC (SEQ ID NO: 208), ATATTTTCTTT (SEQ ID NO: 209), ATGAATATAG (SEQ ID NO: 210), ATGAATATAGATACA (SEQ ID NO: 211), ATGAATATAGATACAG (SEQ ID NO: 212), ATGAATATAGATACAGA (SEQ ID NO: 213), ATATTTTCTTTA (SEQ ID NO: 214), ATATTTTCTTTAA (SEQ ID NO: 215), ATATTTTCTTTAAT (SEQ ID NO: 216), ATGGTGCCAG (SEQ ID NO: 217), ATGGTGCCAGGC (SEQ ID NO: 218), ATGGTGCCAGGCA (SEQ ID NO: 219), ATGGTGCCAGGCAT (SEQ ID NO: 220), ATGGTGCCAGGCATA (SEQ ID NO: 221), ATGGTGCCAGGCATAA (SEQ ID NO: 222), GATGAATATA (SEQ ID NO: 223), GATGAATATAGA (SEQ ID NO: 224), GATGAATATAGAT (SEQ ID NO: 225), GATGAATATAGATA (SEQ ID NO: 226), GATGAATATAGATAC (SEQ ID NO: 227), GATGAATATAGATACA (SEQ ID NO: 228), GTGTTTCCTA (SEQ ID NO: 229), GTGTTTCCTATG (SEQ ID NO: 230), GTGTTTCCTATGA (SEQ ID NO: 231), 336 / 370 B1195.70196WO00 13363760.2GTGTTTCCTATGAT (SEQ ID NO: 232), GTGTTTCCTATGATG (SEQ ID NO: 233), GTGTTTCCTATGATGA (SEQ ID NO: 234), ATAATCCAGG (SEQ ID NO: 235), ATAATCCAGGA (SEQ ID NO: 236), ATAATCCAGGAA (SEQ ID NO: 237), ATAATCCAGGAAA (SEQ ID NO: 238), ATAATCCAGGAAAA (SEQ ID NO: 239), ATAATCCAGGAAAACT (SEQ ID NO: 240), AAACTGAGAA (SEQ ID NO: 241), AAACTGAGAACA (SEQ ID NO: 242), AAACTGAGAACAG (SEQ ID NO: 243), AAACTGAGAACAGA (SEQ ID NO: 244), AAACTGAGAACAGAA (SEQ ID NO: 245), or AAACTGAGAACAGAAT (SEQ ID NO: 246).

14. The method of any one of claims 1-13, wherein the pegRNA comprises a PBS comprising the sequence ATGAATATAGA (SEQ ID NO: 205), ATGAATATAGAT (SEQ ID NO: 206), ATGAATATAGATA (SEQ ID NO: 207), ATGAATATAGATAC (SEQ ID NO: 208), ATATTTTCTTT (SEQ ID NO: 209), ATGAATATAG (SEQ ID NO: 210), ATGAATATAGATACA (SEQ ID NO: 211), ATGAATATAGATACAG (SEQ ID NO: 212), ATGAATATAGATACAGA (SEQ ID NO: 213), ATATTTTCTTTA (SEQ ID NO: 214), ATATTTTCTTTAA (SEQ ID NO: 215), ATATTTTCTTTAAT (SEQ ID NO: 216), ATGGTGCCAG (SEQ ID NO: 217), ATGGTGCCAGGC (SEQ ID NO: 218), ATGGTGCCAGGCA (SEQ ID NO: 219), ATGGTGCCAGGCAT (SEQ ID NO: 220), ATGGTGCCAGGCATA (SEQ ID NO: 221), ATGGTGCCAGGCATAA (SEQ ID NO: 222), GATGAATATA (SEQ ID NO: 223), GATGAATATAGA (SEQ ID NO: 224), GATGAATATAGAT (SEQ ID NO: 225), GATGAATATAGATA (SEQ ID NO: 226), GATGAATATAGATAC (SEQ ID NO: 227), GATGAATATAGATACA (SEQ ID NO: 228), GTGTTTCCTA (SEQ ID NO: 229), GTGTTTCCTATG (SEQ ID NO: 230), GTGTTTCCTATGA (SEQ ID NO: 231), GTGTTTCCTATGAT (SEQ ID NO: 232), GTGTTTCCTATGATG (SEQ ID NO: 233), GTGTTTCCTATGATGA (SEQ ID NO: 234), ATAATCCAGG (SEQ ID NO: 235), ATAATCCAGGA (SEQ ID NO: 236), ATAATCCAGGAA (SEQ ID NO: 237), ATAATCCAGGAAA (SEQ ID NO: 238), ATAATCCAGGAAAA (SEQ ID NO: 239), ATAATCCAGGAAAACT (SEQ ID NO: 240), AAACTGAGAA (SEQ ID NO: 241), AAACTGAGAACA (SEQ ID NO: 242), AAACTGAGAACAG (SEQ ID NO: 243), AAACTGAGAACAGA (SEQ ID NO: 244), AAACTGAGAACAGAA (SEQ ID NO: 245), or AAACTGAGAACAGAAT (SEQ ID NO: 246). 337 / 370 B1195.70196WO00 13363760.

215. The method of any one of claims 1-14, wherein the pegRNA comprises a PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence ATGAATATAGATA (SEQ ID NO: 207).

16. The method of any one of claims 1-15, wherein the pegRNA comprises a PBS comprising the sequence ATGAATATAGATA (SEQ ID NO: 207).

17. The method of any one of claims 1-16, wherein the pegRNA further comprises a structured motif at its 3' end.

18. The method of claim 17, wherein the structured motif is an RNA pseudoknot motif.

19. The method of claim 17 or 18, wherein the structured motif comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 248).

20. The method of claim 19, wherein the structured motif comprises the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 248).

21. The method of any one of claims 1-20, wherein the pegRNA comprises the structure 5'-[spacer]-[backbone scaffold]-[reverse transcription template]-[primer binding site]- [structured motif]-3'.

22. The method of any one of claims 1-21, wherein the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence of any of the pegRNAs in Tables 1 and 2.

23. The method of any one of claims 1-22, wherein the pegRNA comprises a sequence of any of the pegRNAs in Tables 1 and 2. 338 / 370 B1195.70196WO00 13363760.

224. The method of any one of claims 1-23, wherein the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence GTCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTAAAGA AAATATCATCTTTGGTGTTTCCTATGATGAATATAGATACGCGGTTCTATCTAGTTAC GCGTTAAACCAACTAGAA (SEQ ID NO: 250), TCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC TAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTAAAG AAAATATCATCTTTGGTGTGAGTTACGATGAATATAGATACGCGGTTCTATCTAG TTACGCGTTAAACCAACTAGAA (SEQ ID NO: 744), or CTGTATCTATATTCATCATGTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGCCTGGCACCATTAAA GAAAATATCATCTTTGGTGTGTCATACGATGAATATAGATACGCGGTTCTATCTAGTT ACGCGTTAAACCAACTAGAA (SEQ ID NO: 251).

25. The method of any one of claims 1-24, wherein the pegRNA comprises the sequence GTCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTAAAGA AAATATCATCTTTGGTGTTTCCTATGATGAATATAGATACGCGGTTCTATCTAGTTAC GCGTTAAACCAACTAGAA (SEQ ID NO: 250), TCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC TAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTAAAG AAAATATCATCTTTGGTGTGAGTTACGATGAATATAGATACGCGGTTCTATCTAG TTACGCGTTAAACCAACTAGAA (SEQ ID NO: 744), or CTGTATCTATATTCATCATGTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGCCTGGCACCATTAAA GAAAATATCATCTTTGGTGTGTCATACGATGAATATAGATACGCGGTTCTATCTAGTT ACGCGTTAAACCAACTAGAA (SEQ ID NO: 251).

26. The method of any one of claims 1-25, wherein the pegRNA comprises a chemical modification at its 3' end. 339 / 370 B1195.70196WO00 13363760.

227. The method of claim 26, wherein the chemical modification comprises phosphonoacetate (mP).

28. The method of any one of claims 1-27 further comprising providing a nicking guide RNA (ngRNA).

29. The method of claim 28, wherein the ngRNA comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence: TTCACTTCTAATGGTGATTA (SEQ ID NO: 745), TCACTTCTAATGGTGATTAT (SEQ ID NO: 746), AATGGTGATTATGGGAGAAC (SEQ ID NO: 747), GGGAGAACTGGAGCCTTCAG (SEQ ID NO: 748), GGAGAACTGGAGCCTTCAGA (SEQ ID NO: 749), GAGGGTAAAATTAAGCACAG (SEQ ID NO: 750), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), ATTAAAGAAAATATCATCTT (SEQ ID NO: 751), CAAAGCATGCCAACTAGAAG (SEQ ID NO: 752), or CACTACCCAAATTATATATT (SEQ ID NO: 753).

30. The method of claim 28 or 29, wherein the ngRNA comprises a spacer comprising the sequence: TTCACTTCTAATGGTGATTA (SEQ ID NO: 745), TCACTTCTAATGGTGATTAT (SEQ ID NO: 746), AATGGTGATTATGGGAGAAC (SEQ ID NO: 747), GGGAGAACTGGAGCCTTCAG (SEQ ID NO: 748), GGAGAACTGGAGCCTTCAGA (SEQ ID NO: 749), GAGGGTAAAATTAAGCACAG (SEQ ID NO: 750), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), ATTAAAGAAAATATCATCTT (SEQ ID NO: 751), CAAAGCATGCCAACTAGAAG (SEQ ID NO: 752), or CACTACCCAAATTATATATT (SEQ ID NO: 753).

31. The method of claim 29 or 30, wherein the ngRNA spacer further comprises a G at its 5' end. 340 / 370 B1195.70196WO00 13363760.

232. The method of any one of claims 28-31, wherein the ngRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17) or GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 761).

33. The method of any one of claims 28-32, wherein the ngRNA comprises a backbone scaffold comprising the sequence: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17) or GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 761).

34. The method of any one of claims 28-33, wherein the ngRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence of any of the ngRNAs in Table 1.

35. The method of any one of claims 28-34, wherein the ngRNA comprises a sequence of any of the ngRNAs in Table 1.

36. The method of any one of claims 1-35 further comprising providing a dead single guide RNA (dsgRNA).

37. The method of claim 36, wherein providing the dsgRNA facilitates disruption of the chromatin state surrounding a target site of the pegRNA in CFTR, thereby increasing accessibility of the prime editor to the target site.

38. The method of claim 36 or 37, wherein the dsgRNA comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at 341 / 370 B1195.70196WO00 13363760.2least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence CTCCCTCCAAGTTGTCCACG (SEQ ID NO: 764), CTGGAGCCTTCAGA (SEQ ID NO: 765), ATTTTACCCTCTGA (SEQ ID NO: 766), GCCAGGCATAATCC (SEQ ID NO: 767), TCCTGGATTATGCC (SEQ ID NO: 768), TCTTTAATGGTGCC (SEQ ID NO: 769), TTACCTCTTCTAGT (SEQ ID NO: 770), ATGCCAACTAGAAG (SEQ ID NO: 771), GCCAAATATATAATT (SEQ ID NO: 772), or CCGCGCGCGCGAAAAAGCCG (SEQ ID NO: 773).

39. The method of any one of claims 36-38, wherein the dsgRNA comprises a spacer comprising the sequence CTCCCTCCAAGTTGTCCACG (SEQ ID NO: 764), CTGGAGCCTTCAGA (SEQ ID NO: 765), ATTTTACCCTCTGA (SEQ ID NO: 766), GCCAGGCATAATCC (SEQ ID NO: 767), TCCTGGATTATGCC (SEQ ID NO: 768), TCTTTAATGGTGCC (SEQ ID NO: 769), TTACCTCTTCTAGT (SEQ ID NO: 770), ATGCCAACTAGAAG (SEQ ID NO: 771), GCCAAATATATAATT (SEQ ID NO: 772), or CCGCGCGCGCGAAAAAGCCG (SEQ ID NO: 773).

40. The method of any one of claims 36-39, wherein the dsgRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGCTTT (SEQ ID NO: 782), GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 761), or GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17).

41. The method of any one of claims 36-40, wherein the dsgRNA comprises a backbone scaffold comprising the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGCTTT (SEQ ID NO: 782), GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 761), or 342 / 370 B1195.70196WO00 13363760.2GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17).

42. The method of any one of claims 36-41, wherein the dsgRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence of any of the dsgRNAs in Table 1.

43. The method of any one of claims 36-42, wherein the pegRNA comprises a sequence of any of the dsgRNAs in Table 1.

44. The method of any one of claims 1-43, wherein the prime editor comprises a nucleic acid-programmable DNA-binding protein (napDNAbp) and a polymerase.

45. The method of claim 44, wherein the napDNAbp comprises a Cas9 protein.

46. The method of claim 44 or 45, wherein the napDNAbp comprises a Cas9 nickase (nCas9).

47. The method of claim 45 or 46, wherein the Cas9 protein is a Streptococcus pyogenes Cas9 protein, or a variant thereof.

48. The method of any one of claims 44-47, wherein the napDNAbp comprises a Cas9 protein of a PE6 prime editor.

49. The method of any one of claims 44-48, wherein the polymerase is a reverse transcriptase.

50. The method of claim 49, wherein the reverse transcriptase comprises an MMLV reverse transcriptase, a Tf1 reverse transcriptase, or a variant thereof.

51. The method of any one of claims 44-50, wherein the polymerase comprises a reverse transcriptase of a PE6 prime editor. 343 / 370 B1195.70196WO00 13363760.

252. The method of any one of claims 1-51, wherein the prime editor comprises PEmax architecture.

53. The method of any one of claims 1-52 further comprising providing an inhibitor of the cellular mismatch repair (MMR) pathway.

54. The method of claim 53, wherein the inhibitor of the MMR pathway comprises a dominant negative variant of MLH1 (MLH1dn).

55. The method of any one of claims 1-54, wherein the step of contacting corrects an F508del mutation in the CFTR protein.

56. The method of any one of claims 1-55, wherein the step of contacting results in the insertion of the sequence 5′-CTT-3′ into the CFTR gene.

57. The method of any one of claims 1-56, wherein the step of contacting results in the correction of the CFTR gene to a wild-type sequence.

58. The method of any one of claims 1-57, wherein the step of contacting results in at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, or at least 50% correction of the CFTR gene to a wild-type sequence within a cell.

59. The method of any one of claims 1-58, wherein the step of contacting results in an increase in CFTR ion channel function to at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, or at least 50% of wild-type levels within a cell.

60. The method of any one of claims 1-59, wherein the step of contacting results in the installation of one or more silent edits into the CFTR gene.

61. The method of claim 60, wherein the one or more silent edits comprise one or more PAM-disrupting edits and / or one or more MMR pathway-inhibiting edits. 344 / 370 B1195.70196WO00 13363760.

262. The method of claim 60 or 61, wherein the one or more silent edits include one or more of changing the codon encoding CFTR I507 from ATC to ATA; changing the codon encoding CFTR F508 from TTT to TTC; changing the codon encoding CFTR G509 from GGT to GGA; changing the codon encoding CFTR V510 from GTT to GTG, GTC, or GTA; changing the codon encoding CFTR S511 from TCC to AGT, TCA, TCG, AGC, or TCT; and changing the codon encoding CFTR Y512 from TAT to TAC.

63. The method of any one of claims 1-62, wherein the contacting is performed in a cell.

64. The method of claim 63, wherein the cell is an airway epithelial cell.

65. The method of claim 63 or 64, wherein the cell is in a tissue selected from the group consisting of lung tissue, pancreatic tissue, liver tissue, kidney tissue, or intestinal tissue.

66. The method of any one of claims 1-65, wherein the contacting is performed in vivo.

67. The method of any one of claims 1-65, wherein the contacting is performed in vitro.

68. The method of any one of claims 1-66, wherein the contacting is performed in a subject.

69. The method of claim 68, wherein the method is a method of treating cystic fibrosis in a subject.

70. A prime editing guide RNA (pegRNA) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence: TCTGTATCTATATTCATCAT (SEQ ID NO: 1), ACCATTAAAGAAAATATCAT (SEQ ID NO: 2), ATTATGCCTGGCACCATTAA (SEQ ID NO: 3), CTGTATCTATATTCATCATA (SEQ ID NO: 4), TTCATCATAGGAAACACCAA (SEQ ID NO: 5), CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), or CTGTATCTATATTCATCAT (SEQ ID NO: 8). 345 / 370 B1195.70196WO00 13363760.

271. The pegRNA of claim 70, wherein the pegRNA comprises a spacer comprising the sequence TCTGTATCTATATTCATCAT (SEQ ID NO: 1), ACCATTAAAGAAAATATCAT (SEQ ID NO: 2), ATTATGCCTGGCACCATTAA (SEQ ID NO: 3), CTGTATCTATATTCATCATA (SEQ ID NO: 4), TTCATCATAGGAAACACCAA (SEQ ID NO: 5), CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), or CTGTATCTATATTCATCAT (SEQ ID NO: 8).

72. The pegRNA of claim 70 or 71, wherein the pegRNA comprises a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence TCTGTATCTATATTCATCAT (SEQ ID NO: 1) or CTGTATCTATATTCATCAT (SEQ ID NO: 8).

73. The pegRNA of any one of claims 70-72, wherein the pegRNA comprises a spacer comprising the sequence TCTGTATCTATATTCATCAT (SEQ ID NO: 1) or CTGTATCTATATTCATCAT (SEQ ID NO: 8).

74. The pegRNA of any one of claims 70-73, wherein the pegRNA spacer further comprises a G at its 5' end.

75. The pegRNA of any one of claims 70-74, wherein the pegRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17), GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 18), or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTAT CAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 20). 346 / 370 B1195.70196WO00 13363760.

276. The pegRNA of any one of claims 70-75, wherein the pegRNA comprises a backbone scaffold comprising the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17), GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 18), or GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTAT CAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 20).

77. The pegRNA of any one of claims 70-76, wherein the pegRNA comprises a reverse transcription template (RTT) of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

78. The pegRNA of any one of claims 70-77, wherein the pegRNA comprises a reverse transcription template (RTT) comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence AGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 21), TAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 22), CATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 23), CACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 24), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 25), GCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 26), CATAGGAAACACCAAAGATG (SEQ ID NO: 27), AAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 28), AAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 29), TTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 30), ATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 31), CCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 32), ACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 33), CTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 34), CCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 35), TCATTGGTGTTTCCTATG (SEQ ID NO: 36), ATCATTGGTGTTTCCTATG (SEQ ID NO: 347 / 370 B1195.70196WO00 13363760.237), TATCATTGGTGTTTCCTATG (SEQ ID NO: 38), ATATCATTGGTGTTTCCTATG (SEQ ID NO: 39), AATATCATTGGTGTTTCCTATG (SEQ ID NO: 40), AAATATCATTGGTGTTTCCTATG (SEQ ID NO: 41), AAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 42), GAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 43), AGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 44), AAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 45), AAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 46), TAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 47), TTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 48), ATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 49), ACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 50), GCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 51), GGCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 52), TGGCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 53), CATCATAGGAAACACCAAAGATG (SEQ ID NO: 54), ATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 55), TATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 56), ATCTATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 57), TGTATCTATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 58), GGAAACACCAAAGATGATATTTTCTTTA (SEQ ID NO: 59), ATTCATCATAGGAAACACCAAAGATGATATTTTCTTTA (SEQ ID NO: 60), AAAATATCATCTTTGGTGTTTCCTAT (SEQ ID NO: 61), ACCATTAAAGAAAATATCATCTTTGGTGTTTCCTAT (SEQ ID NO: 62), AAAATATCATCTTTG (SEQ ID NO: 63), ACCATTAAAGAAAATATCATCTTTG (SEQ ID NO: 64), GGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGC (SEQ ID NO: 65), TATTCATCATAGGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGC (SEQ ID NO: 66), GGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGCATAATCCAGGA (SEQ ID NO: 67), ATTCATCATAGGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGCATAATCC AGGA (SEQ ID NO: 68), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGTTACG (SEQ ID NO: 69), 348 / 370 B1195.70196WO00 13363760.2TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGTTACG (SEQ ID NO: 70), TGGCACCATTAAAGAAAATATCATCTTCGGTGTGAGTTATG (SEQ ID NO: 71), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGTTATG (SEQ ID NO: 72), TGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 73), TGGCACCATTAAAGAAAATATCATCTTTGGCGTAAGCTACG (SEQ ID NO: 76), TGGCACCATTAAAGAAAATATCATATTTGGTGTTAGCTACG (SEQ ID NO: 77), TGGCACCATTAAAGAAAATATCATATTTGGCGTAAGCTACG (SEQ ID NO: 78), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCAGCTACG (SEQ ID NO: 79), TGGCACCATTAAAGAAAATATCATCTTTGGTGTAAGCTACG (SEQ ID NO: 80), TGGCACCATTAAAGAAAATATCATCTTTGGCGTGAGCTACG (SEQ ID NO: 81), TGGCACCATTAAAGAAAATATCATATTTGGTGTGAGCTACG (SEQ ID NO: 82), TGGCACCATTAAAGAAAATATCATATTTGGTGTCAGCTACG (SEQ ID NO: 83), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCAGCTACG (SEQ ID NO: 84), TGGCACCATTAAAGAAAATATCATATTTGGTGTAAGCTACG (SEQ ID NO: 85), TGGCACCATTAAAGAAAATATCATATTTGGCGTCAGCTACG (SEQ ID NO: 86), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTAGCTACG (SEQ ID NO: 87), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCATACG (SEQ ID NO: 88), TGGCACCATTAAAGAAAATATCATATTTGGCGTGAGCTACG (SEQ ID NO: 89), TGGCACCATTAAAGAAAATATCATATTCGGTGTGAGCTACG (SEQ ID NO: 90), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCATACG (SEQ ID NO: 91), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCATACG (SEQ ID NO: 92), TGGCACCATTAAAGAAAATATCATCTTTGGCGTATCATACG (SEQ ID NO: 93), TGGCACCATTAAAGAAAATATCATCTTTGGCGTAAGTTACG (SEQ ID NO: 94), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCGTACG (SEQ ID NO: 95), TGGCACCATTAAAGAAAATATCATCTTCGGCGTCAGCTACG (SEQ ID NO: 96), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCGTACG (SEQ ID NO: 97), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTAGCTACG (SEQ ID NO: 98), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCGTACG (SEQ ID NO: 99), TGGCACCATTAAAGAAAATATCATCTTTGGGGTATCATACG (SEQ ID NO: 100), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCGTACG (SEQ ID NO: 101), TGGCACCATTAAAGAAAATATCATCTTTGGCGTATCGTACG (SEQ ID NO: 102), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCGTACG (SEQ ID NO: 103), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGTTACG (SEQ ID NO: 70), 349 / 370 B1195.70196WO00 13363760.2TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCGTACG (SEQ ID NO: 104), TGGCACCATTAAAGAAAATATCATCTTCGGTGTATCATACG (SEQ ID NO: 105), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCGTACG (SEQ ID NO: 106), TGGCACCATTAAAGAAAATATCATCTTCGGCGTAAGCTACG (SEQ ID NO: 107), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGCTACG (SEQ ID NO: 108), TGGCACCATTAAAGAAAATATCATCTTTGGCGTTTCGTACG (SEQ ID NO: 109), TGGCACCATTAAAGAAAATATCATCTTTGGTGTAAGTTACG (SEQ ID NO: 110), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCATACG (SEQ ID NO: 111), TGGCACCATTAAAGAAAATATCATATTCGGTGTAAGCTACG (SEQ ID NO: 112), TGGCACCATTAAAGAAAATATCATCTTCGGCGTTAGCTACG (SEQ ID NO: 113), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCATACG (SEQ ID NO: 114), TGGCACCATTAAAGAAAATATCATCTTCGGTGTATCGTACG (SEQ ID NO: 115), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 116), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCAGTTACG (SEQ ID NO: 117), TGGCACCATTAAAGAAAATATCATATTTGGTGTATCATACG (SEQ ID NO: 118), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCATACG (SEQ ID NO: 119), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCGTACG (SEQ ID NO: 120), TGGCACCATTAAAGAAAATATCATATTCGGTGTATCGTACG (SEQ ID NO: 121), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCTTACG (SEQ ID NO: 122), TGGCACCATTAAAGAAAATATCATATTTGGTGTATCGTACG (SEQ ID NO: 123), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGCTACG (SEQ ID NO: 124), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCTTACG (SEQ ID NO: 125), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCTTACG (SEQ ID NO: 126), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCTTACG (SEQ ID NO: 127), TGGCACCATTAAAGAAAATATCATATTCGGCGTCAGCTACG (SEQ ID NO: 128), TGGCACCATTAAAGAAAATATCATCTTTGGGGTATCGTACG (SEQ ID NO: 129), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCATACG (SEQ ID NO: 130), TGGCACCATTAAAGAAAATATCATCTTCGGCGTGAGCTACG (SEQ ID NO: 131), TGGCACCATTAAAGAAAATATCATCTTTGGCGTTAGCTACG (SEQ ID NO: 132), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCTCATACG (SEQ ID NO: 133), TGGCACCATTAAAGAAAATATCATCTTTGGAGTTTCGTACG (SEQ ID NO: 134), TGGCACCATTAAAGAAAATATCATATTCGGTGTTAGCTACG (SEQ ID NO: 135), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCTCGTACG (SEQ ID NO: 136), 350 / 370 B1195.70196WO00 13363760.2TGGCACCATTAAAGAAAATATCATATTTGGTGTAAGTTACG (SEQ ID NO: 137), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCTTACG (SEQ ID NO: 138), TGGCACCATTAAAGAAAATATCATCTTCGGTGTGTCATACG (SEQ ID NO: 139), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCTCGTACG (SEQ ID NO: 140), TGGCACCATTAAAGAAAATATCATCTTCGGCGTATCGTACG (SEQ ID NO: 141), TGGCACCATTAAAGAAAATATCATATTTGGTGTTAGTTACG (SEQ ID NO: 142), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCTTACG (SEQ ID NO: 143), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCTTACG (SEQ ID NO: 144), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCTTACG (SEQ ID NO: 145), TGGCACCATTAAAGAAAATATCATCTTTGGGGTGAGCTACG (SEQ ID NO: 146), AAGAAAATATCATATTTGGAGTAAGTTACG (SEQ ID NO: 147), AAGAAAATATCATATTTGGAGTAAGCTACG (SEQ ID NO: 148), AAGAAAATATCATATTTGGAGTGAGTTACG (SEQ ID NO: 149), AAAGAAAATATCATATTTGGAGTAAGTTACG (SEQ ID NO: 150), AAGAAAATATCATATTTGGTGTTTCGTACG (SEQ ID NO: 151), AAGAAAATATCATATTTGGTGTTAGCTACG (SEQ ID NO: 152), AAAGAAAATATCATATTTGGAGTAAGCTACG (SEQ ID NO: 153), AAGAAAATATCATATTTGGAGTAAGTTATG (SEQ ID NO: 154), AAAGAAAATATCATATTTGGAGTGAGTTACG (SEQ ID NO: 155), AAGAAAATATCATATTTGGTGTAAGCTACG (SEQ ID NO: 156), AAGAAAATATCATATTTGGTGTTTCATACG (SEQ ID NO: 157), AAGAAAATATCATATTTGGAGTCTCATACG (SEQ ID NO: 158), AAGAAAATATCATATTTGGAGTATCATACG (SEQ ID NO: 159), AAGAAAATATCATATTCGGAGTTTCATACG (SEQ ID NO: 160), AAGAAAATATCATATTTGGAGTATCGTACG (SEQ ID NO: 161), AAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 162), AAGAAAATATCATATTTGGAGTTAGTTACG (SEQ ID NO: 163), AAGAAAATATCATATTTGGTGTAAGTTACG (SEQ ID NO: 164), AAGAAAATATCATATTCGGTGTTAGCTACG (SEQ ID NO: 165), AAGAAAATATCATATTTGGAGTGAGCTACG (SEQ ID NO: 166), AAGAAAATATCATATTTGGAGTAAGCTATG (SEQ ID NO: 167), AAGAAAATATCATATTCGGAGTAAGTTACG (SEQ ID NO: 168), AAGAAAATATCATATTTGGCGTTAGCTACG (SEQ ID NO: 169), 351 / 370 B1195.70196WO00 13363760.2AAGAAAATATCATATTTGGTGTTAGTTACG (SEQ ID NO: 170), AAGAAAATATCATATTTGGGGTAAGTTACG (SEQ ID NO: 171), AAGAAAATATCATATTCGGAGTCTCATACG (SEQ ID NO: 172), AAGAAAATATCATATTTGGAGTGAGCTATG (SEQ ID NO: 173), AAGAAAATATCATATTTGGAGTTAGCTACG (SEQ ID NO: 174), AAGAAAATATCATATTTGGGGTAAGTTATG (SEQ ID NO: 175), AAGAAAATATCATATTCGGAGTGAGTTACG (SEQ ID NO: 176), AAGAAAATATCATATTTGGAGTCAGCTACG (SEQ ID NO: 177), AAGAAAATATCATATTTGGTGTCTCGTACG (SEQ ID NO: 178), AAGAAAATATCATATTTGGAGTCTCGTACG (SEQ ID NO: 179), AAGAAAATATCATATTTGGAGTGTCATACG (SEQ ID NO: 180), AAGAAAATATCATATTTGGAGTTTCATACG (SEQ ID NO: 181), AAGAAAATATCATATTTGGTGTCAGCTACG (SEQ ID NO: 182), AAGAAAATATCATATTTGGTGTGAGCTACG (SEQ ID NO: 183), AAGAAAATATCATATTCGGTGTAAGCTACG (SEQ ID NO: 184), AAGAAAATATCATATTCGGTGTTTCGTACG (SEQ ID NO: 185), AAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 186), TAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 187), TTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 188), ATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 189), ACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 190), GCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 191), GGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 192), GCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 193), AAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 194), TAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 195), TTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 196), ATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 197), ACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 198), GCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 199), GGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 200), TGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 201), GCCTGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 202), 352 / 370 B1195.70196WO00 13363760.2or TGCCTGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 203).

79. The pegRNA of any one of claims 70-78, wherein the pegRNA comprises an RTT comprising the sequence AGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 21), TAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 22), CATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 23), CACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 24), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 25), GCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 26), CATAGGAAACACCAAAGATG (SEQ ID NO: 27), AAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 28), AAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 29), TTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 30), ATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 31), CCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 32), ACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 33), CTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 34), CCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 35), TCATTGGTGTTTCCTATG (SEQ ID NO: 36), ATCATTGGTGTTTCCTATG (SEQ ID NO: 37), TATCATTGGTGTTTCCTATG (SEQ ID NO: 38), ATATCATTGGTGTTTCCTATG (SEQ ID NO: 39), AATATCATTGGTGTTTCCTATG (SEQ ID NO: 40), AAATATCATTGGTGTTTCCTATG (SEQ ID NO: 41), AAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 42), GAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 43), AGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 44), AAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 45), AAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 46), TAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 47), TTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 48), ATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 49), ACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 50), GCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 51), 353 / 370 B1195.70196WO00 13363760.2GGCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 52), TGGCACCATTAAAGAAAATATCATTGGTGTTTCCTATG (SEQ ID NO: 53), CATCATAGGAAACACCAAAGATG (SEQ ID NO: 54), ATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 55), TATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 56), ATCTATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 57), TGTATCTATATTCATCATAGGAAACACCAAAGATG (SEQ ID NO: 58), GGAAACACCAAAGATGATATTTTCTTTA (SEQ ID NO: 59), ATTCATCATAGGAAACACCAAAGATGATATTTTCTTTA (SEQ ID NO: 60), AAAATATCATCTTTGGTGTTTCCTAT (SEQ ID NO: 61), ACCATTAAAGAAAATATCATCTTTGGTGTTTCCTAT (SEQ ID NO: 62), AAAATATCATCTTTG (SEQ ID NO: 63), ACCATTAAAGAAAATATCATCTTTG (SEQ ID NO: 64), GGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGC (SEQ ID NO: 65), TATTCATCATAGGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGC (SEQ ID NO: 66), GGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGCATAATCCAGGA (SEQ ID NO: 67), ATTCATCATAGGAAACACCAAAGATGATATTTTCTTTAATGGTGCCAGGCATAATCC AGGA (SEQ ID NO: 68), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGTTACG (SEQ ID NO: 69), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGTTACG (SEQ ID NO: 70), TGGCACCATTAAAGAAAATATCATCTTCGGTGTGAGTTATG (SEQ ID NO: 71), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGTTATG (SEQ ID NO: 72), TGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 73), TGGCACCATTAAAGAAAATATCATCTTTGGCGTAAGCTACG (SEQ ID NO: 76), TGGCACCATTAAAGAAAATATCATATTTGGTGTTAGCTACG (SEQ ID NO: 77), TGGCACCATTAAAGAAAATATCATATTTGGCGTAAGCTACG (SEQ ID NO: 78), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCAGCTACG (SEQ ID NO: 79), TGGCACCATTAAAGAAAATATCATCTTTGGTGTAAGCTACG (SEQ ID NO: 80), TGGCACCATTAAAGAAAATATCATCTTTGGCGTGAGCTACG (SEQ ID NO: 81), TGGCACCATTAAAGAAAATATCATATTTGGTGTGAGCTACG (SEQ ID NO: 82), TGGCACCATTAAAGAAAATATCATATTTGGTGTCAGCTACG (SEQ ID NO: 83), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCAGCTACG (SEQ ID NO: 84), 354 / 370 B1195.70196WO00 13363760.2TGGCACCATTAAAGAAAATATCATATTTGGTGTAAGCTACG (SEQ ID NO: 85), TGGCACCATTAAAGAAAATATCATATTTGGCGTCAGCTACG (SEQ ID NO: 86), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTAGCTACG (SEQ ID NO: 87), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCATACG (SEQ ID NO: 88), TGGCACCATTAAAGAAAATATCATATTTGGCGTGAGCTACG (SEQ ID NO: 89), TGGCACCATTAAAGAAAATATCATATTCGGTGTGAGCTACG (SEQ ID NO: 90), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCATACG (SEQ ID NO: 91), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCATACG (SEQ ID NO: 92), TGGCACCATTAAAGAAAATATCATCTTTGGCGTATCATACG (SEQ ID NO: 93), TGGCACCATTAAAGAAAATATCATCTTTGGCGTAAGTTACG (SEQ ID NO: 94), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCGTACG (SEQ ID NO: 95), TGGCACCATTAAAGAAAATATCATCTTCGGCGTCAGCTACG (SEQ ID NO: 96), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCGTACG (SEQ ID NO: 97), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTAGCTACG (SEQ ID NO: 98), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCGTACG (SEQ ID NO: 99), TGGCACCATTAAAGAAAATATCATCTTTGGGGTATCATACG (SEQ ID NO: 100), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCGTACG (SEQ ID NO: 101), TGGCACCATTAAAGAAAATATCATCTTTGGCGTATCGTACG (SEQ ID NO: 102), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCGTACG (SEQ ID NO: 103), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGTTACG (SEQ ID NO: 70), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCGTACG (SEQ ID NO: 104), TGGCACCATTAAAGAAAATATCATCTTCGGTGTATCATACG (SEQ ID NO: 105), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCGTACG (SEQ ID NO: 106), TGGCACCATTAAAGAAAATATCATCTTCGGCGTAAGCTACG (SEQ ID NO: 107), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGAGCTACG (SEQ ID NO: 108), TGGCACCATTAAAGAAAATATCATCTTTGGCGTTTCGTACG (SEQ ID NO: 109), TGGCACCATTAAAGAAAATATCATCTTTGGTGTAAGTTACG (SEQ ID NO: 110), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCATACG (SEQ ID NO: 111), TGGCACCATTAAAGAAAATATCATATTCGGTGTAAGCTACG (SEQ ID NO: 112), TGGCACCATTAAAGAAAATATCATCTTCGGCGTTAGCTACG (SEQ ID NO: 113), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCATACG (SEQ ID NO: 114), TGGCACCATTAAAGAAAATATCATCTTCGGTGTATCGTACG (SEQ ID NO: 115), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 116), 355 / 370 B1195.70196WO00 13363760.2TGGCACCATTAAAGAAAATATCATCTTTGGCGTCAGTTACG (SEQ ID NO: 117), TGGCACCATTAAAGAAAATATCATATTTGGTGTATCATACG (SEQ ID NO: 118), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCATACG (SEQ ID NO: 119), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCGTACG (SEQ ID NO: 120), TGGCACCATTAAAGAAAATATCATATTCGGTGTATCGTACG (SEQ ID NO: 121), TGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCTTACG (SEQ ID NO: 122), TGGCACCATTAAAGAAAATATCATATTTGGTGTATCGTACG (SEQ ID NO: 123), TGGCACCATTAAAGAAAATATCATCTTCGGGGTGAGCTACG (SEQ ID NO: 124), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCTTACG (SEQ ID NO: 125), TGGCACCATTAAAGAAAATATCATATTTGGTGTTTCTTACG (SEQ ID NO: 126), TGGCACCATTAAAGAAAATATCATATTCGGTGTTTCTTACG (SEQ ID NO: 127), TGGCACCATTAAAGAAAATATCATATTCGGCGTCAGCTACG (SEQ ID NO: 128), TGGCACCATTAAAGAAAATATCATCTTTGGGGTATCGTACG (SEQ ID NO: 129), TGGCACCATTAAAGAAAATATCATATTTGGTGTGTCATACG (SEQ ID NO: 130), TGGCACCATTAAAGAAAATATCATCTTCGGCGTGAGCTACG (SEQ ID NO: 131), TGGCACCATTAAAGAAAATATCATCTTTGGCGTTAGCTACG (SEQ ID NO: 132), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCTCATACG (SEQ ID NO: 133), TGGCACCATTAAAGAAAATATCATCTTTGGAGTTTCGTACG (SEQ ID NO: 134), TGGCACCATTAAAGAAAATATCATATTCGGTGTTAGCTACG (SEQ ID NO: 135), TGGCACCATTAAAGAAAATATCATCTTTGGTGTCTCGTACG (SEQ ID NO: 136), TGGCACCATTAAAGAAAATATCATATTTGGTGTAAGTTACG (SEQ ID NO: 137), TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCTTACG (SEQ ID NO: 138), TGGCACCATTAAAGAAAATATCATCTTCGGTGTGTCATACG (SEQ ID NO: 139), TGGCACCATTAAAGAAAATATCATCTTTGGCGTCTCGTACG (SEQ ID NO: 140), TGGCACCATTAAAGAAAATATCATCTTCGGCGTATCGTACG (SEQ ID NO: 141), TGGCACCATTAAAGAAAATATCATATTTGGTGTTAGTTACG (SEQ ID NO: 142), TGGCACCATTAAAGAAAATATCATATTCGGTGTGTCTTACG (SEQ ID NO: 143), TGGCACCATTAAAGAAAATATCATCTTCGGTGTTTCTTACG (SEQ ID NO: 144), TGGCACCATTAAAGAAAATATCATCTTTGGTGTATCTTACG (SEQ ID NO: 145), TGGCACCATTAAAGAAAATATCATCTTTGGGGTGAGCTACG (SEQ ID NO: 146), AAGAAAATATCATATTTGGAGTAAGTTACG (SEQ ID NO: 147), AAGAAAATATCATATTTGGAGTAAGCTACG (SEQ ID NO: 148), AAGAAAATATCATATTTGGAGTGAGTTACG (SEQ ID NO: 149), 356 / 370 B1195.70196WO00 13363760.2AAAGAAAATATCATATTTGGAGTAAGTTACG (SEQ ID NO: 150), AAGAAAATATCATATTTGGTGTTTCGTACG (SEQ ID NO: 151), AAGAAAATATCATATTTGGTGTTAGCTACG (SEQ ID NO: 152), AAAGAAAATATCATATTTGGAGTAAGCTACG (SEQ ID NO: 153), AAGAAAATATCATATTTGGAGTAAGTTATG (SEQ ID NO: 154), AAAGAAAATATCATATTTGGAGTGAGTTACG (SEQ ID NO: 155), AAGAAAATATCATATTTGGTGTAAGCTACG (SEQ ID NO: 156), AAGAAAATATCATATTTGGTGTTTCATACG (SEQ ID NO: 157), AAGAAAATATCATATTTGGAGTCTCATACG (SEQ ID NO: 158), AAGAAAATATCATATTTGGAGTATCATACG (SEQ ID NO: 159), AAGAAAATATCATATTCGGAGTTTCATACG (SEQ ID NO: 160), AAGAAAATATCATATTTGGAGTATCGTACG (SEQ ID NO: 161), AAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 162), AAGAAAATATCATATTTGGAGTTAGTTACG (SEQ ID NO: 163), AAGAAAATATCATATTTGGTGTAAGTTACG (SEQ ID NO: 164), AAGAAAATATCATATTCGGTGTTAGCTACG (SEQ ID NO: 165), AAGAAAATATCATATTTGGAGTGAGCTACG (SEQ ID NO: 166), AAGAAAATATCATATTTGGAGTAAGCTATG (SEQ ID NO: 167), AAGAAAATATCATATTCGGAGTAAGTTACG (SEQ ID NO: 168), AAGAAAATATCATATTTGGCGTTAGCTACG (SEQ ID NO: 169), AAGAAAATATCATATTTGGTGTTAGTTACG (SEQ ID NO: 170), AAGAAAATATCATATTTGGGGTAAGTTACG (SEQ ID NO: 171), AAGAAAATATCATATTCGGAGTCTCATACG (SEQ ID NO: 172), AAGAAAATATCATATTTGGAGTGAGCTATG (SEQ ID NO: 173), AAGAAAATATCATATTTGGAGTTAGCTACG (SEQ ID NO: 174), AAGAAAATATCATATTTGGGGTAAGTTATG (SEQ ID NO: 175), AAGAAAATATCATATTCGGAGTGAGTTACG (SEQ ID NO: 176), AAGAAAATATCATATTTGGAGTCAGCTACG (SEQ ID NO: 177), AAGAAAATATCATATTTGGTGTCTCGTACG (SEQ ID NO: 178), AAGAAAATATCATATTTGGAGTCTCGTACG (SEQ ID NO: 179), AAGAAAATATCATATTTGGAGTGTCATACG (SEQ ID NO: 180), AAGAAAATATCATATTTGGAGTTTCATACG (SEQ ID NO: 181), AAGAAAATATCATATTTGGTGTCAGCTACG (SEQ ID NO: 182), 357 / 370 B1195.70196WO00 13363760.2AAGAAAATATCATATTTGGTGTGAGCTACG (SEQ ID NO: 183), AAGAAAATATCATATTCGGTGTAAGCTACG (SEQ ID NO: 184), AAGAAAATATCATATTCGGTGTTTCGTACG (SEQ ID NO: 185), AAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 186), TAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 187), TTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 188), ATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 189), ACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 190), GCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 191), GGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 192), GCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 193), AAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 194), TAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 195), TTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 196), ATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 197), ACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 198), GCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 199), GGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 200), TGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 201), GCCTGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 202), or TGCCTGGCACCATTAAAGAAAATATCATATTCGGAGTATCATACG (SEQ ID NO: 203).

80. The pegRNA of any one of claims 70-79, wherein the pegRNA comprises an RTT comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG (SEQ ID NO: 25) or TGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 73).

81. The pegRNA of any one of claims 70-80, wherein the pegRNA comprises an RTT comprising the sequence TGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATG 358 / 370 B1195.70196WO00 13363760.2(SEQ ID NO: 25) or TGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTGTCATACG (SEQ ID NO: 73).

82. The pegRNA of any one of claims 70-81, wherein the pegRNA comprises a primer binding site (PBS) comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence: ATGAATATAGA (SEQ ID NO: 205), ATGAATATAGAT (SEQ ID NO: 206), ATGAATATAGATA (SEQ ID NO: 207), ATGAATATAGATAC (SEQ ID NO: 208), ATATTTTCTTT (SEQ ID NO: 209), ATGAATATAG (SEQ ID NO: 210), ATGAATATAGATACA (SEQ ID NO: 211), ATGAATATAGATACAG (SEQ ID NO: 212), ATGAATATAGATACAGA (SEQ ID NO: 213), ATATTTTCTTTA (SEQ ID NO: 214), ATATTTTCTTTAA (SEQ ID NO: 215), ATATTTTCTTTAAT (SEQ ID NO: 216), ATGGTGCCAG (SEQ ID NO: 217), ATGGTGCCAGGC (SEQ ID NO: 218), ATGGTGCCAGGCA (SEQ ID NO: 219), ATGGTGCCAGGCAT (SEQ ID NO: 220), ATGGTGCCAGGCATA (SEQ ID NO: 221), ATGGTGCCAGGCATAA (SEQ ID NO: 222), GATGAATATA (SEQ ID NO: 223), GATGAATATAGA (SEQ ID NO: 224), GATGAATATAGAT (SEQ ID NO: 225), GATGAATATAGATA (SEQ ID NO: 226), GATGAATATAGATAC (SEQ ID NO: 227), GATGAATATAGATACA (SEQ ID NO: 228), GTGTTTCCTA (SEQ ID NO: 229), GTGTTTCCTATG (SEQ ID NO: 230), GTGTTTCCTATGA (SEQ ID NO: 231), GTGTTTCCTATGAT (SEQ ID NO: 232), GTGTTTCCTATGATG (SEQ ID NO: 233), GTGTTTCCTATGATGA (SEQ ID NO: 234), ATAATCCAGG (SEQ ID NO: 235), ATAATCCAGGA (SEQ ID NO: 236), ATAATCCAGGAA (SEQ ID NO: 237), ATAATCCAGGAAA (SEQ ID NO: 238), ATAATCCAGGAAAA (SEQ ID NO: 239), ATAATCCAGGAAAACT (SEQ ID NO: 240), AAACTGAGAA (SEQ ID NO: 241), AAACTGAGAACA (SEQ ID NO: 242), AAACTGAGAACAG (SEQ ID NO: 243), AAACTGAGAACAGA (SEQ ID NO: 244), AAACTGAGAACAGAA (SEQ ID NO: 245), or AAACTGAGAACAGAAT (SEQ ID NO: 246).

83. The pegRNA of any one of claims 70-82, wherein the pegRNA comprises a PBS comprising the sequence: ATGAATATAGA (SEQ ID NO: 205), ATGAATATAGAT (SEQ ID NO: 206), ATGAATATAGATA (SEQ ID NO: 207), ATGAATATAGATAC (SEQ ID NO: 208), ATATTTTCTTT (SEQ ID NO: 209), ATGAATATAG (SEQ ID NO: 210), 359 / 370 B1195.70196WO00 13363760.2ATGAATATAGATACA (SEQ ID NO: 211), ATGAATATAGATACAG (SEQ ID NO: 212), ATGAATATAGATACAGA (SEQ ID NO: 213), ATATTTTCTTTA (SEQ ID NO: 214), ATATTTTCTTTAA (SEQ ID NO: 215), ATATTTTCTTTAAT (SEQ ID NO: 216), ATGGTGCCAG (SEQ ID NO: 217), ATGGTGCCAGGC (SEQ ID NO: 218), ATGGTGCCAGGCA (SEQ ID NO: 219), ATGGTGCCAGGCAT (SEQ ID NO: 220), ATGGTGCCAGGCATA (SEQ ID NO: 221), ATGGTGCCAGGCATAA (SEQ ID NO: 222), GATGAATATA (SEQ ID NO: 223), GATGAATATAGA (SEQ ID NO: 224), GATGAATATAGAT (SEQ ID NO: 225), GATGAATATAGATA (SEQ ID NO: 226), GATGAATATAGATAC (SEQ ID NO: 227), GATGAATATAGATACA (SEQ ID NO: 228), GTGTTTCCTA (SEQ ID NO: 229), GTGTTTCCTATG (SEQ ID NO: 230), GTGTTTCCTATGA (SEQ ID NO: 231), GTGTTTCCTATGAT (SEQ ID NO: 232), GTGTTTCCTATGATG (SEQ ID NO: 233), GTGTTTCCTATGATGA (SEQ ID NO: 234), ATAATCCAGG (SEQ ID NO: 235), ATAATCCAGGA (SEQ ID NO: 236), ATAATCCAGGAA (SEQ ID NO: 237), ATAATCCAGGAAA (SEQ ID NO: 238), ATAATCCAGGAAAA (SEQ ID NO: 239), ATAATCCAGGAAAACT (SEQ ID NO: 240), AAACTGAGAA (SEQ ID NO: 241), AAACTGAGAACA (SEQ ID NO: 242), AAACTGAGAACAG (SEQ ID NO: 243), AAACTGAGAACAGA (SEQ ID NO: 244), AAACTGAGAACAGAA (SEQ ID NO: 245), or AAACTGAGAACAGAAT (SEQ ID NO: 246).

84. The pegRNA of any one of claims 70-83, wherein the pegRNA comprises a PBS comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence ATGAATATAGATA (SEQ ID NO: 207).

85. The pegRNA of any one of claims 70-84, wherein the pegRNA comprises a PBS comprising the sequence ATGAATATAGATA (SEQ ID NO: 207).

86. The pegRNA of any one of claims 70-85, wherein the pegRNA further comprises a structured motif at its 3' end.

87. The pegRNA of claim 86, wherein the structured motif is an RNA pseudoknot motif. 360 / 370 B1195.70196WO00 13363760.

288. The pegRNA of claim 86 or 87, wherein the structured motif comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 248).

89. The pegRNA of claim 88, wherein the structured motif comprises the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 248).

90. The pegRNA of any one of claims 70-89, wherein the pegRNA comprises the structure 5'-[spacer]-[backbone scaffold]-[reverse transcription template]-[primer binding site]-[structured motif]-3'.

91. The pegRNA of any one of claims 70-90, wherein the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence of any of the pegRNAs in Tables 1 and 2.

92. The pegRNA of any one of claims 70-91, wherein the pegRNA comprises a sequence of any of the pegRNAs in Tables 1 and 2.

93. The pegRNA of any one of claims 70-92, wherein the pegRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence 94. GTCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAA GGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTA AAGAAAATATCATCTTTGGTGTTTCCTATGATGAATATAGATACGCGGTTCTATCTAG TTACGCGTTAAACCAACTAGAA (SEQ ID NO: 250), TCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC TAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTAAAG AAAATATCATCTTTGGTGTGAGTTACGATGAATATAGATACGCGGTTCTATCTAG TTACGCGTTAAACCAACTAGAA (SEQ ID NO: 744), or CTGTATCTATATTCATCATGTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAG 361 / 370 B1195.70196WO00 13363760.2TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGCCTGGCACCATTAAA GAAAATATCATCTTTGGTGTGTCATACGATGAATATAGATACGCGGTTCTATCTAGTT ACGCGTTAAACCAACTAGAA (SEQ ID NO: 251).

95. The pegRNA of any one of claims 70-93, wherein the pegRNA comprises the sequence 96. GTCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAA GGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTA AAGAAAATATCATCTTTGGTGTTTCCTATGATGAATATAGATACGCGGTTCTATCTAG TTACGCGTTAAACCAACTAGAA (SEQ ID NO: 250), TCTGTATCTATATTCATCATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC TAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGGCACCATTAAAG AAAATATCATCTTTGGTGTGAGTTACGATGAATATAGATACGCGGTTCTATCTAG TTACGCGTTAAACCAACTAGAA (SEQ ID NO: 744), or CTGTATCTATATTCATCATGTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGCCTGGCACCATTAAA GAAAATATCATCTTTGGTGTGTCATACGATGAATATAGATACGCGGTTCTATCTAGTT ACGCGTTAAACCAACTAGAA (SEQ ID NO: 251).

97. The pegRNA of any one of claims 70-94, wherein the pegRNA comprises a chemical modification at its 3' end.

98. The pegRNA of claim 95, wherein the chemical modification comprises phosphonoacetate (mP).

99. A nicking guide RNA (ngRNA) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence TTCACTTCTAATGGTGATTA (SEQ ID NO: 745), TCACTTCTAATGGTGATTAT (SEQ ID NO: 746), AATGGTGATTATGGGAGAAC (SEQ ID NO: 747), GGGAGAACTGGAGCCTTCAG (SEQ ID NO: 748), GGAGAACTGGAGCCTTCAGA (SEQ ID NO: 749), GAGGGTAAAATTAAGCACAG (SEQ ID NO: 750), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), 362 / 370 B1195.70196WO00 13363760.2CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), ATTAAAGAAAATATCATCTT (SEQ ID NO: 751), CAAAGCATGCCAACTAGAAG (SEQ ID NO: 752), or CACTACCCAAATTATATATT (SEQ ID NO: 753).

100. The ngRNA of claim 97, wherein the ngRNA comprises a spacer comprising the sequence TTCACTTCTAATGGTGATTA (SEQ ID NO: 745), TCACTTCTAATGGTGATTAT (SEQ ID NO: 746), AATGGTGATTATGGGAGAAC (SEQ ID NO: 747), GGGAGAACTGGAGCCTTCAG (SEQ ID NO: 748), GGAGAACTGGAGCCTTCAGA (SEQ ID NO: 749), GAGGGTAAAATTAAGCACAG (SEQ ID NO: 750), CATTCTGTTCTCAGTTTTCC (SEQ ID NO: 7), CAGTTTTCCTGGATTATGCC (SEQ ID NO: 6), ATTAAAGAAAATATCATCTT (SEQ ID NO: 751), CAAAGCATGCCAACTAGAAG (SEQ ID NO: 752), or CACTACCCAAATTATATATT (SEQ ID NO: 753).

101. The ngRNA of claim 97 or 98, wherein the ngRNA spacer further comprises a G at its 5' end.

102. The ngRNA of any one of claims 97-99, wherein the ngRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17) or GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 761).

103. The ngRNA of any one of claims 97-100, wherein the ngRNA comprises a backbone scaffold comprising the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17) or GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 761). 363 / 370 B1195.70196WO00 13363760.2104. The ngRNA of any one of claims 97-101, wherein the ngRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence of any of the ngRNAs in Table 1.

105. The ngRNA of any one of claims 97-102, wherein the ngRNA comprises a sequence of any of the ngRNAs in Table 1.

106. A dead single guide RNA (dsgRNA) comprising a spacer comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence CTCCCTCCAAGTTGTCCACG (SEQ ID NO: 764), CTGGAGCCTTCAGA (SEQ ID NO: 765), ATTTTACCCTCTGA (SEQ ID NO: 766), GCCAGGCATAATCC (SEQ ID NO: 767), TCCTGGATTATGCC (SEQ ID NO: 768), TCTTTAATGGTGCC (SEQ ID NO: 769), TTACCTCTTCTAGT (SEQ ID NO: 770), ATGCCAACTAGAAG (SEQ ID NO: 771), GCCAAATATATAATT (SEQ ID NO: 772), or CCGCGCGCGCGAAAAAGCCG (SEQ ID NO: 773).

107. The dsgRNA of claim 104, wherein the dsgRNA comprises a spacer comprising the sequence CTCCCTCCAAGTTGTCCACG (SEQ ID NO: 764), CTGGAGCCTTCAGA (SEQ ID NO: 765), ATTTTACCCTCTGA (SEQ ID NO: 766), GCCAGGCATAATCC (SEQ ID NO: 767), TCCTGGATTATGCC (SEQ ID NO: 768), TCTTTAATGGTGCC (SEQ ID NO: 769), TTACCTCTTCTAGT (SEQ ID NO: 770), ATGCCAACTAGAAG (SEQ ID NO: 771), GCCAAATATATAATT (SEQ ID NO: 772), or CCGCGCGCGCGAAAAAGCCG (SEQ ID NO: 773).

108. The dsgRNA of claim 104 or 105, wherein the dsgRNA comprises a backbone scaffold comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGCTTT (SEQ ID NO: 782), GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA 364 / 370 B1195.70196WO00 13363760.2AAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 761), or GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17).

109. The dsgRNA of any one of claims 104-106, wherein the dsgRNA comprises a backbone scaffold comprising the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGCTTT (SEQ ID NO: 782), GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGCTTTT (SEQ ID NO: 761), or GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC (SEQ ID NO: 17).

110. The dsgRNA of any one of claims 104-107, wherein the dsgRNA comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or comprising one, two, three, four, or five mutations relative to, the sequence of any of the dsgRNAs in Table 1.

111. The dsgRNA of any one of claims 104-108, wherein the dsgRNA comprises a sequence of any of the dsgRNAs in Table 1.

112. A composition comprising the pegRNA of any one of claims 70-96, the ngRNA of any one of claims 97-103, and / or the dsgRNA of any one of claims 104-109.

113. The composition of claim 110 further comprising a prime editor.

114. The composition of claim 111, wherein the prime editor comprises a nucleic acid- programmable DNA-binding protein (napDNAbp) and a polymerase.

115. The composition of claim 112, wherein the napDNAbp comprises a Cas9 protein.

116. The composition of claim 112 or 113, wherein the napDNAbp comprises a Cas9 nickase (nCas9). 365 / 370 B1195.70196WO00 13363760.2117. The composition of claim 113 or 114, wherein the Cas9 protein comprises a Streptococcus pyogenes Cas9 protein, or a variant thereof.

118. The composition of any one of claims 110-115, wherein the napDNAbp comprises a Cas9 protein of a PE6 prime editor.

119. The composition of any one of claims 110-116, wherein the polymerase is a reverse transcriptase.

120. The composition of claim 117, wherein the reverse transcriptase is an MMLV reverse transcriptase, a Tf1 reverse transcriptase, or a variant thereof.

121. The composition of any one of claims 110-118, wherein the polymerase comprises a reverse transcriptase of a PE6 prime editor.

122. The composition of any one of claims 110-119, wherein the prime editor comprises PEmax architecture.

123. A system comprising the pegRNA of any one of claims 70-96, the ngRNA of any one of claims 97-103, and / or the dsgRNA of any one of claims 104-109.

124. The system of claim 121 further comprising a prime editor.

125. The system of claim 122, wherein the prime editor comprises a nucleic acid- programmable DNA-binding protein (napDNAbp) and a polymerase.

126. The system of claim 123, wherein the napDNAbp comprises a Cas9 protein.

127. The system of claim 123 or 124, wherein the napDNAbp comprises a Cas9 nickase (nCas9). 366 / 370 B1195.70196WO00 13363760.2128. The system of claim 124 or 125, wherein the Cas9 protein is a Streptococcus pyogenes Cas9 protein, or a variant thereof.

129. The system of any one of claims 124-126, wherein the napDNAbp comprises a Cas9 protein of a PE6 prime editor.

130. The system of any one of claims 124-127, wherein the polymerase is a reverse transcriptase.

131. The system of claim 128, wherein the reverse transcriptase is an MMLV reverse transcriptase, a Tf1 reverse transcriptase, or a variant thereof.

132. The system of any one of claims 123-129, wherein the polymerase comprises a reverse transcriptase of a PE6 prime editor.

133. The system of any one of claims 123-130, wherein the prime editor comprises PEmax architecture.

134. A polynucleotide encoding the pegRNA of any one of claims 70-96, the ngRNA of any one of claims 97-103, and / or the dsgRNA of any one of claims 104-109.

135. One or more polynucleotides encoding the composition of any one of claims 110-120 or the system of any one of claims 121-131.

136. A vector comprising a polynucleotide of claim 132.

137. One or more vectors comprising one or more polynucleotides of claim 133.

138. A pharmaceutical composition comprising the pegRNA of any one of claims 70-96, the ngRNA of any one of claims 97-103, the dsgRNA of any one of claims 104-109, the composition of any one of claims 110-120, the one or more polynucleotides of claim 132 or 133, or the one or more vectors of claim 134 or 135. 367 / 370 B1195.70196WO00 13363760.2139. A cell comprising the pegRNA of any one of claims 70-96, the ngRNA of any one of claims 97-103, the dsgRNA of any one of claims 104-109, the composition of any one of claims 110-120, the one or more polynucleotides of claim 132 or 133, or the one or more vectors of claim 134 or 135.

140. A kit comprising the pegRNA of any one of claims 70-96, the ngRNA of any one of claims 97-103, the dsgRNA of any one of claims 104-109, the composition of any one of claims 110-120, the one or more polynucleotides of claim 132 or 133, or the one or more vectors of claim 134 or 135.

141. Use of the pegRNA of any one of claims 70-96, the ngRNA of any one of claims 97- 103, the dsgRNA of any one of claims 104-109, the composition of any one of claims 110- 120, the one or more polynucleotides of claim 132 or 133, the one or more vectors of claim 134 or 135, or the pharmaceutical composition of claim 136 in the manufacture of a medicament for the treatment of cystic fibrosis.

142. The pegRNA of any one of claims 70-96, the ngRNA of any one of claims 97-103, the dsgRNA of any one of claims 104-109, the composition of any one of claims 110-120, the one or more polynucleotides of claim 132 or 133, the one or more vectors of claim 134 or 135, or the pharmaceutical composition of claim 136 for use in medicine.

143. A pegRNA comprising the sequence CTGTATCTATATTCATCATGTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGCCTGGCACCATTAAA GAAAATATCATCTTTGGTGTGTCATACGATGAATATAGATACGCGGTTCTATCTAGTT ACGCGTTAAACCAACTAGAA (SEQ ID NO: 251).

144. A method of prime editing a cystic fibrosis transmembrane conductance regulator (CFTR) gene comprising contacting a nucleic acid sequence encoding the CFTR gene with a PE6c prime editor and a prime editing guide RNA (pegRNA) comprising the sequence CTGTATCTATATTCATCATGTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTGCCTGGCACCATTAAA 368 / 370 B1195.70196WO00 13363760.2GAAAATATCATCTTTGGTGTGTCATACGATGAATATAGATACGCGGTTCTATCTAGTT ACGCGTTAAACCAACTAGAA (SEQ ID NO: 251). 369 / 370 B1195.70196WO00 13363760.2

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