An evolved and responsive prime editor with improved editing efficiency.

Abstract

This disclosure provides evolved and manipulated reverse transcriptase variants and Cas9 variants having improved properties (e.g., improved editing efficiency when used in the context of a prime editor). For example, fusion proteins containing prime editors comprising the reverse transcriptase variants and Cas9 variants described herein are also provided by this disclosure. This disclosure also provides polynucleotides encoding the reverse transcriptase variants, Cas9 variants, and prime editors provided herein, as well as vectors containing such polynucleotides. Pharmaceutical compositions and cells comprising the reverse transcriptase variants, Cas9 variants, and prime editors described herein are also provided by this disclosure. This disclosure also provides methods and uses involving the reverse transcriptase variants, Cas9 variants, and prime editors described herein.

Description

[Technical Field]

[0001] Related applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application USSN63 / 503,892 filed 23 May 2023; U.S. Provisional Application USSN63 / 506,026 filed 2 June 2023; U.S. Provisional Application USSN63 / 510,078 filed 23 June 2023; U.S. Provisional Application USSN63 / 596,006 filed 3 November 2023; and U.S. Provisional Application USSN63 / 508,616 filed 16 June 2023, each of which is incorporated herein by reference.

[0002] Reference to electronic sequence listings The contents of the electronic sequence listing (B119570180WO00-SEQ-TNG.xml; size: 226,657 bytes; and creation date: May 15, 2024) are incorporated herein by reference in their entirety.

[0003] Government support This invention was made with government support under grants No. UG3AI150551, U01AI142756, R35GM118062, and RM1HG009490 awarded by the National Institutes of Health. The government has certain rights to this invention. [Background technology]

[0004] The ability to implant precise, targeted alterations into living cells and the genomes of organisms is advancing our understanding of biological systems and potentially providing one-time treatments for genetic diseases. Prime editing (PE) is a versatile gene editing technology that can implant any base substitution, insertion, or deletion without generating double-stroke bonds (DSBs). 1 Since >95% of pathogenic substitutions, insertions, deletions, or combinations thereof have a length of ≤50 bp, 2Prime editors (PEs) increase the potential to correct a large proportion of mutations that cause known diseases. PEs require a prime editing guide RNA (pegRNA) and a prime editor protein containing programmed nickase (typically S. pyogenes Cas9 H840A nickase) and reverse transcriptase (RT). The first generation prime editor (PE1) used wild-type Moloney mouse leukemia virus (M-MLV) RT, while subsequent prime editors (PE2-PE5) use engineered quintuplet mutant M-MLV RT (Figure 1A). 1,3 The pegRNA contains a core guide RNA backbone that binds to programmed nickase, a spacer that designates the target site, a primer-binding site (PBS) complementary to the target DNA, and a reverse transcriptase template (RTT) that encodes the desired edit. To set the edit, the prime-editor-pegRNA complex pairs with one strand of the target genomic locus and nicks to the opposing strand, generating an exposed 3' end of the nicked genomic DNA, which binds to the complementary PBS of the pegRNA. RT engages with the resulting primer-template complex and initiates reverse transcription of the RTT, generating a 3' DNA flap containing the desired edit. The newly synthesized 3' flap is incorporated into the genome by the cellular DNA repair pathway, replacing the original DNA sequence and leading to the permanent setting of the desired edit. 1 In the PE3 and PE5 systems, additional sgRNA is used to nick the unedited DNA strand, improving editing efficiency by biasing cellular mismatch repair to prefer the replacement of the unedited strand (Figure 1A). 1,3 .

[0005] From these developments, the PE system was developed to stabilize or circularize pegRNA. 4-6 , changing the Prime Editor architecture 3,4 , and have been improved by manipulating or avoiding cellular mismatch repair to favor the desired editing outcome. 3,9Twin prime editing (twinPE) and related methods have also been developed. These use two pegRNAs to place the edited sequences on both DNA strands and replace the original genomic sequence between the two prime editing nicks with larger (>100bp) programmed insertions and deletions. 10-13,15,16 Prime editing and twinPE have also been used to install site-specific recombinase landing sites, enabling recombinase-mediated targeted insertions or inversions of gene sizes (>5,000bp). 10 Prime editing, twin prime editing, and prime editors are further described, for example, in International Patent Application No. PCT / US2020 / 023721, filed March 19, 2020, published as WO 2020 / 191239; International Patent Application No. PCT / US2021 / 031439, filed May 7, 2021, published as WO 2021 / 226558; and International Patent Application No. PCT / 2021 / 052097, filed September 24, 2021, published as WO 2022 / 067130; the contents of each of these are incorporated herein by reference.

[0006] Despite this progress, the reverse transcriptase at the core of prime editors has proven difficult to improve through protein engineering. Many of the prime editing systems reported to date, including the current PE4max and PE5max systems, use M-MLV RT engineered on PE2. The five M-MLV RT mutations in PE2 were identified through decades of in vitro screening of improved RT variants 18-21 , followed by screening of many combinations of M-MLV RT mutants to optimize prime editing efficiency. 1These mutations are significant for prime editing efficiency, yet only a few similar mutations have been described for other RTs tested in prime editing experiments. Prime editor proteins using non-M-MLV RTs could, in principle, offer the significant advantage of encompassing smaller sizes that could facilitate in vivo prime editor delivery, mRNA production, or ribonucleoprotein (RNP) preparation. Just as different deaminases have provided a diverse collection of base editors that greatly increases the probability of finding one ideally suited to a particular application, different RT enzymes could also improve the properties of PEs, such as editing efficiency, suitability for longer or shorter prime edits, or compatibility with placing sequences of different compositions. 22 Despite these potential benefits, all previously reported prime editors that did not use manipulated M-MLV RT in PE2 showed substantially lower prime editing efficiency than PE2 for most target sequences, even after extensive protein engineering. 4,17,24 Further improvement of highly manipulated M-MLV RT in PE2 has also proven difficult, because all reported variants of this RT have either not resulted in any significant improvement in prime editing efficiency in mammalian cells. 17,24 Similarly, Cas9 mutations known to improve nuclease performance have also been shown to increase prime editing efficiency. 3 No Cas9 variants have yet been identified as specifically improving prime editing. Therefore, additional RT and Cas9 variants evolved and / or manipulated for the purpose of improving prime editing efficiency would advance this field. [Overview of the Initiative]

[0007] As presented herein, phage-assisted continuous evolution (PACE) for prime editing 26A selection process was developed, and PE PACE and protein engineering were used to generate novel polymerases (e.g., reverse transcriptases or "RTs") and Cas9 variants that enhance prime editing efficiency and in vivo delivery potential. First, natural RTs were screened from a wide variety of organisms and found that most exhibit negligible prime editing activity in mammalian cells. Two weakly active RTs, Escherichia coli Ec48 retron, were identified. 27 and Schizosaccharomyces pombe Tf1 retrotransposon 28 These were evolved to create the next generation of prime editors (PE6a and PE6b). These are 516–810 bp smaller than PE2, while offering mammalian prime editing efficiency comparable to or higher than PE2 for many target sites and editing types. Commonly used prime editor variants used in dual AAV delivery systems. 4,23,29-31 It was discovered that the reduced RT processing efficiency of PEmaxΔRNaseH (i.e., PEmax containing MMLV reverse transcriptase with a shortened C-terminal RNaseH domain) results in lower performance in long edits with a high degree of secondary structure. To generate dual AAV-compatible RTs capable of accommodating longer edits or edits requiring RT templates with a high degree of secondary structure, PE PACE and protein engineering were used to produce PE6c and PE6d. These RT variants offer a significant advantage in editing efficiency compared to PEmaxΔRNaseH for edits requiring structured pegRNA RT templates. PE6a-PE6d RTs also offer improved editing efficiency and lower indel frequencies compared to full-length PEmax. Finally, PE PACE was used to evolve the Cas9 nickase domain of the prime editor to create PE6e-PE6g. These further improve prime editing efficiency. The improved RTs and Cas9 nickase domains of the PE6 variants interact with each other as well as mismatch repair escape strategies. 3 , epegRNA5 , and PEmax architecture 3 Combined with other technologies, this can provide cumulative benefits in various contexts, including patient-derived fibroblasts and primary human T cells. Finally, PE6c and PE6d are demonstrated to uniquely enable long prime editing and twinPE in vivo. After dual AAV delivery of the PE6 system, an average 12–183-fold improvement in prime editing efficiency was achieved in mouse cortex compared to previous technology-level systems for 38–42 bp edit placement, resulting in targeted placement of loxP sequences in 62% of transdextrins in mouse cortex.

[0008] The improved Prime Editors PE6a to PE6g described herein include the following amino acid substitutions for specific wild-type reverse transcriptase or Cas9 proteins: [Table 1-1] [Table 1-2]

[0009] In some embodiments, the Disclosure provides a prime editor comprising PE6a reverse transcriptase (or a reverse transcriptase that is 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 PE6a reverse transcriptase) and a nucleic acid programmed DNA-binding protein (napDNAbp) (e.g., Cas9 protein). In some embodiments, the Disclosure provides a prime editor comprising PE6b reverse transcriptase (or a reverse transcriptase that is 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 PE6b reverse transcriptase) and napDNAbp (e.g., Cas9 protein). In some embodiments, the Disclosure provides a prime editor comprising PE6c reverse transcriptase (or a reverse transcriptase that is 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 PE6c reverse transcriptase) and napDNAbp (e.g., Cas9 protein). In some embodiments, the Disclosure provides a prime editor comprising PE6d reverse transcriptase (or a reverse transcriptase that is 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 PE6d reverse transcriptase) and napDNAbp (e.g., Cas9 protein) and napDNAbp (e.g., Cas9 protein).

[0010] In some embodiments, the Disclosure provides a prime editor comprising the Cas9 protein of PE6e (or a Cas9 protein that is 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 Cas9 protein of PE6e) and a polymerase (e.g., reverse transcriptase). In some embodiments, the Disclosure provides a prime editor comprising the Cas9 protein of PE6f (or a Cas9 protein that is 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 Cas9 protein of PE6f) and a polymerase (e.g., reverse transcriptase). In some embodiments, the disclosure provides a prime editor comprising PE6g Cas9 protein (or a Cas9 protein that is 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 PE6g Cas9 protein) and a polymerase (e.g., reverse transcriptase).

[0011] In certain embodiments, the Disclosure provides a prime editor (PE6a-e) comprising PE6a reverse transcriptase and PE6e Cas9 protein. In certain embodiments, the Disclosure provides a prime editor (PE6a-f) comprising PE6a reverse transcriptase and PE6f Cas9 protein. In certain embodiments, the Disclosure provides a prime editor (PE6a-g) comprising PE6a reverse transcriptase and PE6g Cas9 protein. In certain embodiments, the Disclosure provides a prime editor (PE6b-e) comprising PE6b reverse transcriptase and PE6e Cas9 protein. In certain embodiments, the Disclosure provides a prime editor (PE6b-f) comprising PE6b reverse transcriptase and PE6f Cas9 protein. In certain embodiments, the Disclosure provides a prime editor (PE6b-g) comprising PE6b reverse transcriptase and PE6g Cas9 protein. In certain embodiments, the Disclosure provides a prime editor (PE6c-e) comprising PE6c reverse transcriptase and PE6e Cas9 protein. In certain embodiments, the Disclosure provides a prime editor (PE6c-f) comprising PE6c reverse transcriptase and PE6f Cas9 protein. In certain embodiments, the Disclosure provides a prime editor (PE6c-g) comprising PE6c reverse transcriptase and PE6g Cas9 protein. In certain embodiments, the Disclosure provides a prime editor (PE6d-e) comprising PE6d reverse transcriptase and PE6e Cas9 protein. In certain embodiments, the Disclosure provides a prime editor (PE6d-f) comprising PE6d reverse transcriptase and PE6f Cas9 protein. In certain embodiments, the Disclosure provides a prime editor (PE6d-g) comprising PE6d reverse transcriptase and PE6g Cas9 protein.

[0012] In one aspect, the present disclosure provides a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 1 (Tf1 reverse transcriptase), wherein the reverse transcriptase variant includes amino acid substitutions at positions 70, 72, 87, 102, 106, 118, 128, 158, 269, 363, 413, and 492 relative to SEQ ID NO: 1, or corresponding substitutions in homologous sequences. In some embodiments, the reverse transcriptase variant further includes amino acid substitutions at positions 188, 260, 297, and 288 relative to SEQ ID NO: 1.

[0013] In another aspect, the Disclosure provides reverse transcriptase variants 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% sequence identity with SEQ ID NO: 30 (MMLV reverse transcriptase), wherein the reverse transcriptase variant includes amino acid substitutions at positions 128 and 200 of SEQ ID NO: 30, or corresponding substitutions in homologous sequences. In some embodiments, the reverse transcriptase variant further includes amino acid substitutions at positions 223, 306, 313, and 330 of SEQ ID NO: 30, or corresponding substitutions in homologous sequences. In some embodiments, the reverse transcriptase variant includes a shortening of the RNaseH domain of SEQ ID NO: 30 (e.g., a shortening at D497 of SEQ ID NO: 30).

[0014] In another aspect, the present disclosure provides reverse transcriptase variants 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% sequence identity with SEQ ID NO: 30, wherein the reverse transcriptase variants include amino acid substitutions T128N and V223M; T128N and V223Y; T128F and V223M; or D200C and V223M, or corresponding substitutions in homologous sequences with respect to SEQ ID NO: 30.

[0015] In another aspect, the present disclosure provides a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 30 (MMLV reverse transcriptase), wherein the reverse transcriptase variant includes amino acid substitutions at positions 128, 129, 196, 200, and 223 with respect to SEQ ID NO: 30, or corresponding substitutions in homologous sequences.

[0016] In another aspect, the present disclosure provides Cas9 variants 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% sequence identity with SEQ ID NO: 2 (Streptococcus pyogenes Cas9 nickase), wherein the Cas9 variants include amino acid substitutions at positions 775 and 918 with respect to SEQ ID NO: 2, or corresponding substitutions in homologous sequences.

[0017] In another aspect, the present disclosure provides Cas9 variants 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% sequence identity with SEQ ID NO: 2 (Streptococcus pyogenes Cas9 nickase), wherein the Cas9 variants include amino acid substitutions at positions 99, 471, 632, 645, and 721 relative to SEQ ID NO: 2, or corresponding substitutions in homologous sequences.

[0018] In another aspect, the present disclosure provides Cas9 variants 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% sequence identity with SEQ ID NO: 2 (Streptococcus pyogenes Cas9 nickase), wherein the Cas9 variants include amino acid substitutions at positions 99, 471, and 632 with respect to SEQ ID NO: 2, or corresponding substitutions in homologous sequences.

[0019] In another aspect, the present disclosure provides Cas9 variants 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% sequence identity with SEQ ID NO: 2 (Streptococcus pyogenes Cas9 nickase), wherein the Cas9 variants include amino acid substitutions at positions 471 and 918 with respect to SEQ ID NO: 2, or corresponding substitutions in homologous sequences.

[0020] In another aspect, the present disclosure provides Cas9 variants 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% sequence identity with SEQ ID NO: 2 (Streptococcus pyogenes Cas9 nickase), wherein the Cas9 variants include amino acid substitutions at positions 753 and 1151 with respect to SEQ ID NO: 2, or corresponding substitutions in homologous sequences.

[0021] In another aspect, the present disclosure provides Cas9 variants 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% sequence identity with SEQ ID NO: 2 (Streptococcus pyogenes Cas9 nickase), wherein the Cas9 variants include one or more amino acid substitutions at positions selected from the group consisting of 260, 298, 395, 769, 778, 1014, 1034, 1100, 1106, 1138, 1152, and 1320 relative to SEQ ID NO: 2, or corresponding substitutions in homologous sequences.

[0022] In another aspect, the present disclosure provides Cas9 variants 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% sequence identity with SEQ ID NO: 2 (Streptococcus pyogenes Cas9 nickase), wherein the Cas9 variants include amino acid substitutions at positions 23 and 754 with respect to SEQ ID NO: 2, or corresponding substitutions in homologous sequences.

[0023] In another aspect, the Disclosure provides a prime editor comprising (i) any of the reverse transcriptase variants provided herein, and (ii) a napDNAbp, such as a Cas9 protein (e.g., Cas9 nickase, or any of the Cas9 variants provided herein (which may also be Cas9 nickase), or a Cas9 nuclease or nuclease-inactivated Cas9 (dCas9)).

[0024] In another aspect, the Disclosure provides a prime editor comprising (i) any of the Cas9 variants provided herein, and (ii) a polymerase (e.g., a reverse transcriptase such as any of the reverse transcriptase variants provided herein). In some embodiments, the reverse transcriptase comprises a sequence 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% sequence identity with SEQ ID NO: 7 (Ec48 reverse transcriptase), wherein the reverse transcriptase comprises amino acid substitutions at positions 60, 87, 165, 243, 267, 279, 318, and 343, or at corresponding positions in the homologous sequence, relative to SEQ ID NO: 7.

[0025] In some embodiments, the reverse transcriptase variants provided herein include an amino acid sequence identical to any one of SEQ ID NOs. 25-27 or 50, or an amino acid sequence that is 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 any one of SEQ ID NOs. In some embodiments, the Cas9 variants provided herein include an amino acid sequence identical to any one of SEQ ID NOs. 28, 48, or 49, or an amino acid sequence that is 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 any one of SEQ ID NOs. In some embodiments, the prime editors provided herein include a Cas9 variant comprising an amino acid sequence identical to any one of the amino acid sequences of SEQ ID NOs. 28, 48, or 49, or an amino acid sequence identical to 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%, and a reverse transcriptase variant comprising an amino acid sequence identical to any one of the amino acid sequences of SEQ ID NOs. 25-27 or 50, or an amino acid sequence identical to 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%.

[0026] In another aspect, the present disclosure provides a fusion protein comprising one of the Cas9 variants provided herein and an effector domain. In certain embodiments, the effector domain includes nuclease activity, nickas activity, recombinase activity, deaminase activity, methyltransferase activity, methylase activity, acetylase activity, acetyltransferase activity, transcriptional activating activity, transcriptional repressing activity, or polymerase activity.

[0027] In another aspect, the Disclosure provides a complex comprising one of the prime editors or other fusion proteins provided herein and prime editing guide RNA (pegRNA).

[0028] In some respects, this disclosure provides polynucleotides encoding any of the reverse transcriptase variants, Cas9 variants, fusion proteins, or prime editors provided herein. In other respects, this disclosure provides vectors comprising any of the polynucleotides provided herein.

[0029] In another aspect, the Disclosure provides adeno-associated virus (AAV) particles comprising any of the reverse transcriptase variants, Cas9 variants, prime editors, fusion proteins, complexes, polynucleotides, and / or vectors provided herein.

[0030] In another aspect, the present disclosure provides cells comprising any of the reverse transcriptase variants, Cas9 variants, prime editors, fusion proteins, complexes, polynucleotides, vectors, and / or AAV particles provided herein.

[0031] In another aspect, the present disclosure provides pharmaceutical compositions comprising any of the reverse transcriptase variants, Cas9 variants, prime editors, fusion proteins, complexes, polynucleotides, vectors, AAV particles, and / or cells provided herein.

[0032] In another aspect, this disclosure provides a method for editing nucleic acid molecules by prime editing, comprising contacting the nucleic acid molecule with one of the prime editors or complexes provided herein. In certain embodiments, the editing in the nucleic acid molecule includes one or more nucleotide insertions, one or more nucleotide substitutions, one or more nucleotide deletions, or a combination thereof. In certain embodiments, the method is a twin-prime editing method (also known as dual-flap prime editing). In some embodiments, this disclosure provides a method for using the prime editors, complexes, polynucleotides, or vectors provided herein for veterinary use. In some embodiments, this disclosure provides a method for using the prime editors, complexes, polynucleotides, or vectors provided herein for agricultural use.

[0033] In another aspect, the Disclosure provides kits comprising any of the reverse transcriptase variants, Cas9 variants, prime editors, fusion proteins, complexes, polynucleotides, vectors, AAV particles, and / or cells provided herein.

[0034] In another aspect, this disclosure provides for the use in the manufacture of any of the reverse transcriptase variants, Cas9 variants, prime editors, fusion proteins, complexes, polynucleotides, vectors, AAV particles, and / or cells provided herein.

[0035] In another aspect, this disclosure provides pharmaceutical uses of any of the reverse transcriptase variants, Cas9 variants, prime editors, fusion proteins, complexes, polynucleotides, vectors, AAV particles, and / or cells provided herein.

[0036] In some aspects, the present disclosure provides a system for phage-assisted continuous and discontinuous evolution (PACE and PANCE) of a prime editor. In certain embodiments, the present disclosure provides a system comprising i) a first polynucleotide encoding pegRNA and gIII genes; ii) a second polynucleotide encoding a Cas9 protein fused to an N-intane; iii) a third polynucleotide encoding RNA polymerase; iv) a fourth polynucleotide encoding a protein that can be mutagenerated into a phage, optionally, wherein the fourth polynucleotide comprises an MP6 plasmid; and v) a fifth polynucleotide encoding reverse transcriptase fused to a C-intane. In certain embodiments, the present disclosure provides a system comprising i) a first polynucleotide encoding pegRNA and gIII genes; ii) a second polynucleotide encoding a prime editor; iii) a third polynucleotide encoding RNA polymerase; and iv) a fourth polynucleotide encoding a protein that can be mutagenerated into a phage, optionally, wherein the fourth polynucleotide comprises an MP6 plasmid.

[0037] The concepts described above and any additional concepts discussed below can be arranged in any preferred combination, as this disclosure is not limited in this respect. Furthermore, other advantages and novel features of this disclosure will become apparent from the following detailed description in various non-limiting embodiments, when considered together with the accompanying figures. [Brief explanation of the drawing]

[0038] The following figures form part of this specification and are included to further demonstrate certain aspects of this disclosure. They can be better understood by referring to one or more of these figures in conjunction with the detailed descriptions of the specific embodiments presented herein. [Figure 1A]Identification of reverse transcriptases and manipulation of novel prime editor candidates. Figure 1A shows an overview of prime editing using the PE1, PE2, and PE3 systems. All three systems use a prime editor protein containing SpCas9(H840A) nickase fused to a reverse transcriptase (RT) enzyme. The PE1 system uses RT from Moloney's mouse leukemia virus (M-MLV), and the PE2 system uses an engineered quintuplet mutant variant of M-MLV RT having D200N, L603W, T306K, W313F, and T330P. An additional single guide RNA (sgRNA) is used in the PE3 system to nick the unedited strand. PBS = primer binding site. RT template = reverse transcriptase template. [Figure 1B] Figure 1B shows the phylogenetic classification of all RTs (circles) tested for prime editing in this specification. The enzymes that show activity in the PE system (dark gray circles) belong to four different RT classes. [Figure 1C] Figure 1C shows that 20 different RT enzymes other than M-MLV RT exhibit activity in the prime editing system at endogenous sites in HEK293T cells. Bars reflect the mean of n=3 independent replicates. Dots indicate the values ​​of individual replicates. In all figures, the prime editing efficiency shown reflects the frequency of the intended prime editing outcome, which does not involve indels or other changes at the target site. [Figure 1D-1F]Figure 1D shows a comparison of wild-type (WT) Tf1 RT, PE2ΔRNaseH (i.e., including a shortening of the C-terminal RNaseH domain of MMLV reverse transcriptase between amino acids D497 and I498 in MMLV reverse transcriptase of sequence number 30, for example), and PE2 in three longer, more complex PE (HEK3) or twinPE (CCR5 and IDS) edits of HEK293T cells. Bars reflect the mean of n=3 independent replicates. Dots indicate the values ​​of individual replicates. Figure 1E shows a comparison of prime editors containing manipulated retroviral RT variants with their WT counterparts in HEK293T cells. rdPERV = Porcine endogenous retrovirus RT D200N, T306K, W313F, E330P, L603W. rdAVIRE = Avian reticuloendotheliosis virus RT D200N, T306K, W313F, G330P, L603W. rdKORV = Koala retrovirus RT D198N, T304K, W311F, E328P, L600W. rdWMSV = Wooliesal sarcoma virus RT D198N, T304K, W311F, E328P, L600W. All values ​​from n=3 independent repeats are shown. Horizontal bars indicate the mean. Figure 1F shows that the mutated residues to improve editing in the Tf1 RT prime editor correspond to V188, R118, L258, M281, and V286 (red) in Ty3 RT (blue). V188 and R118 are closely proximal to the RNA (green) substrate and correspond to K118 and S188 on Tf1, respectively. L258, M281, and V286 are close to the DNA (yellow) substrate and correspond to I260, S297, and R288 on Tf1, respectively. [Figure 1G-1H]Figure 1G shows that a rationally designed Tf1 quintuple mutant variant (rdTf1) exhibits improved editing in HEK293T cells compared to its WT counterpart. The bars reflect the mean of n=3 independent replicates. The dots indicate the values ​​of individual replicates. All edits are PE edits, with the exception of the AAVS1 site, which is twinPE. rdTf1 = Tf1 RT K118R, S188K, I260L, S297Q, R288Q. Figure 1H shows that a rationally designed Ec48 triple mutant variant (rdEc48) exhibits improved editing for five edits in HEK293T cells compared to its WT counterpart. The bars reflect the mean of n=3 independent replicates. The dots indicate the values ​​of individual replicates. rdEc48 = Ec48 RT R315K, L182N, T189N. [Figure 1I-1J] Figure 1I shows a comparison of prime editors containing manipulated RT variants with PE2 in HEK293T cells. All values ​​from n=3 independent replicates are shown. Horizontal bars indicate the mean. All edits use single-flap prime editing, with the exception of the AAVS1 site which uses twinPE. Figure 1J shows a comparison of rdTf1 with PE2 and its WT counterpart in three longer, more complex PE (HEK3) or twinPE (CCR5 and IDS) edits in HEK293T cells. Bars reflect the mean of n=3 independent replicates. Dots indicate values ​​for individual replicates. [Figure 2A-2B]Development and validation of prime-edited PACE selection. Figure 2A is a schematic diagram of the PE PACE selection circuit. Infection of host E. coli cells with selection phage (SP, blue) causes NpuN intein and NpuC intein (pink) to mediate protein splicing to reconstitute the two halves (purple and pink) of the PE2 prime editor. The prime editor then engages with pegRNA (dark green) and corrects the frameshift of T7 RNAP (orange) via prime editing. The functional T7 RNAP then transcribes gIII (light green), which enables SP proliferation. Figure 2B shows evaluation of the v1 PE PACE circuit. Phage replication levels from overnight proliferation of empty phage (red), NpuC-PE2-RT phage (purple), and T7-RNAP phage (green) by host cells possessing the PE PACE circuit before pegRNA optimization. Bars reflect the mean of n=3 independent replicates. The dots indicate the values ​​of each iteration. [Figure 2C-2D] Figure 2C shows the screening of pegRNAs for the v1 PE PACE circuit. The values ​​for overnight growth of empty phage (red), NpuC-PE2-RT phage (purple), and T7-RNAP phage (green) are shown. Each dot reflects the mean of n=3 independent biological replicates for a different pegRNA. Individual replicates are shown in Figure 9C. Figure 2D shows the overnight growth of empty phage (red), NpuC-PE1-RT phage (light purple), NpuC-PE2-RT phage (dark purple), and T7-RNAP phage (green) in the v1 pegRNA-optimized circuit. The bars reflect the mean of n=3 independent replicates. The dots indicate the values ​​for individual replicates. [Figure 2E-2F]Figure 2E shows the PANCE titer in the evolution of the NpuC-PE1-RT phage. The gray shading indicates the passage of evolutionary drift. Here, the phage was supplied with gIII in the absence of selection, allowing for free mutagenerative replication. The titers of four repeat lagoons are shown. Figure 2F shows the mutation table for RT clones enriched during the PANCE of the NpuC-PE1-RT phage. Four clones were sequenced from each lagoon (L1-L4; clones are in lagoon order). Light purple indicates conserved mutations. Dark purple indicates conserved mutations that were also present in previously manipulated RTs on PE2. [Figure 2G-2H] Figure 2G shows a schematic diagram of PE-PACE selection for the evolution of a full-length prime editor containing the Cas9 domain. The P1 plasmid (green) and P3 plasmid (orange) are identical to those used in Figure 2A. Figure 2H shows a PANCE experiment to compare the outcomes of selection using v1 (requiring a 1 bp insertion) and v2 (requiring a 20 bp insertion) selection circuits. The full-length editor phage was divided into 16 separate lagoons. Eight lagoons were evolved by the v1 circuit (yellow), and eight lagoons were evolved by the v2 circuit (blue). After 31 passages, clones from each selection were Sanger-sequenced, and the resulting mutations were compared to generate Figures 2D-2F. From top to bottom, the sequences are sequence numbers 134-137. [Figure 2I-2J] Figure 2I shows a violin plot indicating the number of mutations per clone for the M-MLV domain of full-length editor phages evolved by either the v1 (yellow) or v2 (blue) circuit. Data are shown as individual values, with one dot representing one sequenced phage. The mean is shown by the dotted line. Figure 2J shows the predicted locations of mutant residues on the M-MLV from the v1 (yellow) or v2 (blue) PANCE. The structure is from the highly homologous XMRV (PDB:4HKQ). [Figure 2K]Figure 2K shows the overnight growth of a pool of wild-type and evolved RT phages in their corresponding or non-corresponding host cell selection lines. The phages were from PANCE via the v1 circuit (yellow bars), from PANCE via the v2 circuit (blue bars), or wild-type PE2 phage (gray bars). Growth was then measured in either the v1 circuit (left) or the v2 circuit (right). The bars reflect the mean of n=3 independent replicates. The dots indicate the values ​​of individual replicates. [Figure 3A] Phage-assisted evolution of compact RTs for prime editing. Figure 3A outlines evolutionary campaigns for NpuC-RT phages encoding Gs RT, Ec48 RT, or Tf1 RT. Shades indicate which selection circuit (v1 is yellow, v2 is blue, v3 is purple) was used. It is specified whether a given evolution was PANCE or PACE: the number in parentheses after the PANCE or PACE label indicates how many passages (p) of PANCE or how many hours (h) of PACE were performed. Arrowheads indicate that evolution was stopped and stringency was increased without mammalian characterization; variants characterized in mammalian cells are indicated and labeled with dots. Finally, evolutions using extra manipulation to increase stringency are labeled in pink and reflect either a change in PBS or a change in the expression of the target T7 RNAP gene. [Figure 3B-3D]Figure 3B shows the locations of residues on Gs RT (PDB: 6AR1) that are close to post-evolutionally mutated DNA and RNA substrates, mapped onto the structure of Gs RT (PDB: 6AR1). Mutated residues after PANCE in the v2 circuit are shown in red, mutated residues after PANCE and PACE in the v1 circuit are shown in blue, DNA substrates are shown in green, and RNA substrates are shown in yellow. Figure 3C shows the predicted locations of residues (E60, E279, and K318) on Ec48 RT that are close to post-PANCE mutated DNA and RNA substrates in the v1 and v2 circuits. The residues are mapped onto the AlphaFold predicted structure of Ec48 superimposed on the substrate of XMRV RT (PDB: 4HKQ). Mutated residues after PANCE in the v1 circuit are shown in blue, mutated residues after PANCE in the v2 circuit are shown in red, DNA substrates are shown in green, and RNA substrates are shown in yellow. Figure 3D shows the predicted locations of several conserved residues on Tf1 RT mutated after PANCE in the v1, v2, and v3 circuits. The residues are mapped onto the AlphaFold predicted structure of Tf1 RT superimposed with the substrate of Ty3 RT (PDB: 4OL8). Residues S492, K413, I128, and K118 are all predicted to be close to the substrate, while residues P70, G72, M102, and K106 adorn the surface of the enzyme, which may be important for its interaction with pegRNA RTT. Residues mutated after PANCE in the v1 circuit are shown in blue, residues mutated after PANCE in the v2 circuit are shown in red, and residues mutated after PANCE in the v3 circuit are shown in orange. DNA substrates are shown in green, and RNA substrates are shown in yellow. [Figure 3E] Figure 3E shows prime editing in HEK293T cells using prime editors containing wild-type (gray) Gs, Ec48, and Tf1 RTs, evolved Gs-RT (evoGs, green), evolved Ec48 RT (evoEc48, blue), and evolved Tf1 RT (evoTf1, yellow). Bars reflect the mean of n=3 independent replicates. Dots indicate the values ​​for individual replicates. In all figures, the shown prime editing efficiency reflects the frequency of the intended prime editing outcome without indels at the target site. [Figure 3F] Figure 3F shows a comparison of the prime editor of an optimized PEmax architecture containing one of the engineered quintuplet mutants Marathon RT (Marathon penta, red), evoEc48 (blue), or evoTf1 (yellow) in HEK293T cells with PEmax (gray). The bars reflect the mean of n=3 independent replicates. The dots indicate the values ​​of individual replicates. [Figure 3G] Figure 3G shows prime editing in primary human T cells at commonly edited test loci. The bars reflect the average of n=4 independent replicates. The dots indicate the values ​​of individual replicates. Indel-free edits are shown in blue or pink, while indel-containing edits are shown in gray. [Figure 3H] Figure 3H shows the modification of the HEXA 1278insTATC mutation causing Tay-Sachs disease in a previously engineered HEK293T cell line model (left) and patient-derived fibroblasts (right). The bars reflect the mean of n=3 independent repeats for HEK293T cells in the model. The bars reflect n=2 independent repeats for patient-derived fibroblasts. The dots indicate the values ​​of individual repeats. [Figure 4A-4B] An overview of the evolved prime editor preference and the RT evolution campaigns described herein. Figure 4A shows an overview of the evolutionary and manipulative campaigns used to generate PE6c and PE6d. Figure 4B shows conserved mutations from M-MLV RT evolution. The structure of XMRV RT (PDIB 4HKQ), which is highly homologous to M-MLV, shows that the PACE-evolved residues (blue) are located near the enzyme active site (dark gray) and the DNA / RNA double-stranded substrate (pink / purple). The incoming dNTPs are shown in yellow. Below, the pink line indicates the location on M-MLV RT where the PACE-evolved mutations shortened the protein. [Figure 4C-4D]Figure 4C shows the multiplier changes in editing efficiency relative to PEmax for PEmaxΔRNaseH, PE6c, and PE6d in HEK293T cells. Individual repeats are plotted. n=3 biological repeats per edit. Figure 4D shows the editing efficiency of PEmaxΔRNaseH and PE6d for HEK3 +1 loxP insertion edit (pink) and HEK3 +1 FLAG insertion edit (orange) in HEK293T cells. The bars reflect the average of n=3 independent repeats. The dots indicate the values ​​for individual repeats. NUPACK predicted structures of RTT and PBS extension for each edit are shown. [Figure 4E-4F] Figure 4E shows the results of a TdT assay by HEK3 +1 loxP insertion editing in HEK293T cells. The y-axis indicates the percentage of total RT products of a given length, and the x-axis indicates the length of the product in base pairs. PEmaxΔRNaseH is shown in gray, and PE6d is shown in blue. The lines are the mean values ​​from n=3 biological repeats. Pink boxes indicate the DNA bases by the template of the structured portion of the pegRNA. Figure 4F shows the editing efficiency of PEmaxΔRNaseH (gray) and PE6d (blue) in an example of manipulated hairpin editing in HEK293T cells and its corresponding pinless control. The sequences of the RTTs are shown (sequences 138 and 139 from top to bottom), and point mutations in the pinless control are shown in red. The bars reflect the mean of n=3 independent repeats. Dots indicate the values ​​of individual repeats. The NUPACK predicted structures of the RTT and PBS extensions for each edit are shown. [Figure 4G] Figure 4G shows the relationship between pegRNA RTT / PBS secondary structure and PE6d improvement. The y-axis reflects the fold improvement of PE6d compared to PEmaxΔRNaseH. The x-axis is the absolute value of the free energy of pegRNA folding as measured by NUPACK. Each dot represents one edit in HEK293T cells, calculated from the mean value from n=3 biological repeats. See Figure 11D for individual edit values ​​and edit identities. [Figure 4H-4J]Figure 4H shows a comparison of evolved and engineered RT to PEmaxΔRNaseH in typical twinPE editing in HEK293T cells. The bars reflect the average indel-free editing efficiency of n=3 independent replicates. The dots indicate the values ​​for individual replicates. Solid bars indicate editing efficiency. Striped bars indicate indels. Figure 4I shows a twinPE-mediated insertion of a 38bp attB sequence into the Rosa26 locus in N2a cells. Indel-free editing is shown in yellow, and indels are shown in gray. The bars reflect the average of n=3 independent replicates. The dots indicate the values ​​for individual replicates. Figure 4J shows a PE-mediated insertion of a 42bp sequence containing loxP into the Dnmt1 locus in N2a cells. Indel-free editing is shown in yellow, and indels are shown in gray. The bars reflect the average of n=3 independent replicates. The dots indicate the values ​​for individual replicates. [Figure 5A] Characterization of PE6 variants compared to PEmax. Figure 5A shows the prime editing efficiencies of PE6c, PE6d, and PEmax in difficult twin PE editing in HEK293T cells. Bars reflect the indel-free editing efficiencies of n=3 independent replicates. Dots indicate the values ​​for individual replicates. [Figure 5B] Figure 5B shows the edit-to-indel ratios of PE6c, PE6d, and PEmax at the site indicated in 5A in HEK293T cells. The bars reflect the mean of n=3 independent replicates. The dots indicate the values ​​of individual replicates. [Figure 5C-5D]Figure 5C shows twin-prime editing in primary human T cells at the CCR5 safe harbor locus. Indel-free edits are shown in red, and indel-containing edits are shown in gray. Bars reflect the average of n=4 independent replicates. Dots indicate the values ​​for individual replicates. Figure 5D shows the edit-to-indel ratio of PE6b and PEmaxΔRNaseH normalized to PEmax in HEK293 T cells. Individual replicates are plotted. There are n=3 biological replicates per edit. Lines reflect the average between all edits and replicates. Individual edit efficiencies and indel levels are shown in Figures 12D–12E. [Figure 5E] Figure 5E shows the edit-to-indel ratio of prime editors at the endogenous HEK293T site. Editors with the highest edit-to-indel ratio were selected and plotted alongside PEmax for each specific editor. The bars reflect the average of n=3 independent iterations. The dots indicate the values ​​for individual iterations. Individual edit efficiencies and indel levels are shown in Figures 12D–12E. [Figure 5F] Figure 5F shows the prime editing efficiencies of PE6b and PE6c, normalized to PEmax editing efficiency, for 77 edits that placed pathogenic alleles at endogenous sites in HEK293T cells. No nicking gRNA was used, and the MLH1dn plasmid was transfected concurrently with the prime editor plasmid for all conditions. All values ​​from n=3 repeats are shown. The line reflects the mean across all edits and repeats. Prime editing efficiencies for edits where PE6b or PE6c performed more than 1.5 times better than PEmax are shown on the right. The bars reflect the mean of n=3 independent repeats. The dots indicate the values ​​for individual repeats. The prime editing efficiency used is the frequency of the intended prime editing outcome, which does not involve indels or other changes at the target site. [Figure 5G]Figure 5G shows the modification of pathogenic mutations involved in Crigler-Nadjar syndrome, Bloom syndrome, and Pompe disease in HEK293T cell models using PEmax, PE6b, and PE6c. The bars reflect the mean of n=3 independent replicates. The dots indicate the values ​​of individual replicates. [Figure 5H] Figure 5H shows the correction of mutations associated with Crigler-Nadjar syndrome (UGT1A1) and Bloom syndrome (RECQL3) in patient-derived fibroblasts using PE6c and PEmax. Bars reflect the mean of n=3 independent replicates in the treated samples and n=1–3 replicates in the untreated control for edits (red) and indels (gray). Dots indicate the values ​​for individual replicates. [Figure 6A] An overview of the evolution and manipulation of the improved Cas9 domain for prime editing, as well as the use of PE6. Figure 6A shows an overview of the evolutionary campaign for the full-length PE2 phage. The color indicates which circuit (v1 is yellow, v2 is blue, v3 is purple) the evolution was carried out in. The green color indicates a reversion analysis. It is specified whether a given evolution was PANCE or PACE: the number in parentheses after the PANCE or PACE label indicates how many passages (p) of PANCE or how many hours (h) of PACE were carried out. Arrowheads indicate that the evolution was stopped and stringency was increased without mammalian characterization; mutants characterized in mammalian cells are shown and labeled with dots. Finally, evolutions utilizing extra manipulation to increase stringency are labeled in pink and reflect either a change in PBS or a change in the expression of the target T7 RNAP gene. [Figure 6B-6C]Figure 6B shows the evaluation of PACE-evolved clones in HEK293T cells. Bars reflect the mean of n=3 independent replicates. Dots indicate the values ​​of individual replicates. EvoCas9-1 to evoCas9-4 were isolated from low-stringency evolution. EvoCas9-5 and evoCas9-6 were isolated from high-stringency evolution. Figure 6C shows the evaluation of individual Cas9 mutations by prime editing efficiency at two test sites. The y-axis shows the editing efficiency for Pcsk9 +3 C→G and +6 G→C in N2a cells. The x-axis shows the editing efficiency for RNF2 +5 G→T editing in HEK293T cells. Mutants incorporated into the final Cas9 variant are shown in green. Mutants previously shown to reduce Cas9 binding or structurally predicted to do so are shown in chestnut brown. PEmaxΔRNaseH is shown in orange. [Figure 6D] Figure 6D shows a comparison of combined Cas9 variants against PEmaxΔRNaseH in HEK293T and N2a cells. The editing efficiency of the variants is normalized against the editing efficiency produced by PEmaxΔRNaseH. Individual repeats are plotted. There are n=3 biological repeats per edit. [Figure 6E-6F] Figure 6E shows a comparison of PEmax, PE6a, and PE6a / e at two sites in HEK293T cells. The bars reflect the mean of n=3 independent replicates. The dots indicate the values ​​of individual replicates. Figure 6F shows a comparison of PEmaxΔRNaseH, PE6c, and PE6g in HEK293T cells. The bars reflect the mean of n=3 independent replicates. The dots indicate the values ​​of individual replicates. [Figure 6G] Figure 6G shows a decision tree for selecting a PE6 variant. [Figure 7A]The PE6 variant enables a new class of in vivo prime editing. Figure 7A is a schematic diagram showing a dual AAV delivery system for twinPE (v3em twinPE-AAV). In the N-terminal AAV, the production of the N-terminal portion of Cas9 (yellow) fused to the N-terminal Npu split intein (orange) is regulated by the Cbh promoter (green) and the SV40 late polyA signal (yellowish-brown). In the C-terminal AAV, the C-terminal Npu split intein (dark green) is fused to the remainder of the prime editor (Cas9 yellow, RT purple). The SV40 late polyA signal (yellowish-brown), two epegRNAs (light and dark blue), and AAV ITR (black) are also shown. [Figure 7B] Figure 7B shows the injection routes and twinPE editing efficiencies of PEmaxΔRNaseH and PE6d viruses for twinPE-mediated insertion of a 38 bp attB sequence in mouse Rosa26 in the mouse cortex. N and C-terminal twinPE viruses are administered via ICV injection (total 4 × 10¹⁰ vg) in combination with GFP-KASH virus. Editing efficiencies (light and dark blue) and indel frequencies (black and gray) are shown on the right. Bars reflect the mean of n=3–4 mice. Dots represent individual mice. [Figure 7C] Figure 7C shows the injection routes and PE editing efficiencies of PEmaxΔRNaseH and PE6d viruses for the placement of a 42bp insertion containing loxP at the Dnmt1 locus in mouse cortex. (Left) The C-terminal virus is modified to contain one epegRNA and one nicking sgRNA to encode PE editing rather than twinPE editing. (Right) Editing efficiency (light / dark pink) and indel rate (black / gray). Bars reflect the average of n=3 mice. Dots indicate individual mice. [Figures 8A-8B]Characterization and manipulation of reverse transcriptases for prime editing related to Figures 1A–1J. Figure 8A shows that the small native RT enzyme demonstrates poor activity in the prime editing system (HEK293T cells, HEK3 + 5 G→T editing). Manipulated RT enzymes in Figures 1A–1J are highlighted in green, and the WT M-MLV RT used in the PE1 system is highlighted in black. All other enzymes are red. The dots reflect the average of n=3 independent repeats. Figure 8B shows an overview of twinPE. The prime editor protein (gray and blue) targets opposing strands of DNA using two pegRNAs (dark blue and blue-green). The prime editor generates two 3' flaps (red) that are complementary to each other. These newly synthesized 3' flaps anneal, and after the original DNA sequence on the 5' flap is degraded, the edited sequence on the flap is permanently placed at the target DNA site. [Figure 8C] Figure 8C shows that incorporating each of the five mutations similar to those on PE2 improves the activity of four retroviral RT enzymes in HEK293T cells: PERV = porcine endogenous retrovirus RT, AVIRE = avian reticuloendotheliosis virus RT, KORV = koala retrovirus RT, and WMSV = woolysal sarcoma virus RT. Combining all five mutations together (Penta) further improves the activity of each enzyme. All values ​​from n=3 independent replicates are shown. Horizontal bars indicate the mean value. [Figure 8D-8E]Figure 8D shows that a rational manipulation guided by the structure of Tf1 RT identifies five mutations that improve prime editing in HEK293T cells. The elucidated structure of the Tf1 RT homolog, Ty3 RT, was used to predict mutations that could increase RT contact with its DNA-RNA substrate (PDB:4OL8). All values ​​from n=3 independent repeats are shown. Horizontal bars indicate the mean values ​​across all sites and repeats. Figure 8E shows that combining all mutations identified from the structure-guided rational manipulation improves the activity of the Tf1 RT prime editor in HEK293T cells. The final rationally designed Tf1 variant (rdTf1) is a combination of five mutations: K118R, S188K, I260L, R288Q, and S297Q. All values ​​from n=3 independent repeats are shown. Horizontal bars indicate the mean values. [Figure 8F-8H] Figure 8F shows the AlphaFold predicted structure of the Ec48 RT enzyme. Figure 8G shows that aligning the AlphaFold predicted structure of Ec48 RT (blue) with the RT from a closely related heterotropic mouse leukemia virus-associated virus (XMRV, PDB 4HKQ, yellow) suggests that the T189 residue on Ec48 RT is similar to the D200 residue on M-MLV RT. Figure 8H shows that a rational operation guided by the structure of Ec48 RT identifies six mutations that improve prime editing. The AlphaFold predicted structure of Ec48 RT was superimposed with the structure of the RT from heterotropic mouse leukemia virus-associated virus (XMRV) (PDB:4HKQ) to perform structure-guided mutation introduction. All values ​​from n=3 independent repeats are shown. Horizontal bars indicate the mean value. [Figure 8I]Figure 8I shows the proximal residues (red) to the substrates mutated to improve the activity of the Ec48 RT prime editor. The residues are mapped onto the predicted AlphaFold structure of Ec48 RT aligned with the elucidated substrate of XMRV RT (PDB: 4HKQ). L182 and T385 are proximal to the DNA substrate (green), R315 and K307 are proximal to the RNA substrate (yellow), and R378 is proximal to both the DNA and RNA substrates. [Figure 8J] Figure 8J shows that combining the top three mutations identified from structure-guided manipulation improves the activity of the Ec48 RT prime editor in HEK293T cells. The final rationally designed Ec48 RT variant (rdEc48) contains three mutations: L182N, T189N, and R315K. All values ​​from n=3 independent replicates are shown. Horizontal bars indicate the mean value. [Figure 9A] Design and validation of the PE PACE circuit related to Figures 2A-2K. Figure 9A shows an overview of phage-assisted continuous evolution (PACE). Host E. coli (gray) carrying the relevant selection circuit plasmids (green, pink, and orange) and mutation plasmids (MP, black) continuously flow into a fixed-volume lagoon (left). The addition of arabinose induces the expression of the mutagenic gene on the MP. Selection phages (blue) carrying the NpuC-RT transgene (purple) infect E. coli and induce mutation. If the mutated RT is inactive (red, bottom / right), prime editing does not induce gIII expression and pIII production, and the phage cannot proliferate. These phages encoding inactive RT are washed out of the lagoon by the continuous flow. If the mutated RT is active (green, center), prime editing leads to pIII production, and the phage encoding that RT can proliferate faster than they are diluted from the lagoon. [Figure 9B]Figure 9B outlines phage-assisted discontinuous evolution (PANCE). The same principle shown in Figure 9A is used in PANCE, with the exception that periodic individual dilution steps are used instead of continuous flow to dilute the selection culture. A logarithmic metaphase culture of selection E. coli is infected with phages, and arabinose is added to induce mutagenesis (left). After overnight incubation, the culture is centrifuged to pellet the bacteria and allow isolation of growing phages from the supernatant (center). A small volume of the supernatant (typically a 1:50 dilution) is used to infect a new lagoon of the logarithmic metaphase selection strain (right). This process is repeated until the phage titer stabilizes (i.e., when the overnight phage growth is greater than or equal to the dilution). [Figure 9C] Figure 9C shows the effect of pegRNA optimization on PE2 phage proliferation. Overnight growth of empty phage (natural control, red), PE2 phage (purple), and T7 RNAP phage (positive control, green) in strains containing different PBS and RTT length pegRNAs. Bars reflect the mean of n=3 independent replicates. Dots indicate the values ​​of individual replicates. This data was used to generate Figure 2C. [Figure 9D] Figure 9D shows a luciferase assay for screening pegRNA for the v2 PE PACE circuit. Selection strains encoding luxAB, transcriptionally coupled to gIII, infected either empty phages (red) or PE2 phages (purple). Four hours after infection, OD600-normalized luminescence was measured as an indicator of circuit activation. Bars reflect the mean of n=3 independent replicates. Dots indicate the values ​​of individual replicates. Strains in which PE2 phages outperformed empty phages were used for v2 evolution. [Figure 9E]Figure 9E shows the overnight growth of wild-type RT and evolved RT phage pools in their corresponding or non-corresponding host cell selection lines. An additional evolved pool of phages is shown here, in addition to those provided in Figure 2K. The phages were from PANCE via the v1 circuit (yellow bars), from PANCE via the v2 circuit (blue bars), or wild-type PE2 phage (gray bars). Growth was then measured in either the v1 circuit (left) or the v2 circuit (right). The bars reflect the mean of n=3 independent replicates. The dots indicate the values ​​of individual replicates. [Figure 9F] Figure 9F shows the design of the v3 circuit, as well as improvements compared to the v1 and v2 designs. Long insert editing (20bp insert editing with 60bp RTT) was used to select a highly prosecutable, highly active prime editor. Unlike the v1 and v2 circuits, the v3 pegRNA (gray) targets the non-coding strand of the T7 RNAP; this shortens the time between prime editing and wild-type T7 RNAP production. In addition to the 20bp insert (green) required to restore the T7 RNAP frame, the v3 pegRNA also codes for seed editing (blue) and silent PAM editing (chestnut). This prevents subsequent ligation and nicking of the edited sequence. From top to bottom, the sequences are sequence numbers 140 and 141. [Figure 10A-10B] Figures 3A-3H show the evolution and characterization of compact RTs for prime editing. Figure 10A shows the overnight proliferation of phages encoding dead M-MLV RT (red), Gs (blue), or PE2 (purple) RT in the NpuC-RT phage architecture in the pegRNA-optimized v1 PE PACE circuit. Bars reflect the mean of n=3 independent replicates. Dots indicate the values ​​of individual replicates. Figure 10B shows the phage titers during PANCE for NpuC-Gs-RT phages. The gray shading indicates the passage of evolutionary drift. Here, the phages were fed gIII in the absence of selection, allowing for free mutagenerative replication. The titers of four replicate lagoons are shown. [Figure 10C-10D] Figure 10C shows the PACE of the NpuC-Gs-RT phage. The left y-axis and the pink and blue lines show the SP titers of three different repeat lagoons at various time points. The right y-axis and the gray dotted line show the flow rate in volume per hour. Figure 10D shows the indel frequencies in HEK293T cells for PEmax (gray) and prime editors of the optimized PEmax architecture containing either the engineered quintuplet mutant Marathon RT (Marathon penta, red), evoEc48 (blue), or evoTf1 (yellow). The corresponding edit frequencies are shown in Figure 3F. The bars reflect the mean of three independent repeats. The dots indicate the values ​​of individual repeats. [Figures 10E-10F] Figure 10E shows the performance of PE6a and PE6b in the presence and absence of epegRNA in HEK293T cells. All values ​​from n=3 independent replicates are shown. Horizontal bars indicate the mean. Figure 10F shows a comparison of PE6a, PE6b, and PEmax in three longer, more complex edits in HEK293T cells. The bars reflect the mean of n=3 independent replicates. Dots indicate the values ​​of individual replicates. [Figure 11A] Development and characterization of highly processy dual AAV-compatible RTs. Figure 11A shows the editing efficiency of a prime editor containing a single M-MLV mutant in HEK293T cells. The prime editing efficiency used is the frequency of the intended prime editing outcome, which does not involve indels or other changes at the target site. The line reflects the mean of n=2 independent replicates per edit. The dots indicate the values ​​for individual replicates. [Figure 11B-11C]Figure 11B shows an overview of the terminal deoxynucleotidyltransferase (TdT) assay for sequencing newly reverse-transcribed DNA flaps not integrated into the genome. Shortly after treatment with prime editor and pegRNA, cells are lysed and DNA is purified. The terminal transferase enzyme (yellow) adds poly-G sequences to all DNA 3' ends. PCR amplification for high-throughput DNA sequencing is performed using locus-specific forward primers and poly-C reverse primers. Figure 11C shows the results of the TdT assay with HEK3 +1 FLAG insertion editing in HEK293T cells. The y-axis indicates the percentage of total RT products of a given length, and the x-axis represents the length of the product in base pairs. PEmaxΔRNaseH is shown in gray, and PE6d is shown in blue. The line represents the mean value from n=3 biological replicates. [Figure 11D] Figure 11D shows the editing efficiencies of PE6b-d, PEmax, and PEmaxΔRNaseH for edits manipulated to contain various levels of secondary structure. "UC" indicates the pinless control compared to the corresponding hairpin edit. These values ​​were used to generate the free energy versus magnification improvement plot in Figure 4G. All edits were performed in HEK293T cells. Individual replicates are shown with n=3 replicates per condition. [Figure 11E] Figure 11E shows the editing efficiency (left) and indel rate (right) of PE6d and PEmaxΔRNaseH for a series of prime edits using short, unstructured pegRNAs in HEK293T cells. The bars reflect the mean of n=3 independent replicates. The dots indicate the values ​​for individual replicates. PEmaxΔRNaseH is shown on the right for each edit site, and PE6d is shown on the left for each edit site. [Figure 11F]Figure 11F shows the results of a TdT assay by RNF2 + 5 G→T editing in HEK293T cells. Note that the x-axis differs from other TdT plots shown herein: instead of bases from correctly placed RTT templates, it quantifies the number of bases from abnormally placed sgRNA backbone templates (e.g., x=1 indicates the addition of one extra base from a backbone template). The y-axis indicates the percentage of edit-containing flaps having a given number of bases from a backbone template. For each prime editor, the line reflects the mean of n=3 independent replicates. The pie chart indicates the percentage of edit-containing flaps having either ≤2 bp (solid color) or >2 bp (striped) bases from a backbone template. The data shown are the mean of three independent biological replicates. [Figure 11G] Figure 11G shows a unique molecular identifier (UMI) analysis of prime editing efficiency for twinPE editing in N2a cells (left) and HEK293T cells (center, right). The UMI protocol was applied to remove PCR bias. The trends are consistent with the data shown in Figures 4A–4J. The bars reflect the mean of n=3 independent replicates. The dots indicate the values ​​for individual replicates. For the "edit" and "indel" cases, PEmaxΔRNaseH is shown on the left, PE6c on the center, and PE6d on the right. [Figure 12A-12C]Figures 5A-5H show a comparison of PE6 variants with PEmax related to the relevant data. Figure 12A shows the prime editing efficiency of the best-performing PE6 variant (either PE6c or PE6d), normalized to the editing efficiency of PEmax at the site tested in Figure 5A. All values ​​from n=3 independent replicates are shown. Editing was performed in HEK293 T cells. Horizontal bars indicate the mean. Figure 12B shows the indel frequencies of PEmax, PE6c, and PE6d in the edits tested in Figure 5A. This data was used in Figure 5B. Bars reflect the mean of three independent replicates. Editing was performed in HEK293 T cells. Dots indicate the values ​​of individual replicates. Figure 12C shows screening of PE6 variants for attB insertion into the CCR5 locus in primary human T cells. Bars reflect the mean of n=4 independent replicates for edits (red) and indels (gray). Dots indicate the values ​​of individual replicates. [Figure 12D] Figure 12D shows the absolute prime editing efficiency of the PE6 variant, PEmaxΔRNaseH, and PEmax in HEK293T cells used in the data plots in Figures 5D–5E. The prime editing efficiency used is the frequency of the intended prime editing outcome, which does not involve indels or other changes at the target site. The bars reflect the mean of three independent replicates. The dots indicate the values ​​of individual replicates. [Figure 12E] Figure 12E shows the indel frequencies of the PE6 variant, PEmaxΔRNaseH, and PEmax in HEK293T cells used to plot the data in Figures 5D–5E. The bars reflect the mean of three independent replicates. The dots indicate the values ​​of individual replicates. [Figure 12F] Figure 12F shows the percentage of sequencing reads containing PE6 variants, PEmaxΔRNaseH, and PEmax-based pegRNA backbone insertions after prime editing in HEK293T cells. These reads contribute to the total indel frequency. Bars reflect the mean of n=3 independent replicates. Dots indicate the values ​​for individual replicates. [Figure 12G]Figure 12G shows prime editing efficiency for edits where PE6b or PE6c outperformed PEmax using nicking gRNA. Bars reflect the mean of n=3 independent replicates. Dots indicate the value for individual replicates. The prime editing efficiency used is the frequency of the intended prime editing outcome, which does not involve indels or other changes at the target site in HEK293T cells. [Figure 12H] Figure 12H shows the indel frequencies of PE6 variants and PEmax in HEK293T cells at the site shown in Figure 5F. The bars reflect the mean of n=3 independent replicates. The dots indicate the values ​​of individual replicates. [Figure 12I-12J] Figure 12I shows the correction of Pompe disease-associated mutations in patient-derived fibroblasts using PE6c and PEmax. Bars reflect the mean of n=3 independent repeats for editing (red) and indels (gray). Dots indicate the values ​​of individual repeats. Figure 12J shows the distribution of edit outcomes after correction of Pompe disease-associated pathogenic mutations in patient-derived fibroblasts using PE6c. Patients were heterozygous. Indel genotypes are shown. From top to bottom, the sequences are SEQ ID NOs: 142, 143, 144, 144, 142, 144, 31, 143, and 144. [Figure 13A] Evolution and manipulation of Cas9 mutants for PE related to Figures 6A-6G. Figure 13A shows a representative PACE campaign for the v1 circuit. Different colored lines represent lagoons of different replicates. PACE experiments with fewer than four lagoons shown experienced either cheating (probably activity-independent phage proliferation from rare gene III recombination onto SP) or washout (complete loss of viable phages) for one or more lagoons. The upper graph represents phage titer during the PACE experiment. The lower graph shows flow rate at the corresponding time. [Figure 13B-13C]Figure 13B shows the reversion analysis of EvoCas9-4 in HEK293T cells. Editing efficiency was normalized to values ​​obtained using PE2. Data are shown as individual data points for n=3 biological repeats and as the overall mean across the four sites tested. Figure 13C shows the structural analysis of mutations that impair mammalian prime editing activity. (Left) Structure of wild-type SpCas9 (gray) bound to its guide RNA (purple) and DNA substrate (yellow / orange) (PDB:4UN3). Residue K1151 is shown in dark pink. (Right) Structure of wild-type SpCas9 (gray) bound to its guide RNA (purple) and DNA substrate (orange) (PDB:4OO8). Wild-type residues K1003, K1014, and A1034 are shown in dark pink. [Figure 13D] Figure 13D shows a circuit for investigating the editing-independent effect of a prime editor on the PE PACE circuit. E. coli carries a modified T7 RNAP, a luxAB gene under the T7 promoter, and pegRNA used during selection. The prime editor variant is introduced onto the plasmid under the control of an arabinose-inducible promoter. After induction, OD-normalized luminescence was used for n=3 biological replicates to measure circuit switch-on. Bars reflect the mean of n=3 independent replicates. Dots indicate values ​​for individual replicates. [Figure 13E] Figure 13E shows the prime editing efficiency of N2a cells (left, Ctnnb1~Pcks9) and HEK293T cells (right, CXCR4~RNF2) used to generate the magnification changes reported in Figure 6D. Individual repeats are plotted. n=3 biological repeats per edit. [Figure 13F] Figure 13F shows the structure (PDB:4UN3) of Cas9 (gray) bound to the sgRNA (purple). The residue H721, which is mutated into Tyr during evolution, is shown by a green bar. The dotted lines indicate the predicted polar contacts between H721 and other atoms. [Figure 14A]Figures 7A-7C illustrate in vivo prime editing by PE6c and PE6d delivered via dual AAV. Figure 14A shows an analysis of the truncated PE6c variant. Edits (yellow) and indels (gray) are shown for the placement of the attB sequence at the mouse Rosa26 locus in N2a cells. The bars reflect the average of n=3 independent repeats. The dots indicate the value of individual repeats. The number below each variant indicates the number of DNA bases deleted from the C-terminal end of the Tf1 gene. [Figure 14B] Figure 14B shows a representative flowchart for the isolation of unsorted and sorted nuclei from mouse cortex. Left: Scatter plot of all events. Gate A was set up to collect nuclei. Center: Selection of single-nucleus droplets at Gate B. Right: FITC signaling was used to collect unsorted cells (Gate C) and transduced GFP-positive cells (Gate D). [Figure 14C] Figure 14C shows the twinPE editing efficiency of PEmaxΔRNaseH and PE6c viruses in mouse cortex. N and C-terminal twinPE viruses are administered via ICV injection (total 4 × 10¹⁰ vg) in combination with GFP-KASH virus. Editing efficiency (light and dark blue) and indel (black / gray) rates are shown on the right. Bars reflect the mean of n=3–4 mice. Dots indicate individual mice. [Figure 14D] Figure 14D shows the injection routes and PE editing (Dnmt1 loxP insertion) efficiencies of PEmaxΔRNaseH and PE6d viruses at low viral doses (total 2 × 10¹⁰ vg) in mouse cortex. (Left) The C-terminal virus is modified to contain one epegRNA and one nicking sgRNA to encode PE editing rather than twinPE editing. (Right) Editing efficiency (light / dark pink) and indel rate (black / gray). Bars reflect the mean of n=3 mice. Dots indicate individual mice. [Figure 14E]Figure 14E shows off-target edits from AAV-treated and untreated mice. Bars reflect the mean of n=3 mice. Dots indicate individual mice. Bulk (light pink) and transduced (dark pink) values ​​of PE6d were either less than 0.1% on average or not statistically significant from the untreated control (light gray). For both ns notes, p=0.08. Analysis was performed by unpaired t-tests with Welch correction. The y-axis indicates aggregated off-target edits and indels (see Methods for Calculation). OT6 could not be amplified. All treated samples are from high AAV dose conditions. [Figure 15A] A table of mutations from the evolution of v1 PE PA(N)CE Gs related to Figures 3A-3H. These are clonal mutations that emerged from evolution. Lagoon 2 cheated during PACE and was not sequenced. Silent mutations have been omitted for clarity. [Figure 15B-1] A table of mutations from the evolution of v1 PE PA(N)CE Gs related to Figures 3A-3H. These are clonal mutations that emerged from evolution. Lagoon 2 cheated during PACE and was not sequenced. Silent mutations have been omitted for clarity. [Figure 15B-2] A table of mutations from the evolution of v1 PE PA(N)CE Gs related to Figures 3A-3H. These are clonal mutations that emerged from evolution. Lagoon 2 cheated during PACE and was not sequenced. Silent mutations have been omitted for clarity. [Figure 16] Table of variations from the evolution of v1 PE PANCE Tf1 related to Figures 3A-3H. Clonal variations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 17-1] Table of variations from the evolution of v1 PE PANCE Ec48 related to Figures 3A-3H. These are clonal variations that emerged from evolution. Silent variations have been omitted for clarity. [Figure 17-2]Table of variations from the evolution of v1 PE PANCE Ec48 related to Figures 3A-3H. These are clonal variations that emerged from evolution. Silent variations have been omitted for clarity. [Figure 18] Table of variations from the evolution of v1 PE PANCE Vc95 related to Figures 3A-3H. Clonal variations that have emerged from evolution. Silent mutations have been omitted for clarity. [Figures 19A-19B] Variation table from the v1 full-length editor evolution related to Figures 4A-4G. Clonal mutations that appeared during evolution. Blue indicates amino acid changes, and orange indicates shortened mutations of either stop codons (*) or frameshifts (FS). Silent mutations have been omitted for clarity. [Figure 19C-1] Variation table from the v1 full-length editor evolution related to Figures 4A-4G. Clonal mutations that appeared during evolution. Blue indicates amino acid changes, and orange indicates shortened mutations of either stop codons (*) or frameshifts (FS). Silent mutations have been omitted for clarity. [Figure 19C-2] Variation table from the v1 full-length editor evolution related to Figures 4A-4G. Clonal mutations that appeared during evolution. Blue indicates amino acid changes, and orange indicates shortened mutations of either stop codons (*) or frameshifts (FS). Silent mutations have been omitted for clarity. [Figure 19D] Variation table from the v1 full-length editor evolution related to Figures 4A-4G. Clonal mutations that appeared during evolution. Blue indicates amino acid changes, and orange indicates shortened mutations of either stop codons (*) or frameshifts (FS). Silent mutations have been omitted for clarity. [Figure 19E] Variation table from the v1 full-length editor evolution related to Figures 4A-4G. Clonal mutations that appeared during evolution. Blue indicates amino acid changes, and orange indicates shortened mutations of either stop codons (*) or frameshifts (FS). Silent mutations have been omitted for clarity. [Figure 19F]Variation table from the v1 full-length editor evolution related to Figures 4A-4G. Clonal mutations that appeared during evolution. Blue indicates amino acid changes, and orange indicates shortened mutations of either stop codons (*) or frameshifts (FS). Silent mutations have been omitted for clarity. [Figure 19G-1] Variation table from the v1 full-length editor evolution related to Figures 4A-4G. Clonal mutations that appeared during evolution. Blue indicates amino acid changes, and orange indicates shortened mutations of either stop codons (*) or frameshifts (FS). Silent mutations have been omitted for clarity. [Figure 19G-2] Variation table from the v1 full-length editor evolution related to Figures 4A-4G. Clonal mutations that appeared during evolution. Blue indicates amino acid changes, and orange indicates shortened mutations of either stop codons (*) or frameshifts (FS). Silent mutations have been omitted for clarity. [Figure 19H-1] Variation table from the v1 full-length editor evolution related to Figures 4A-4G. Clonal mutations that appeared during evolution. Blue indicates amino acid changes, and orange indicates shortened mutations of either stop codons (*) or frameshifts (FS). Silent mutations have been omitted for clarity. [Figure 19H-2] Variation table from the v1 full-length editor evolution related to Figures 4A-4G. Clonal mutations that appeared during evolution. Blue indicates amino acid changes, and orange indicates shortened mutations of either stop codons (*) or frameshifts (FS). Silent mutations have been omitted for clarity. [Figure 19I-19J] Variation table from the v1 full-length editor evolution related to Figures 4A-4G. Clonal mutations that appeared during evolution. Blue indicates amino acid changes, and orange indicates shortened mutations of either stop codons (*) or frameshifts (FS). Silent mutations have been omitted for clarity. [Figure 20-1]Variation tables from PANCE full-length editor evolution comparing v1 and v2, related to Figures 4A-4G. Clonal variation arising from evolution. Variations from v1 are shown in yellow, and variations from v2 evolution are shown in blue. Silent variation is omitted for clarity. Frameshift variation is not shown. [Figure 20-2] Variation tables from PANCE full-length editor evolution comparing v1 and v2, related to Figures 4A-4G. Clonal variation arising from evolution. Variations from v1 are shown in yellow, and variations from v2 evolution are shown in blue. Silent variation is omitted for clarity. Frameshift variation is not shown. [Figure 20-3] Variation tables from PANCE full-length editor evolution comparing v1 and v2, related to Figures 4A-4G. Clonal variation arising from evolution. Variations from v1 are shown in yellow, and variations from v2 evolution are shown in blue. Silent variation is omitted for clarity. Frameshift variation is not shown. [Figure 20-4] Variation tables from PANCE full-length editor evolution comparing v1 and v2, related to Figures 4A-4G. Clonal variation arising from evolution. Variations from v1 are shown in yellow, and variations from v2 evolution are shown in blue. Silent variation is omitted for clarity. Frameshift variation is not shown. [Figure 20-5] Variation tables from PANCE full-length editor evolution comparing v1 and v2, related to Figures 4A-4G. Clonal variation arising from evolution. Variations from v1 are shown in yellow, and variations from v2 evolution are shown in blue. Silent variation is omitted for clarity. Frameshift variation is not shown. [Figure 20-6] Variation tables from PANCE full-length editor evolution comparing v1 and v2, related to Figures 4A-4G. Clonal variation arising from evolution. Variations from v1 are shown in yellow, and variations from v2 evolution are shown in blue. Silent variation is omitted for clarity. Frameshift variation is not shown. [Figure 21]Table of variations from the evolution of v2 PE PANCE Gs related to Figures 4A-4G. These are clonal variations that emerged from evolution. Silent variations have been omitted for clarity. [Figure 22] Table of variations from the evolution of v2 PE PANCE Ec48 related to Figures 4A-4G. These are clonal variations that emerged from evolution. Silent variations have been omitted for clarity. [Figure 23] Table of variations from the evolution of v2 high-stringency PE PANCE Ec48 related to Figures 4A-4G. Clonal variations that emerged from evolution. Silent variations have been omitted for clarity. [Figure 24] Table of variations from the evolution of v2 PE PANCE Tf1 related to Figures 4A-4G. Clonal variations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 25-1] Figures 4A-4G show a table of mutations from the evolution of v3 PE PANCE Tf1. These are clonal mutations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 25-2] Figures 4A-4G show a table of mutations from the evolution of v3 PE PANCE Tf1. These are clonal mutations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 25-3] Figures 4A-4G show a table of mutations from the evolution of v3 PE PANCE Tf1. These are clonal mutations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 25-4] Figures 4A-4G show a table of mutations from the evolution of v3 PE PANCE Tf1. These are clonal mutations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 25-5] Figures 4A-4G show a table of mutations from the evolution of v3 PE PANCE Tf1. These are clonal mutations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 25-6]Figures 4A-4G show a table of mutations from the evolution of v3 PE PANCE Tf1. These are clonal mutations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 26] Figures 4A-4G show a table of mutations from the evolution of v1 PE PACE PE2 RT. These are clonal mutations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 27-1] Figures 4A-4G show a table of mutations from the evolution of v2 PE PANCE PE2 RT. These are clonal mutations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 27-2] Figures 4A-4G show a table of mutations from the evolution of v2 PE PANCE PE2 RT. These are clonal mutations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 27-3] Figures 4A-4G show a table of mutations from the evolution of v2 PE PANCE PE2 RT. These are clonal mutations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 27-4] Figures 4A-4G show a table of mutations from the evolution of v2 PE PANCE PE2 RT. These are clonal mutations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 28A] Figures 4A-4G show a table of mutations from the evolution of v3 PE PANCE PE2 RT. These are clonal mutations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 28B] Figures 4A-4G show a table of mutations from the evolution of v3 PE PANCE PE2 RT. These are clonal mutations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 28C] Figures 4A-4G show a table of mutations from the evolution of v3 PE PANCE PE2 RT. These are clonal mutations that emerged from evolution. Silent mutations have been omitted for clarity. [Figure 29A-1]Variation table from the full-length editor evolution of v1-v3 related to Figures 6A-6F. Clonal variation that emerged from evolution. Silent variation has been omitted for clarity. [Figure 29A-2] Variation table from the full-length editor evolution of v1-v3 related to Figures 6A-6F. Clonal variation that emerged from evolution. Silent variation has been omitted for clarity. [Figure 29B] Variation table from the full-length editor evolution of v1-v3 related to Figures 6A-6F. Clonal variation that emerged from evolution. Silent variation has been omitted for clarity. [Figure 29C-1] Variation table from the full-length editor evolution of v1-v3 related to Figures 6A-6F. Clonal variation that emerged from evolution. Silent variation has been omitted for clarity. [Figure 29C-2] Variation table from the full-length editor evolution of v1-v3 related to Figures 6A-6F. Clonal variation that emerged from evolution. Silent variation has been omitted for clarity. [Figure 29D] Variation table from the full-length editor evolution of v1-v3 related to Figures 6A-6F. Clonal variation that emerged from evolution. Silent variation has been omitted for clarity. [Figure 29E] Variation table from the full-length editor evolution of v1-v3 related to Figures 6A-6F. Clonal variation that emerged from evolution. Silent variation has been omitted for clarity. [Figure 29F] Variation table from the full-length editor evolution of v1-v3 related to Figures 6A-6F. Clonal variation that emerged from evolution. Silent variation has been omitted for clarity. [Figure 30] Figure 30 shows the delivery of PEmaxΔRNaseH or PE6 variants to the CNS of 12 mice via neonatal ventricle (P0 ICV) injection (n=4 for each of the three groups). [Figure 31] Figure 31 shows in vivo liver editing after ICV injection. The PE6 editor substantially enhances liver editing (30% editing in the liver after ICV injection). [Modes for carrying out the invention]

[0039] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by practitioners of the art to which this invention pertains. The following references provide to those skilled in the art many common definitions of terms used herein: 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). The following terms used herein have the meanings attributed to them unless otherwise specified.

[0040] Adeno-associated virus (AAV) Adeno-associated virus, or AAV, is a virus that infects humans and several other primate species. The wild-type AAV genome is a single-stranded deoxyribonucleic acid (ssDNA) that is either positive or negative sense. The genome contains two inverted end repeats (ITRs), one at each end of the DNA strand, as well as two open reading frames (ORFs) between the ITRs: rep and cap. The rep ORF contains four overlapping genes encoding the Rep protein required for the AAV lifecycle. The cap ORF contains overlapping genes encoding the capsid proteins: VP1, VP2, and VP3, which interact together to form the viral capsid. VP1, VP2, and VP3 are translated from a single mRNA transcript, which can be spliced ​​in two different ways: either a longer or shorter intron is excised, resulting in the formation of two mRNA isoforms: approximately 2.3 kb and approximately 2.6 kb in length. The capsid forms a supramolecular assembly of approximately 60 individual capsid protein subunits into a non-enveloped T-1 icosahedral lattice capable of protecting the AAV genome. The mature capsid is composed of VP1, VP2, and VP3 (with molecular masses of approximately 87, 73, and 62 kDa, respectively) in a ratio of approximately 1:1:10.

[0041] Recombinant AAV (rAAV) particles may comprise a nucleic acid vector (e.g., a recombinant genome) which may comprise at least: (a) one or more heterogeneous nucleic acid regions containing a sequence encoding a target protein or polypeptide (e.g., a split-prime editor) or a target RNA (e.g., gRNA), or one or more nucleic acid regions containing a sequence encoding a Rep protein; and (b) one or more regions containing inverted end repeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITR sequences) that flank one or more nucleic acid regions (e.g., heterogeneous nucleic acid regions). In some embodiments, the nucleic acid vector is between 4kb and 5kb in size (e.g., 4.2–4.7kb). In some embodiments, the nucleic acid vector further comprises a region encoding a Rep protein. In some embodiments, the nucleic acid vector is circular. In some embodiments, the nucleic acid vector is single-stranded. In some embodiments, the nucleic acid vector is double-stranded. In some embodiments, a double-stranded nucleic acid vector may be a self-complementary vector containing, for example, a region of the nucleic acid vector that is complementary to another region of the nucleic acid vector that initiates the formation of the double-stranded state of the nucleic acid vector.

[0042] In some embodiments, AAV is used to deliver any of the following: reverse transcriptase variants, Cas9 variants, fusion proteins, prime editors, and / or polynucleotides or vectors encoding them.

[0043] Cas9 The term “Cas9” or “Cas9 nuclease” refers to a nuclease guided by RNA containing a Cas9 domain or fragment thereof (e.g., a protein containing the active or inactive DNA cleavage domain of Cas9 and / or the gRNA binding domain of Cas9). As used herein, “Cas9 domain” refers to a protein fragment containing the active or completely or partially inactive cleavage domain of Cas9 and / or the gRNA binding domain of Cas9. “Cas9 protein” refers to the full-length Cas9 protein. Cas9 nucleases may also refer to cason1 nucleases or CRISPR( C stestered R egularly I nterspaced S hort P alindromic RAlso known as epeat-associated nucleases. CRISPR is an adaptive immune system that provides protection from mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain sequences, spacers, that are complementary to the ancestral mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, the correct processing of precrRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 domain. The tracrRNA acts as a guide for the processing of precrRNA, assisted by ribonuclease 3. Subsequently, Cas9 / crRNA / tracrRNA endo-cleaves linear or circular dsDNA targets complementary to the spacers. Strands on target DNA that are not complementary to the crRNA are first endo-cut and then 3'-5' exo-trimmed. In nature, DNA binding and cleavage typically require proteins and both RNAs. However, single guide RNAs ("sgRNAs," or simply "gRNAs") can be manipulated to incorporate aspects of both crRNAs and tracrRNAs into a single RNA species. See, for example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012). The entire content of this is incorporated herein by reference. Cas9 recognizes short motifs (PAMs or protospacer-adjacent motifs) on CRISPR repeat sequences to help distinguish between self and non-self.Cas9 nuclease sequences and structures are well known to those skilled in the art (e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc.Natl.Acad.Sci.USA98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma CM, Gonzales K., Chao See Y., Pirzada ZA, Eckert MR, 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 JA, Charpentier E. Science 337:816-821 (2012). The contents of each of these are incorporated herein by reference. Cas9 orthologs have been described in various species, including but not limited to S. pyogenes and S. thermophilus. Additional preferred Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure.Such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immune systems” (2013) RNA Biology 10:5, 726-737; the entire contents of this are incorporated herein by reference. In some embodiments, the Cas9 nucleases include one or more mutations that partially impair or inactivate the DNA cleavage domain.

[0044] A Cas9 domain with an inactivated nuclease can be interchangeably referred to as the “dCas9” protein (from the nuclease “dead” Cas9). Methods for generating Cas9 domains (or fragments thereof) with 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 these are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the strand non-complementary. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, 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, proteins containing fragments of the Cas9 protein are provided. For example, in some embodiments, the protein contains one of two Cas9 domains: (1) the gRNA-binding domain of Cas9; or (2) the DNA-cleaving domain of Cas9. In some embodiments, proteins containing Cas9 or its fragments are referred to as "Cas9 variants." Cas9 variants share homology to Cas9 or its fragments.For example, a Cas9 variant is at least approximately 70% identical, at least approximately 80% identical, at least approximately 90% identical, at least approximately 95% identical, at least approximately 96% identical, at least approximately 97% identical, at least approximately 98% identical, at least approximately 99% identical, at least approximately 99.5% identical, at least approximately 99.8% identical, or at least approximately 99.9% identical to a wild-type Cas9 (e.g., SpCas9 with number 6). In some embodiments, Cas9 variants 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 of SEQ ID NO: 6). In some embodiments, the Cas9 variant includes a fragment of Cas9 of SEQ ID NO: 6 (e.g., a gRNA-binding domain or a DNA-cleaving domain) such that the fragment is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 6). 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%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of the corresponding wild-type Cas9 (e.g., SpCas9 of SEQ ID NO: 6).

[0045] In some embodiments, the Cas9 protein comprises any of the amino acid substitutions described herein. In certain embodiments, the Cas9 protein comprises the amino acid substitutions K775R and K918A relative to wild-type Streptococcus pyogenes Cas9 or Streptococcus pyogenes Cas9 nickase (SEQ ID NO: 2). In certain embodiments, the Cas9 protein comprises the amino acid substitutions H99R, E471K, I632V, D645N, R654C, and H721Y relative to wild-type Streptococcus pyogenes Cas9 or Streptococcus pyogenes Cas9 nickase (SEQ ID NO: 2). In certain embodiments, the Cas9 protein comprises the amino acid substitutions H99R, E471K, I632V, D645N, H721Y, and K918A relative to wild-type Streptococcus pyogenes Cas9 or Streptococcus pyogenes Cas9 nickase (SEQ ID NO: 2).

[0046] CRISPR CRISPR is a family of DNA sequences (i.e., CRISPR clusters) in bacteria and archaea that represent snippets of preceding infections caused by viruses that have invaded prokaryotes. These DNA snippets are used by prokaryotic cells to detect and destroy DNA from subsequent attacks by similar viruses, and together with various CRISPR-related proteins (including Cas9 and its homologs) and CRISPR-related RNAs, they effectively constitute the prokaryotic immune defense system. In nature, CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In certain types of CRISPR systems (e.g., type II CRISPR systems), the correct processing of precrRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. tracrRNA acts as a guide for the processing of precrRNA, assisted by ribonuclease 3. Subsequently, Cas9 / crRNA / tracrRNA endo-cleaves linear or circular dsDNA targets complementary to the RNA. Specifically, DNA strands on targets not complementary to crRNA are first endo-cut and then 3'-5' exo-trimmed. In nature, DNA binding and cleavage typically require both proteins and both RNAs. However, a single guide RNA ("sgRNA" or simply "gRNA") can be manipulated to incorporate both aspects of crRNA and tracrRNA into a single RNA species, the guide RNA. See, for example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012). The entire content of this is incorporated here by reference. Cas9 recognizes short motifs (PAM or protospacer adjacency motifs) on CRISPR repeat sequences to help distinguish between self and non-self.CRISPR biology and Cas9 nuclease sequences and structures are well known to those skilled in the art (e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc.Natl.Acad.Sci.USA98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma CM, Gonzales See K., Chao Y., Pirzada ZA, Eckert MR, 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 JA, Charpentier E. Science 337:816-821 (2012). The contents of each of these are incorporated herein by reference. Cas9 orthologs have been described in various species, including but not limited to S. pyogenes and S. thermophilus. Additional preferred Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure.Such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immune systems” (2013) RNA Biology 10:5, 726-737; the entire contents of this document are incorporated herein by reference.

[0047] Generally, the “CRISPR system” refers collectively to the transcripts and other elements involved in or driving the expression of CRISPR-related (“Cas”) genes, and includes the sequence encoding the Cas gene, the tracr (trans-activated CRISPR) sequence (e.g., tracrRNA or active partial tracrRNA), the tracr mate sequence (which, in the context of the endogenous CRISPR system, encompasses “direct repeats” and tracrRNA-processed partial direct repeats), the guide sequence (also called a “spacer”), or other sequences and transcripts from the CRISPR locus. The tracrRNA of the system is (fully or partially) complementary to the tracr mate sequence present on the guide RNA.

[0048] Edited chains and unedited chains The terms “edited strand” and “unedited strand” are terms that may be used when describing the mechanism of a prime editing system on a double-stranded DNA substrate. “Edited strand” refers to the strand of DNA that is nicked by the prime-editor complex to form the 3' end. This is then extended as a newly synthesized single-stranded DNA (also referred herein to as a newly synthesized 3' DNA flap). This contains the desired edit, ultimately displacing and replacing a single-stranded region of DNA immediately downstream of the nick, thereby establishing a 3' DNA flap containing the desired edit downstream of the nick on the “edited strand.” In some embodiments, the newly synthesized 3' DNA flap containing the nucleotide edit pairs with an unedited strand that does not contain the nucleotide edit on the heteroduplex, thereby creating a mismatch. In some embodiments, the mismatch is recognized by DNA repair and / or replication mechanisms, such as endogenous DNA repair mechanisms. In some embodiments, through DNA repair, the intended nucleotide edit is incorporated into both strands of the target double-stranded DNA substrate. The present invention may also refer to the “edited strand” as the “protospacer strand” or “PAM strand” because these elements are present on the strand. The “edited strand” may also be called the “non-target strand” because the edited strand is not the strand that anneals to the spacer of the pegRNA molecule, but rather the complement of the strand that anneals to the spacer of the pegRNA. The “non-edited” strand is not directly edited by the PE system. Rather, the desired edits made by the PE system on the 3' DNA flap are incorporated into the “non-edited strand” through DNA replication and / or repair. In some embodiments, the “non-edited strand” is the strand that anneals to the spacer of the pegRNA and is therefore also called the “target strand.”

[0049] fusion protein As used herein, the term “fusion protein” means a hybrid polypeptide comprising protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion or the carboxy-terminal (C-terminal) portion of the fusion protein, thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. The protein may include different domains, e.g., a nucleic acid programmed DNA-binding domain (e.g., the gRNA-binding domain of Cas9 that guides the binding of the protein to a target site) and a reverse transcriptase (i.e., a prime editor). In some embodiments, the fusion protein comprises one of the reverse transcriptase variants provided herein fused to at least one other domain (e.g., the Cas9 protein disclosed herein (e.g., the Cas9 variants provided herein), NLS, or any other domain). In some embodiments, the fusion protein comprises one of the Cas9 variants provided herein fused to at least one other domain (e.g., the reverse transcriptase disclosed herein (e.g., the reverse transcriptase variants provided herein), NLS, or any other domain). In certain embodiments, the fusion protein comprises one of the reverse transcriptase variants provided herein and one of the Cas9 variants provided herein. Any of the fusion proteins provided herein may be produced by any method known in the art. For example, the prime editor fusion protein provided herein may be produced via recombinant protein expression and purification. This is particularly suitable for fusion proteins containing a peptide linker. Methods for recombinant protein expression and purification are well known and are incorporated herein by reference in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th This includes what is described in ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012).

[0050] Genetic elements of AAV particle vectors The nucleic acids of this disclosure (e.g., nucleic acids delivered by AAV particles described herein) may comprise one or more gene elements. A “gene element” means a specific nucleotide sequence that has a role in nucleic acid expression (e.g., a promoter, enhancer, or terminator) or codes for a specific product of the engineered nucleic acid (e.g., a nucleotide sequence that codes for guide RNA and / or protein).

[0051] A “promoter” refers to a regulatory region of a nucleic acid sequence in which the initiation and rate of transcription of the rest of the nucleic acid sequence are controlled. A promoter may also contain subregions to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. A promoter may be constitutive, inducible, activatable, repressible, tissue-specific, or a combination thereof. A promoter drives the expression or transcription of the nucleic acid sequence it regulates. In this specification, a promoter is considered “operatably linked” when it is in the correct functional location and orientation in relation to the nucleic acid sequence it regulates in order to control (“drive”) the transcription initiation and / or expression of that sequence.

[0052] A promoter may be naturally associated with a gene or sequence, and can be obtained by isolating a 5' non-coding sequence located upstream of the coding fragment of a given gene or sequence. Such a promoter is called an “endogenous promoter.” In some embodiments, coding nucleic acid sequences may be under the control of recombinant or heterologous promoters. These refer to promoters that are not normally associated with the sequence they encode in their natural environment. Such promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that do not “occur naturally,” such as those containing mutations that modulate different elements and / or expression of different transcriptional regulatory regions through genetic engineering methods known in the art. In addition to the synthetic production of promoter and enhancer nucleic acid sequences, sequences may be produced using nucleic acid amplification technologies, including recombinant cloning and / or polymerase chain reaction (PCR).

[0053] In some embodiments, the promoters used pursuant to this disclosure are “inducible promoters.” These are promoters characterized by modulating (e.g., initiating or activating) transcriptional activity in the presence of, being influenced by, or being in contact with an inducible signal. An inducible signal may be an endogenous or normally exogenous condition (e.g., light), a compound (e.g., a drug or non-drug compound), or a protein that comes into contact with the inducible promoter in such a way that it is active in modulating transcriptional activity from the inducible promoter. Thus, a “transcriptional modulating signal” of a nucleic acid means an inducible signal that acts on an inducible promoter. A transcriptional modulating signal may activate or inactivate transcription, depending on the regulatory system used. Activation of transcription may involve acting directly on the promoter to drive transcription, or indirectly on the promoter by inactivating a repressor that prevents the promoter from driving transcription. Conversely, deactivation of transcription may involve acting directly on the promoter to prevent transcription, or indirectly on the promoter by activating a repressor, which then acts on the promoter.

[0054] A "transcriptional terminator" is a nucleic acid sequence that causes transcription to stop. Transcriptional terminators can be unidirectional or bidirectional. They consist of DNA sequences involved in the specific termination of RNA transcripts by RNA polymerase. Transcriptional terminator sequences prevent transcriptional activation of downstream nucleic acid sequences by upstream promoters. Transcriptional terminators may be necessary in vivo to achieve desired expression levels or to avoid transcription of certain sequences. A transcriptional terminator is considered "operably ligated" to a nucleotide sequence if it can terminate the transcription of the sequence to which it is ligated.

[0055] The most commonly used type of terminator is the forward terminator. When placed downstream of the nucleic acid sequence to be transcribed, a forward transcription terminator will cause transcription to terminate. In some embodiments, bidirectional transcription terminators are provided, which typically cause transcription to terminate on both the forward and reverse strands. In some embodiments, reverse transcription terminators are provided, which typically terminate transcription only on the reverse strand.

[0056] In prokaryotic systems, terminators are typically divided into two categories: (1) Rho-independent terminators and (2) Rho-dependent terminators. Rho-independent terminators generally consist of palindrome sequences that form a stem-loop rich in GC base pairs followed by several T bases. Although not constrained by theory, conventional models of transcription termination posit that the stem-loop causes RNA polymerase to pause, and transcription of the poly(A) tail causes the RNA:DNA double helix to unwind and dissociate from RNA polymerase.

[0057] In eukaryotic systems, the terminator region may contain a specific DNA sequence that allows site-specific cleavage of a new transcript by exposing a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch (poly-A) of approximately 200 A residues to the 3' end of the transcript. RNA molecules modified by this poly-A tail appear more stable and are translated more efficiently. Therefore, in some embodiments involving eukaryotes, the terminator may contain a signal for RNA cleavage. In some embodiments, the terminator signal promotes polyadenylation of the message. The terminator and / or polyadenylation site elements may serve to enhance output nucleic acid levels and / or minimize inter-nucleic acid read-through.

[0058] Terminators for use pursuant to this disclosure include any transcription terminators described herein or known to those skilled in the art. Examples of terminators include, without limitation, gene termination sequences such as the bovine growth hormone terminator, as well as viral termination sequences such as the SV40 terminator, spy, yejM, secG-leuU, thrLABC, rrnB T1, hisLGDCBHAFI, metZWV, rrnC, xapR, aspA, and arcA terminators. In some embodiments, the termination signal may be an untranscribeable or untranslatable sequence, such as one resulting from sequence shortening.

[0059] Linker As used herein, the term "linker" refers to a molecule that connects two other molecules or parts. In the case of a peptide linker that connects two domains of a fusion protein, the linker may be an amino acid sequence. For example, napDNAbp (e.g., Cas9) can be fused to a reverse transcriptase by an amino acid linker sequence. The linker may also be a nucleotide sequence in the case of connecting two nucleotide sequences together (e.g., on gRNA). For example, in this case, a conventional guide RNA is linked to the RNA extension of a prime editing guide RNA, which may contain an RT template sequence and an RT primer binding site, via a spacer or linker nucleotide sequence. In other embodiments, the linker may be an organic molecule, a group, a polymer, or a chemical part. In some embodiments, the linker is 5 to 200 amino acids long, 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 long. Longer or shorter linkers are also conceived.

[0060] napDNAbp As used herein, the term “nucleic acid-programmed DNA-binding protein” or “napDNAbp” refers to a protein that uses RNA:DNA hybridization to target and bind to a specific sequence on a DNA molecule, with Cas9 being an example. Each napDNAbp is associated with at least one guide nucleic acid (e.g., guide RNA). This localizes the napDNAbp to a DNA sequence containing a DNA strand (i.e., the target strand) that is complementary to the guide nucleic acid or a portion of it (e.g., a protospacer of the guide RNA). In other words, the guide nucleic acid “programs” the napDNAbp (e.g., Cas9 or its equivalent) to localize and bind to a complementary sequence.

[0061] While not constrained by theory, the binding mechanism of the napDNAbp-guide RNA complex generally involves a step of forming an R-loop. This induces the napDNAbp to unwind the double-stranded DNA target, thereby separating the strands on the region bound by the napDNAbp. The guide RNA protospacer then hybridizes with the "target strand," which replaces the "non-target strand" complementary to the target strand, forming the single-stranded region of the R-loop. In some embodiments, the napDNAbp contains one or more nuclease activities that then cut the DNA, leaving various types of damage. For example, the napDNAbp may contain nuclease activities that cut the non-target strand at a first location and / or the target strand at a second location. Depending on the nuclease activity, the target DNA may be cut to form a "double-strand break," thereby cutting both strands. In other embodiments, the target DNA may be cut at only a single site; that is, the DNA is "nicked" on one strand. Exemplary napDNAbp having different nuclease activities include “Cas9 nickase” (“nCas9”) and deactivated Cas9 (“dead Cas9” or “dCas9”) that lacks nuclease activity. Exemplary sequences of these and other napDNAbp are provided herein. In some embodiments, napDNAbp have nickase activity in the RuvC domain and / or the HNH domain. In some embodiments, napDNAbp have nickase activity in either the RuvC domain or the HNH domain.

[0062] Nikkaze As used herein, "nickase" refers to a napDNAbp (e.g., Cas protein) capable of cleaving only one of the two complementary strands of a double-stranded target DNA sequence, thereby generating a nick on that strand. In some embodiments, the nickase cleaves the non-target strand of the double-stranded target DNA sequence. In some embodiments, the nickase comprises an amino acid sequence having one or more mutations on the catalytic domain of a classical napDNAbp (e.g., Cas protein), where one or more mutations reduce or eliminate the nuclease activity of the catalytic domain. In some embodiments, the nickase is Cas9 having one or more mutations on the RuvC-like domain relative to the wild-type Cas9 sequence or to the equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, the nickase is Cas9 having one or more mutations on the HNH-like domain relative to the wild-type Cas9 sequence or to the equivalent amino acid position in other Cas9 variants or Cas9 equivalents. In some embodiments, nickase is Cas9 containing an aspartic acid-to-alanine substitution (D10A) on the RuvC I catalytic domain of Cas9, relative to the classical Cas9 sequence or equivalent amino acid positions in other Cas9 variants or Cas9 equivalents. In some embodiments, nickase is Cas9 containing H840A, N854A, and / or N863A mutations, relative to the classical SpCas9 sequence or equivalent amino acid positions in other Cas9 variants or Cas9 equivalents. In some embodiments, the term “Cas9 nickase” refers to Cas9 in which one of the two nuclease domains is inactivated. This enzyme can cleave only one strand of target DNA. In some embodiments, nickase is a Cas protein that is not Cas9 nickase.

[0063] In some embodiments, the napDNAbp of the prime editing complex comprises an endonuclease having nucleic acid-programmed DNA-binding ability. In some embodiments, napDNAbp comprises an active endonuclease capable of cleaving both strands of a double-stranded target DNA. In some embodiments, napDNAbp is a nuclease-active endonuclease, e.g., a nuclease-active Cas protein, capable of cleaving both strands of a double-stranded target DNA by generating nicks on each strand. For example, a nuclease-active Cas protein can generate cleavage (nicks) on each strand of a double-stranded target DNA. In some embodiments, the two nicks on both strands are staggered and are generated by a napDNAbp containing, for example, Cas12a or Cas12b1. In some embodiments, the two nicks on both strands are at the same genomic location and are generated by a napDNAbp containing, for example, a nuclease-active Cas9. In some embodiments, napDNAbp comprises an endonuclease that is a nickase. For example, in some embodiments, napDNAbp comprises an endonuclease containing one or more mutations that reduce the nuclease activity of the endonuclease, making it a nickase. In some embodiments, napDNAbp comprises an inactive endonuclease. For example, in some embodiments, napDNAbp comprises an endonuclease containing one or more mutations that cause loss of nuclease activity. In various embodiments, napDNAbp is a Cas9 protein or a variant thereof. napDNAbp may also be a nuclease-active Cas9, a nuclease-inactive Cas9 (dCas9), or a Cas9 nickase (nCas9). In a preferred embodiment, napDNAbp is a Cas9 nickase (nCas9) that nicks only one strand.In another embodiment, napDNAbp may 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 Argonaut, and optionally have nickas activity such that only one strand is cut. In some embodiments, napDNAbp is selected from Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f(Cas14), Cas12f1, Cas12j(CasΦ), and Argonaut, and optionally has nickase activity such that one DNA strand is preferentially cut relative to the other DNA strand.

[0064] Nuclear localization sequence (NLS) The term “nuclear localization sequence” or “NLS” refers to an amino acid sequence that facilitates the transport of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and will be obvious to those skilled in the art. For example, an NLS sequence is described in Plank et al., International PCT Application PCT / EP2000 / 011690, filed November 23, 2000, published May 31, 2001, as WO / 2001 / 038547. Its contents are incorporated herein by reference with respect to its disclosure of exemplary nuclear localization sequences. In some embodiments, the NLS is incorporated on a fusion protein (for example, on a prime editor as described herein). In certain embodiments, NLS includes the amino acid sequences PKKKRKV (SEQ ID NO: 94), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 99), KRTADGSEFESPKKKRKV (SEQ ID NO: 97), KRTADGSEFEPKKKRKV (SEQ ID NO: 106), NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 107), PAAKRVKLD (SEQ ID NO: 98), RQRRNELKRSF (SEQ ID NO: 108), or NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 109).

[0065] nucleic acid As used herein, the term “nucleic acid” refers to polymers of nucleotides. Polymers include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C5 bromolidine, C5 fluorouridine, C5 iodouridine, C5 propynyluridine, C5 propynylcytidine, C5 methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methyl The nucleic acids may include anine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudridine, 1-methyladenosine, 1-methylguanosine, N6-methyladenosine, 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 5'N phosphoramidite linkages). In some embodiments, the nucleic acid is pegRNA or epegRNA. In some embodiments, the nucleic acid is, for example, a target nucleic acid to be edited on a genome.

[0066] PEgRNA As used herein, the terms “prime-editing guide RNA,” “PEgRNA,” “pegRNA,” or “extended guide RNA” refer to a specialized form of guide RNA modified to include one or more additional sequences for implementing the prime-editing described herein. The prime-editing guide RNA described herein includes one or more “extension regions,” also referred herein to as “extension arms” of nucleic acid sequences. The extension regions may, but are not limited to, single-stranded RNA or DNA. Furthermore, the extension regions may occur at the 3' end of the conventional guide RNA. In other configurations, the extension regions may occur at the 5' end of the conventional guide RNA. In yet another configuration, the extension regions may occur in the intramolecular region of the conventional guide RNA, for example, on a gRNA core region that binds to and / or conjugates to napDNAbp. The extension region includes a “DNA synthesis template” or “reverse transcriptase template” that encodes single-stranded DNA (by the polymerase / reverse transcriptase of the prime editor), which in turn (a) is designed to be homologous to the endogenous target DNA to be edited, and (b) includes at least one desired nucleotide change (e.g., a transition, transversion, deletion, or insertion) to be introduced or incorporated onto the endogenous target DNA. The extension region may also include other functional sequence elements, such as but not limited to “primer binding sites” and “linker” sequences, or other structural elements, such as but not limited to aptamers, stem-loops, hairpins, toe-loops (e.g., 3' toe-loops), or RNA-protein recruitment domains (e.g., MS2 hairpins). As used herein, a “primer binding site” includes a sequence that hybridizes to a single-stranded DNA sequence having a 3' end, generated from the R-loop-nicked DNA.

[0067] In certain embodiments, the pegRNA has a 3' extension arm, a spacer, and a gRNA core. The 3' extension arm further comprises a DNA synthesis template, a primer binding site, and a linker in the 5' to 3' direction. Where the polymerase of the prime editor described herein is a different type of polymerase rather than RT, the DNA synthesis template may also be more broadly referred to as the "DNA synthesis template."

[0068] In certain other embodiments, the pegRNA has a 5' extension arm, a spacer, and a gRNA core. The 5' extension further comprises a DNA synthesis template, a primer binding site, and a linker in the 5' to 3' direction. The DNA synthesis template may also be more broadly referred to as the "DNA synthesis template," where the polymerase of the prime editor described herein is a different type of polymerase rather than RT.

[0069] In yet another embodiment, the pegRNA has a spacer, a gRNA core, and an extension arm in the 5' to 3' direction. The extension arm is located at the 3' end of the pegRNA. The extension arm further includes a homology arm, an editing template, and a primer binding site in the 5' to 3' direction. The extension arm may also include optional modification regions at the 3' and 5' ends. These may be the same sequence or different sequences. In addition, the 3' end of the pegRNA may include a transcriptional terminator sequence. These sequence elements of the pegRNA are further described and defined herein.

[0070] In yet another embodiment, the pegRNA has an extension arm, a spacer, and a gRNA core in the 5' to 3' direction. The extension arm is located at the 5' end of the pegRNA. The extension arm further includes a primer binding site, an editing template, and a homology arm in the 3' to 5' direction. The extension arm may also include optional modification regions at the 3' and 5' ends. These may be the same sequence or different sequences. The pegRNA may also include a transcriptional terminator sequence at the 3' end. These sequence elements of the pegRNA are further described and defined herein.

[0071] In some embodiments, the spacer sequence of pegRNA is approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In certain embodiments, the spacer sequence of pegRNA is approximately 20 nucleotides in length. In some embodiments, the prime-binding site is approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides in length. In some embodiments, the homology arm of pegRNA is approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the DNA synthesis template is approximately 5 to 58 nucleotides long, approximately 10 to 16 nucleotides long, or approximately 12 to 17 nucleotides long. In certain embodiments, the DNA synthesis template is less than 15 nucleotides long.

[0072] In some embodiments, pegRNA is “engineered pegRNA” (“epegRNA”). Compared to pegRNA, epegRNA includes, for example, an additional structured motif attached to its 3' end. Such an additional structured motif can stabilize the pegRNA or otherwise prevent it from being degraded. Preferred structured motifs include, but are not limited to, toe loops, hairpins, stem loops, pseudoknots, aptamers, G quadruples, tRNAs, riboswitches, and ribozymes. In some embodiments, the 3' structured motif includes evopreq1.

[0073] pegRNA is further included, for example, in the following international patent applications: PCT / US2020 / 023721, filed on March 19, 2020, published as WO 2020 / 191239; PCT / US2021 / 031439, filed on May 7, 2021, published as WO 2021 / 226558; PCT / 2021 / 052097, filed on September 24, 2021, published as WO 2022 / 067130; PCT / US2022 / 012054, filed on January 11, 2022, published as WO 2022 / 150790; WO This is described in International Patent Application No. PCT / US2022 / 078655, filed on 25 October 2022 and published as 2023 / 076898; and International Patent Application No. PCT / US2022 / 074628, filed on 5 August 2022 and published as WO 2023 / 015309; the contents of each of these are incorporated herein by reference.

[0074] PE1 As used herein, "PE1" refers to a prime edit composition comprising 1) a fusion protein containing the Cas9 protein variant Cas9(H840A) having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(wt)]-NLS and wild-type MMLV RT, and 2) a desired PEgRNA, wherein the fusion protein (referred to as the PE1 protein) has the amino acid sequence of SEQ ID NO: 3 shown below. [ka] [ka]

[0075] PE2 As used herein, "PE2" refers to a prime editing composition comprising 1) a fusion protein containing the Cas9 protein variant Cas9(H840A) and the variant MMLV RT having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)]-NLS, and 2) a desired PEgRNA, wherein the fusion protein (referred to as the PE2 protein) has the amino acid sequence of SEQ ID NO: 4 shown below: [ka] [ka]

[0076] PE3 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 to induce preferred replacement of the edited strand and introduce a nick onto the unedited DNA strand.

[0077] PE3b 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 to introduce a nick onto an unedited 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 placement of the desired edit. This is achieved by designing a second-strand nicking guide RNA having a spacer sequence that includes complementarity to the edited strand after the placement of the desired nucleotide edit(s), rather than to an endogenous target DNA sequence, and hybridizes only with it. Using this strategy, the mismatch between the nicking guide RNA spacer and the unedited target DNA should prevent sgRNA nicking until after the editing event on the PAM strand occurs.

[0078] PE4 As used herein, “PE4” refers to a prime-edited composition comprising PE2 and further comprising an MLH1 dominant-negative protein variant (i.e., wild-type MLH1 with amino acids 754-756 shortened; this 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, the PE4 system comprises a fusion protein containing a PE2 protein and an MLH1 dominant-negative protein linked via an arbitrary linker.

[0079] PE5 and PE5b 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 shortened; this may be referred to as “MLH1 Δ754-756” or “MLH1dn”). ​​The MLH1 dominant-negative variant may be expressed in trans in some embodiments. In some embodiments, the PE5 system comprises a fusion protein containing a PE2 protein and an MLH1 dominant-negative protein linked via an arbitrary linker. “PE5b” refers to a prime-editing composition comprising a PE3 and an MLH1 dominant-negative protein, wherein a second-strand nicking guide RNA is designed for temporal control such that the second-strand nicks are not introduced until after the placement of the desired edit. This is achieved by designing a second-strand nickeling guide RNA that has a spacer sequence that includes complementarity to the edited strand after the placement of the desired nucleotide edit(s), and hybridizes only with that, rather than to an endogenous target DNA sequence.

[0080] PE6 The term “PE6” refers to the suite of next-generation prime editors described herein (PE6a, PE6b, PE6c, PE6d, PE6e, PE6f, and PE6g) that include improved reverse transcriptase and / or Cas9 variants. The improved reverse transcriptase and Cas9 domains of the PE6 variants may also be combined with each other to provide cumulative benefits. For example, a PE6 prime editor containing the improved reverse transcriptase variant of PE6a and the improved Cas9 variant of PE6e is referred to herein as the prime editor “PE6a-e” (or “PE6e-a”). Any possible combination of the PE6 prime editors is contemplated herein and includes, 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.

[0081] Each PE6 prime editor contains a Cas9 domain, e.g., a Cas9 variant, and a reverse transcriptase domain, e.g., a reverse transcriptase variant. PE6a contains reverse transcriptase variants with amino acid substitutions E60K, K87E, E165D, D243N, R267I, E279K, K318E, and K343N relative to Ec48 reverse transcriptase (SEQ ID NO: 7). PE6b contains reverse transcriptase variants with amino acid substitutions P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, and S492N relative to Tf1 reverse transcriptase (SEQ ID NO: 1). PE6c includes reverse transcriptase variants with amino acid substitutions P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, S188K, I260L, F269L, R288Q, S297Q, A363V, K413E, and S492N relative to Tf1 reverse transcriptase (SEQ ID NO: 1). PE6d includes reverse transcriptase variants with amino acid substitutions T128N, D200C, and V223Y relative to MMLV reverse transcriptase (SEQ ID NO: 30) which has a shortened C-terminal RNaseH domain (e.g., between D497 and I498 in SEQ ID NO: 30) (as well as substitutions T306K, W313F, and T330P used in MMLV reverse transcriptase of PE2 and PEmax). PE6e contains Cas9 variants including amino acid substitutions K775R and K918A compared to wild-type Streptococcus pyogenes Cas9 or Streptococcus pyogenes Cas9 nickase (SEQ ID NO: 2). PE6f contains Cas9 variants including amino acid substitutions H99R, E471K, I632V, D645N, H721Y, and K918A compared to wild-type Streptococcus pyogenes Cas9 or Streptococcus pyogenes Cas9 nickase (SEQ ID NO: 2).PE6g contains Cas9 variants including amino acid substitutions H99R, E471K, I632V, D645N, R654C, and H721Y relative to wild-type Streptococcus pyogenes Cas9 or Streptococcus pyogenes Cas9 nickase (SEQ ID NO: 2). The Cas9 domain and reverse transcriptase variant of the PE6 prime editor described herein may be covalently linked or conjugated. For example, they may be directly fused to each other or linked via a linker peptide to form a fusion protein. Alternatively, the Cas9 domain and reverse transcriptase may be provided in trans, i.e., not covalently linked. Furthermore, any of the PE6 prime editor fusion proteins provided herein may include prime editor fusion protein architectures described herein or known in the art, such as the PE2 protein architecture or the PEmax protein architecture. Exemplary PEmax protein components, sequences, and corresponding architectures are provided below. In some embodiments, any of the PE6 prime editors provided herein may further include additional amino acid mutations, such as those included in PEmax provided below.

[0082]

[0083]

[0084]

[0085]

[0086] In some embodiments, the PE6d Prime Editor comprises a fusion protein comprising Cas9 having amino acid substitutions R221K, N394K and (i.e., R221K, N394K, and H840A substitutions relative to SEQ ID NO: 2) and MMLV-RT having amino acid substitutions T128N, D200C, V223Y, T306K, W313F, and T330P relative to SEQ ID NO: 30, as well as a C-terminal shortening between D497 and I498. In some embodiments, the PE6d Prime Editor comprises a fusion protein comprising a reverse transcriptase variant defined in SEQ ID NO: 27 and a Cas9 variant defined in SEQ ID NO: 11. In some embodiments, the PE6d prime editor comprises a fusion protein having the following configuration ("PEmax architecture"): [binoderm NLS]-[Cas9(R221K)(N394K)(H840A)]-[linker]-[MMLV_RT(T128N)(D200C)(V223Y)(T306K)(T330P)(D497 / I498 C-terminal shortening))]-[binoderm NLS]-[NLS]. In some embodiments, the PE6d prime editor comprises a fusion protein having the following sequence: [ka] [ka]

[0087] In some embodiments, the PE6d Prime Editor comprises a fusion protein comprising Cas9 having the amino acid sequence defined in SEQ ID NO: 10 (i.e., H840A substitution relative to wild-type Cas9) and MMLV-RT having the amino acid substitutions T128N, D200C, V223Y, T306K, W313F, and T330P relative to SEQ ID NO: 30, as well as a C-terminal shortening between D497 and I498. In some embodiments, the PE6d Prime Editor comprises a fusion protein comprising the reverse transcriptase defined in SEQ ID NO: 27 and the Cas9 variant defined in SEQ ID NO: 10. In some embodiments, the PE6d prime editor comprises a fusion protein having the following configuration ("PE2 architecture"): [binodermous NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(T128N)(D200C)(V223Y)(T306K)(T330P)(D497 / I498 C-terminal shortening)]-[NLS]. In some embodiments, the PE6d prime editor comprises a fusion protein having the following sequence:

[0088] [ka]

[0089] PE7 The term "PE7" refers to PE6 Prime Editor Plus Second Strand Nicking Guide RNA. For example, "PE7a" refers to PE6a Prime Editor Plus Second Strand Nicking Guide RNA provided herein.

[0090] PEmax As used herein, "PEmax" refers to a prime editing composition comprising 1) a fusion protein containing the Cas9 protein variant Cas9(R221K N394K H840A) and the variant MMLV RT having the following structure: [binodelux NLS]-[Cas9(R221K)(N394K)(H840A)]-[linker]-[MMLV_RT(D200N)(T306K)(W313F)(T330P)(L603W)]-[binodelux NLS]-[NLS] and 2) a desired PEgRNA, wherein the fusion protein (referred to as the PEmax protein) has the amino acid sequence of SEQ ID NO: 5 shown below: [ka] [ka]

[0091] PACE As used herein, the term "phage-assisted continuous evolution (PACE)" refers to continuous evolution using phages as viral vectors. The general concept of PACE technology is illustrated by, for example, the international PCT application PCT / US2009 / 056194 filed on September 8, 2009, published on March 11, 2010 as WO 2010 / 028347; the international PCT application PCT / US2011 / 066747 filed on December 22, 2011, published on June 28, 2012 as WO 2012 / 088381; the US application US Patent No. 9,023,594 issued on May 5, 2015; the international PCT application PCT / US2015 / 012022 filed on January 20, 2015, published on September 11, 2015 as WO 2015 / 134121; and the WO This is described in the international PCT application PCT / US2016 / 027795, filed on April 15, 2016, published as 2016 / 168631, and the entire contents of each of these are incorporated herein by reference.

[0092] PANCE As used herein, “phage-assisted discontinuous evolution (PANCE)” refers to discontinuous evolution using phages as viral vectors. PANCE is a simplified technique for rapid in vivo-directed evolution, using stepwise flask subculturing of evolving “selection phages” (SPs) containing the target genes to be evolved between new E. coli host cells, thereby allowing the genes contained in the SPs to evolve continuously while the genes within the host E. coli remain constant. Stepwise flask subculturing has long served as a widely accessible approach for the laboratory evolution of microorganisms, and more recently, similar approaches have been developed for bacteriophage evolution. The PANCE system features lower stringency than the PACE system.

[0093] polymerase As used herein, the term “polymerase” means an enzyme that synthesizes nucleotide chains and can be used in connection with the prime editor delivery systems described herein. A polymerase may be a “template-dependent” polymerase (i.e., a polymerase that synthesizes nucleotide chains based on the order of nucleotide bases of a template chain). A polymerase may also be a “template-independent” polymerase (i.e., a polymerase that synthesizes nucleotide chains without the requirement of a template chain). A polymerase may also be further classified as a “DNA polymerase” or an “RNA polymerase”. In various embodiments, the prime editor system includes a DNA polymerase. In various embodiments, the DNA polymerase may be a “DNA-dependent DNA polymerase” (i.e., the template molecule is a strand of DNA). In such a case, the DNA template molecule may be pegRNA, where the extension arms include a strand of DNA. In such cases, pegRNA may be described as a chimeric or hybrid pegRNA containing an RNA portion (i.e., a guide RNA component encompassing a spacer and gRNA core) and a DNA portion (i.e., an extension arm). In various other embodiments, DNA polymerase may be an "RNA-dependent DNA polymerase" (i.e., according to this, the template molecule is a strand of RNA). In such cases, pegRNA is RNA, i.e., it encompasses RNA extension. The term "polymerase" may also refer to an enzyme that catalyzes the polymerization of nucleotides (i.e., polymerase activity). Generally, the enzyme will initiate synthesis at the 3' end of a primer annealed to a polynucleotide template sequence (e.g., a primer sequence annealed to the primer binding site of pegRNA, for example) and proceed toward the 5' end of the template strand. "DNA polymerase" catalyzes the polymerization of deoxynucleotides. The term DNA polymerase as used herein in reference to DNA polymerase encompasses "its functional fragment".The “functional fragment” refers to a portion of either wild-type or mutant DNA polymerase that encapsulates less than the entire amino acid sequence of the polymerase and retains the ability to catalyze polynucleotide polymerization under the conditions of at least one set of factors. Such a functional fragment may exist as a separate entity or it may be a component of a larger polypeptide, such as a fusion protein.

[0094] Prime Edit As used herein, the term “prime editing” refers to an approach for gene editing that uses napDNAbp, polymerase (e.g., reverse transcriptase), and specialized guide RNA. It encompasses a primer binding site and a DNA synthesis template for encoding (or deleting) desired new genetic information, which is then incorporated onto a target DNA sequence. Prime editing is described in Anzalone, AV 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. Also, see the international PCT application PCT / US2020 / 023721, filed March 19, 2020, published as WO 2020 / 191239, which is incorporated herein by reference.

[0095] Prime Editing represents a platform for genome editing, a versatile and precise method for directly writing new genetic information to a designated DNA site using a nucleic acid-programmable DNA-binding protein ("napDNAbp") that works in conjunction with polymerase (i.e., provided in the form of a fusion protein, or otherwise in trans with napDNAbp), where the Prime Editing system is programmed by a Prime Editing (PE) guide RNA ("pegRNA"), which both designates the target site and serves as a template for the synthesis of the desired edit in the form of a replacement DNA strand as an manipulated extension (either DNA or RNA) on the guide RNA (e.g., at the 5' or 3' end or in the interior of the guide RNA). The replacement strand containing the desired edit (e.g., a single nucleic acid substitution) shares (or is homologous to) the same sequence as (or homologous to) the endogenous strand immediately downstream of the nick site at the target site to be edited (with the exception that it contains the desired edit). Through DNA repair and / or replication mechanisms, the endogenous strand downstream of the nick site is replaced by the newly synthesized replacement strand containing the desired edit. In some cases, prime editing can be considered a “search and replace” genome editing technology, because the prime editors described herein not only search and locate the desired target site to be edited, but also simultaneously encode a replacement strand containing the desired edit that is placed in place of the corresponding endogenous DNA strand at the target site. The prime editors of this disclosure relate in part to the discovery that the mechanism of reverse transcription (TPRT) or “prime editing” primed by a target can be utilized or adapted to perform precise CRISPR / Cas-based genome editing with high efficiency and gene flexibility. TPRT is naturally used by mobile DNA factors such as mammalian non-LTR retrotransposons and bacterial group II introns.A Cas protein-reverse transcriptase fusion or related system is used to target a specific DNA sequence by guide RNA, generate a single-strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of the manipulated DNA synthesis template incorporated into the guide RNA. However, while the concept begins with a prime editor that uses reverse transcriptase as a DNA polymerase component, the prime editors described herein are not limited to reverse transcriptase and may substantially encompass the use of any DNA polymerase. In fact, this application may consistently refer to a prime editor having “reverse transcriptase,” but it is hereby defined that reverse transcriptase is only one type of DNA polymerase that can act in prime editing. Therefore, wherever this specification refers to “reverse transcriptase,” those skilled in the art should understand that any suitable DNA polymerase may be used instead of reverse transcriptase. Therefore, in one aspect, a prime editor may include Cas9 (or 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 on the target DNA (a sequence complementary to an endogenous protospacer sequence). The pegRNA also contains novel genetic information in the form of an extension encoding a replacement strand of DNA containing the desired nucleotide change, which is used to replace the corresponding endogenous DNA strand at the target site. To transfer the information from the pegRNA to the target DNA, the mechanism of prime editing involves nicking the target site on one strand of DNA to expose a 3' hydroxyl group. The exposed 3' hydroxyl group can then be used to prime DNA polymerization directly from the edit-encoding extension on the pegRNA to the target site. In various embodiments, the extension providing a template for polymerization of the replacement strand containing the edit may be formed from RNA or DNA. In the case of RNA extension, the polymerase of the prime editor may be an RNA-dependent DNA polymerase (e.g., reverse transcriptase).In the case of DNA extension, the polymerase of the prime editor may be a DNA-dependent DNA polymerase. The newly synthesized strand formed by the prime editor (i.e., the replacement DNA strand containing the desired nucleotide edits) will be homologous to the genomic target sequence (i.e., will have the same sequence), with the exception of the inclusion of one or more desired nucleotide changes (e.g., a single nucleotide substitution, deletion, or insertion, or a combination thereof). The newly synthesized (or replacement) strand of DNA may also be called a single-stranded DNA flap. It will compete for hybridization with a complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. The resolution of a hybridized intermediate (also known as a heteroduplex, which includes a single-stranded DNA flap synthesized by a reverse transcriptase hybridized to the endogenous DNA strand, with the exception of a mismatch at the site where the desired nucleotide edit is placed on the edited strand) may include the removal of the strand-substituted flap resulting from the endogenous DNA (e.g., by the 5' end DNA flap endonuclease FEN1), ligation of the synthesized single-stranded DNA flap onto 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 (in which a nucleotide sequence change is desired) with a nucleic acid-programmed DNA-binding protein (napDNAbp) complexed with a prime editing guide RNA (pegRNA). In various embodiments, the prime editing guide RNA (pegRNA) contains an extension at the 3' or 5' end of the guide RNA or at an intramolecular location on the guide RNA, encoding the desired nucleotide change (e.g., single base substitution, insertion, or deletion). First, the napDNAbp / extended gRNA complex contacts the DNA molecule, and the extended gRNA guides the napDNAbp to bind to the target locus. Next, a nick is introduced on one of the DNA strands of the target locus (e.g., by a nuclease or chemical agent), thereby creating an available 3' end on one of the strands of the target locus. In certain embodiments, the nick is created on the strand of DNA corresponding to the R loop strand, i.e., the strand that is not hybridized to the guide RNA sequence, i.e., the "non-target strand". However, the nick can be introduced on either strand. In other words, the nick can be introduced onto the R-loop “target strand” (i.e., the strand that hybridizes to the protospacer of the extended gRNA) or the “non-target strand” (i.e., the strand that forms the single-stranded portion of the R-loop and is complementary to the target strand). In the next step, the 3' end of the DNA strand (formed by the nick) interacts with the extended portion of the guide RNA to prime the 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., a “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 from the 3' end of the primed site toward the 5' end of the prime-editing guide RNA to synthesize a single strand of DNA. The DNA polymerase (e.g., reverse transcriptase) can be fused to napDNAbp or, alternatively, provided trans toward napDNAbp.This involves forming a single-stranded DNA flap containing the desired nucleotide change (e.g., a single base change, insertion, or deletion, or a combination thereof) that is otherwise homologous to the endogenous DNA at or adjacent to the nicking site. In the next step, napDNAbp and guide RNA are released. The last two steps involve the dissolution of the single-stranded DNA flap so that the desired nucleotide change is incorporated into the target locus. This process can be driven toward the formation of the desired product by removing the corresponding 5' endogenous DNA flap that forms once the 3' single-stranded DNA flap invades and hybridizes with the endogenous DNA sequence. Although not constrained by theory, the cell's endogenous DNA repair and replication processes resolve mismatched DNA and incorporate nucleotide changes (one or more) to form the desired modulated product. The process can also be driven toward product formation by "second-strand nicking." This process can introduce at least one of the following genetic changes: transversion, transition, deletion, and insertion.

[0097] Prime Editor The term “prime editor” means a polypeptide or polypeptide component involved in prime editing as described herein. In some embodiments, the prime editor comprises a fusion construct comprising napDNAbp (e.g., Cas9 nickase and / or any of the Cas9 variants provided herein) and reverse transcriptase (e.g., any of the reverse transcriptase variants provided herein). In some embodiments, the prime editor can perform prime editing on a target nucleotide sequence in the presence of pegRNA (or “extended guide RNA”). In some embodiments, the prime editor comprises trans-provided napDNAbp (e.g., Cas9 nickase) and reverse transcriptase; that is, the napDNAbp and reverse transcriptase are not fused. The trans-provided napDNAbp and reverse transcriptase may be anchored via non-peptide bonds, e.g., an MS2 RNA-protein binding RNA sequence and an MS2 coat protein fused to either the napDNAbp or the reverse transcriptase, or they may be recruited simply by pegRNA without being linked to each other. In some embodiments, the prime editor compositions, systems, or complexes provided herein include a fusion protein, or a fusion protein complexed with pegRNA and / or further complexed with a second-strand nickeling sgRNA. In some embodiments, the prime editor system may also refer to a complex comprising a fusion protein (reverse transcriptase fused to napDNAbp), pegRNA, and a conventional guide RNA capable of guiding the second-site nickeling step of the unedited strand, as described herein.

[0098] Proteins, peptides, and polypeptides The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to polymers of amino acid residues linked together by peptide (amide) bonds. The terms refer to proteins, peptides, or polypeptides 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 amino acids on a protein, peptide, or polypeptide may be modified, for example, by the addition of chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, conjugations, functionalizations, or linkers for other modifications. A protein, peptide, or polypeptide may also be a single molecule or a multimolecular complex. A protein, peptide, or polypeptide may be merely a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, synthetic, or a combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein can be produced through recombinant protein expression and purification. This is particularly suitable for fusion proteins containing peptide linkers. Methods for recombinant protein expression and purification are well known and include those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). The contents of this manual are incorporated herein by reference.

[0099] Protospacer As used herein, the term "protospacer" refers to a sequence (e.g., approximately 20 bp) on DNA adjacent to a PAM (protospacer adjacent motif) sequence. Protospacers share the same sequence as the spacer sequence of the guide RNA (with the exception that protospacers contain thymine, while spacer sequences contain uracil). The guide RNA anneals to the complement of the protospacer sequence on the target DNA (specifically, one of its strands, i.e., the "target strand" relative to the "non-target strand" of the target DNA sequence). In some embodiments, for the Cas nickase component of a prime editor to function, it also requires a specific protospacer adjacent motif (PAM), which varies depending on the Cas protein component itself, e.g., the type of Cas protein and the bacterial species from which it originates. The most commonly used Cas9 nuclease, derived from S. pyogenes, recognizes a PAM sequence called NGG that is directly downstream of the protospacer sequence on the non-target strand of genomic DNA.

[0100] Protospacer adjacent motif (PAM) As used herein, the terms “protospacer adjacent motif” or “PAM” refer to a DNA sequence (e.g., a sequence of approximately 2–6 nucleotides) that is an important targeting component of a Cas nuclease, such as Cas9. For example, in some embodiments of the Cas9 nuclease, the PAM sequence is located on either strand and downstream from the Cas9 cut site in the 5' to 3' direction. A classical PAM sequence (i.e., a PAM sequence associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5'-NGG-3', where “N” is any nucleic acid base followed by two guanine ("G") nucleic acid bases. In some embodiments, SpCas9 may also recognize additional non-classical PAMs (e.g., NAG and NGA).

[0101] Different PAM sequences may be associated with different Cas9 nucleases or equivalent proteins from different organisms. In addition, any given Cas9 nuclease, such as SpCas9, can be modified to modulate its PAM specificity by causing it to recognize alternative PAM sequences.

[0102] reverse transcriptase The term “reverse transcriptase” describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require primers to synthesize DNA transcripts from an RNA template. Historically, reverse transcriptases have been primarily used to transcribe mRNA into cDNA, which can then be cloned onto vectors for further manipulation. Avian myoblastosis virus (AMV) reverse transcriptase was the first widely used RNA-dependent DNA polymerase (Verma, Biochim. Biophys. Acta 473:1 (1977)). The enzyme possesses 5'-3' RNA-directed DNA polymerase activity, 5'-3' DNA-directed DNA polymerase activity, and RNaseH activity. RNaseH is a processive 5' and 3' ribonuclease specific to the RNA strand of RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). Known viral reverse transcriptases lack the 3'-5' exonuclease activity necessary for proofreading, so transcriptional errors cannot be corrected by reverse transcriptase (Saunders and Saunders, Microbial Genetics Applied to Biotechnology, London: Croom Helm (1987)). Detailed studies of AMV reverse transcriptase activity and its associated RNaseH activity are presented by Berger et al., Biochemistry 22:2365-2372 (1983). Another reverse transcriptase widely used in molecular biology is that from Moloney's mouse leukemia virus (M-MLV or "MMLV"). See, for example, Gerard, GR, DNA 5:271-279 (1986) and Kotewicz, ML, et al., Gene 35:249-258 (1985). M-MLV reverse transcriptases that substantially lack RNaseH activity have also been described. For example, see USPat. No. 5,244,797.The present invention intends to utilize any such reverse transcriptase, or a variant or variant thereof.

[0103] In some embodiments, the prime editor provided herein includes MMLV RT, or a variant or fragment of MMLV RT. In some embodiments, the prime editor provided herein includes Ec48 RT, or a variant or fragment of Ec48 RT. In some embodiments, the prime editor provided herein includes Tf1 RT, or a variant or fragment of Tf1 RT.

[0104] In certain embodiments, the reverse transcriptase includes amino acid substitutions E60K, K87E, E165D, D243N, R267I, E279K, K318E, and K343N relative to Ec48 reverse transcriptase (SEQ ID NO: 7). In certain embodiments, the reverse transcriptase includes amino acid substitutions P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, and S492N relative to Tf1 reverse transcriptase (SEQ ID NO: 1). In certain embodiments, the reverse transcriptase includes amino acid substitutions P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, S188K, I260L, F269L, R288Q, S297Q, A363V, K413E, and S492N relative to Tf1 reverse transcriptase (SEQ ID NO: 1). In certain embodiments, the reverse transcriptase includes amino acid substitutions T128N, D200C, and V223Y relative to MMLV reverse transcriptase (SEQ ID NO: 30) having a shortened C-terminal RNaseH domain.

[0105] Reverse transcription As used herein, the term “reverse transcription” refers to the ability of an enzyme to synthesize a DNA strand (i.e., complementary DNA or cDNA) using RNA as a template. In some embodiments, reverse transcription may be “error-prone reverse transcription.” This refers to the characteristic of certain reverse transcriptases that their DNA polymerization activity is prone to errors.

[0106] Spacer array As used herein in relation to guide RNA or pegRNA, the term “spacer sequence” refers to a portion of guide RNA or pegRNA of approximately 20 nucleotides containing a nucleotide sequence that shares the same sequence as the protospacer sequence on the target DNA sequence. The spacer sequence anneals to the complement of the protospacer sequence to form an ssRNA / ssDNA hybrid structure at the target site and a corresponding R-loop ssDNA structure of the endogenous DNA strand.

[0107] replacement As used herein, the term “substitution” means the replacement of a residue in a sequence, such as a nucleic acid or amino acid sequence, by another residue, or the deletion or insertion of one or more residues in a sequence. The term “mutation” may also be used in this disclosure to mean substitution (i.e., “nucleic acid mutation” or “amino acid mutation”). Substitutions are typically described herein by identifying the original residue, followed by the location of the residue in the sequence and the identity of the newly mutated / substituted residue. Various methods for making amino acid substitutions provided herein are well known in the art, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 thProvided by (ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)). In some embodiments, the substitution is located on a reverse transcriptase, e.g., MMLV reverse transcriptase, Ec48 reverse transcriptase, or Tf1 reverse transcriptase. In some embodiments, the substitution is located on a Cas9 protein, e.g., a SpCas9 protein.

[0108] variant As used herein, the term “variant” should be understood to mean the manifestation of a quality that has a pattern deviating from that which occurs naturally. The term “variant” encompasses homologous proteins that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 975%, at least 98%, or at least 99% identity with the reference sequence and have the same or substantially the same functional activity (single or multiple) as the reference sequence. The term also encompasses variants, shortens, or domains of the reference sequence that exhibit the same or substantially the same functional activity (single or multiple) as the reference sequence.

[0109] In some embodiments, the variant contains one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions relative to the wild-type sequence. In some embodiments, the variant is a reverse transcriptase variant. In certain embodiments, the reverse transcriptase variant contains one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions relative to the wild-type reverse transcriptase sequence (e.g., wild-type MMLV reverse transcriptase, wild-type Ec48 reverse transcriptase, or wild-type Tf1 reverse transcriptase). In certain embodiments, a Cas9 variant comprises one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions relative to a wild-type Cas9 sequence (e.g., wild-type SpCas9) or Cas9 nickase (e.g., SpCas9 nickase).

[0110] vector As used herein, the term “vector” means a nucleic acid that can be modified to encode a gene of interest, enter a host cell, mutate and replicate within the host cell, and then transfer the replicated form of the vector to another host cell. Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phages, as well as conjugative plasmids. Additional suitable vectors will be apparent to those skilled in the art based on this disclosure.

[0111] Wild type As used herein, the terms “wild type” or “WT” are articulate terms as understood by those skilled in the art, and mean the typical form of an organism, strain, gene, or feature as it occurs naturally, distinct from mutant or variant forms.

[0112] Embodiments for carrying out the invention This disclosure describes the use of directed evolution and protein engineering to generate novel reverse transcriptases and Cas9 variants that enhance editing efficiency when used in the context of prime editors. In particular, next-generation prime editors (PE6a and PE6b) have been developed that are approximately 500–800 bp smaller than PE2 while providing mammalian prime editing efficiency comparable to or higher than that of PE2. In addition, highly active and processable prime editors (PE6c and PE6d) using either M-MLV RT or Tf1 RT have also been developed. These evolved and engineered reverse transcriptases provide substantial improvements compared to previously used prime editors, e.g., increased editing efficiency for longer edits. Evolved variants of the Cas9 nickasase domain of prime editors (PE6e–PE6g) have also been created to further improve prime editing efficiency.

[0113] Therefore, this disclosure provides evolved and manipulated reverse transcriptase variants and Cas9 variants having improved properties (e.g., improved editing efficiency when used in the context of a prime editor). For example, fusion proteins containing prime editors comprising the reverse transcriptase variants and Cas9 variants described herein are also provided by this disclosure. This disclosure also provides polynucleotides encoding the reverse transcriptase variants, Cas9 variants, fusion proteins, and prime editors provided herein, as well as vectors containing such polynucleotides. Pharmaceutical compositions, AAVs, and cells comprising the reverse transcriptase variants, Cas9 variants, and prime editors (and / or polynucleotides or vectors encoding them) described herein are also provided by this disclosure. This disclosure also provides methods and uses involving the reverse transcriptase variants, Cas9 variants, and prime editors described herein.

[0114] PE6 prime editor, Cas9 variant, reverse transcriptase variant, and fusion protein Several aspects of this disclosure provide evolved and / or manipulated reverse transcriptases and Cas9 proteins having various improved properties (e.g., smaller size for increased delivery efficiency (e.g., using AAV), improved prime editing efficiency (e.g., for editing requiring a structured pegRNA RT template), reduced indel frequency, etc.), as well as prime editors comprising them. In some embodiments, the structure and folding prediction of pegRNA, encompassing the free energy of folding of pegRNA components, e.g., RT templates or extension arms, can be measured by NUPACK free energy prediction as described in Zadeh, JNet al., (2011). NUPACK: Analysis and design of nucleic acid systems. J. Comput.Chem. 32, 170-173, which is incorporated herein by reference.

[0115] The variants provided by this disclosure include variants of Escherichia coli Ec48 reverse transcriptase, Schizosaccharomyces pombe Tf1 reverse transcriptase, Moloney's mouse leukemia virus (MMLV) reverse transcriptase, and Streptococcus pyogenes Cas9, as well as variants comprising amino acid substitutions disclosed herein at corresponding positions on homologous proteins.

[0116] In one aspect, this disclosure provides reverse transcriptase variants comprising various amino acid substitutions to the amino acid sequence of Schizosaccharomyces pombe Tf1 reverse transcriptase provided below: ISSSKHTLSQMNKVSNIVKEPELPDIYKEFKDITADTNTEKLPKPIKGLEFEVELTQENYRLPIRNYPLPPGKMQAMNDEINQGLKSGIIRESKAINACPVMFVPKKEGTLRMVVDYKPLNKYVKPNI YPLPLIEQLLAKIQGSTIFTKLDLKSAYHLIRVRKGDEHKLAFRCPRGVFEYLVMPYGISTAPAHFQYFINTILGEAKESHVVCYMDDILIHSKSESEHVKHVKDVLQKLKNANLIINQAKCEFHQSQ VKFIGYHISEKGFTPCQENIDKVLQWKQPKNRKELRQFLGSVNYLRKFIPKTSQLTHPLNKLLKKDVRWKWTPTQTQAIENIKQCLVSPPVLRHFDFSKKILLETDASDVAVGAVLSQKHDDDKYYPVGYYSAKMSKAQLNYSVSDKEMLAIIKSLKHWRHYLESTIEPFKILTDHRNLIGRITNESEPENKRLARWQLFLQDFNFEINYRPGSANHIADALSRIVDETEPIPKDSEDNSINFVNQISI(Sequence ID 1).

[0117] In some embodiments, the Disclosure provides a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 1, wherein the reverse transcriptase variant includes amino acid substitutions at positions 70, 72, 87, 102, 106, 118, 128, 158, 269, 363, 413, and 492 with respect to SEQ ID NO: 1, or corresponding substitutions in homologous sequences. In some embodiments, the amino acid substitution at position 70 is a P70X substitution, where X is any amino acid other than the wild type. In some embodiments, the amino acid substitution at position 70 is a P70T substitution. In some embodiments, the amino acid substitution at position 72 is a G72X substitution, where X is any amino acid other than the wild type. In some embodiments, the amino acid substitution at position 72 is a G72V substitution. In some embodiments, the amino acid substitution at position 87 is an S87X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 87 is an S87G substitution. In some embodiments, the amino acid substitution at position 102 is an M102X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 102 is an M102I substitution. In some embodiments, the amino acid substitution at position 106 is a K106X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 106 is a K106R substitution. In some embodiments, the amino acid substitution at position 118 is a K118X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 118 is a K118R substitution. In some embodiments, the amino acid substitution at position 128 is an I128X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 128 is an I128V substitution. In some embodiments, the amino acid substitution at position 158 is an L158X substitution, where X is any amino acid other than the wild-type amino acid.In certain embodiments, the amino acid substitution at position 158 is an L158Q substitution. In some embodiments, the amino acid substitution at position 269 is an F269X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 269 is an F269L substitution. In some embodiments, the amino acid substitution at position 363 is an A363X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 363 is an A363V substitution. In some embodiments, the amino acid substitution at position 413 is a K413X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 413 is a K413E substitution. In some embodiments, the amino acid substitution at position 492 is an S492X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 492 is an S492N substitution. In certain embodiments, the reverse transcriptase variant includes substitutions P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, and S492N relative to SEQ ID NO: 1. In some embodiments, the reverse transcriptase variant further includes amino acid substitutions at positions 188, 260, 297, and 288 relative to SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 188 is an S188X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 188 is an S188K substitution. In some embodiments, the amino acid substitution at position 260 is an I260X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 260 is an I260L substitution. In some embodiments, the amino acid substitution at position 297 is an S297X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 297 is an S297Q substitution. In some embodiments, the amino acid substitution at position 288 is an R288X substitution, where X is any amino acid other than the wild type.In certain embodiments, the amino acid substitution at position 288 is the R288Q substitution. In certain embodiments, the reverse transcriptase variant further comprises substitutions S188K, I260L, S297Q, and R288Q relative to SEQ ID NO: 1.

[0118] In another aspect, this disclosure provides reverse transcriptase variants comprising various amino acid substitutions to the amino acid sequence of the MMLV reverse transcriptase provided below: (Sequence ID 30).

[0119] In some embodiments, the disclosure provides a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 30, wherein the reverse transcriptase variant comprises amino acid substitutions at positions 128 and 200, or corresponding substitutions in homologous sequences, relative to SEQ ID NO: 30. In some embodiments, the substitution at position 128 is a T128X substitution, where X is any amino acid other than the wild type. In some embodiments, the substitution at position 128 is a T128N substitution. In some embodiments, the substitution at position 200 is a D200X substitution, where X is any amino acid other than the wild type. In some embodiments, the substitution at position 200 is a D200C substitution. In some embodiments, the reverse transcriptase variant comprises the amino acid substitutions T128N and D200C. In some embodiments, the reverse transcriptase variant further comprises an amino acid substitution at position 223 relative to SEQ ID NO: 30. In some embodiments, the amino acid substitution at position 223 is a V223X substitution, where X is any amino acid other than the wild-type amino acid. In certain embodiments, the amino acid substitution at position 223 is a V223Y substitution. In some embodiments, the reverse transcriptase variant further includes amino acid substitutions from the MMLV reverse transcriptase used for PE2 and PEmax (e.g., amino acid substitutions T306K, W313F, and T330P). In certain embodiments, the reverse transcriptase variant includes shortening of all or part of the C-terminal RNaseH domain of the MMLV reverse transcriptase (e.g., shortening at amino acid positions 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, or 510 of SEQ ID NO: 30). In certain embodiments, the reverse transcriptase variant includes a shortening between positions D497 and I498 of SEQ ID NO: 30.

[0120] In some embodiments, the disclosure provides a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 30, wherein the reverse transcriptase variant includes amino acid substitutions T128N and V223M, or corresponding substitutions in homologous sequences, relative to SEQ ID NO: 30. In some embodiments, the disclosure provides a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 30, wherein the reverse transcriptase variant includes amino acid substitutions T128N and V223Y, or corresponding substitutions in homologous sequences, relative to SEQ ID NO: 30. In some embodiments, the disclosure provides a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 30, wherein the reverse transcriptase variant includes amino acid substitutions T128F and V223M, or corresponding substitutions in homologous sequences, relative to SEQ ID NO: 30. In some embodiments, the disclosure provides a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 30, wherein the reverse transcriptase variant includes amino acid substitutions D200C and V223M, or corresponding substitutions in homologous sequences, relative to SEQ ID NO: 30.

[0121] In some embodiments, the Disclosure provides a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 30, wherein the reverse transcriptase variant includes amino acid substitutions at positions 128, 129, 196, 200, and 223 with respect to SEQ ID NO: 30, or corresponding substitutions in homologous sequences. In some embodiments, the amino acid substitution at position 128 is a T128X substitution, where X is any amino acid other than the wild type. In some embodiments, the amino acid substitution at position 128 is a T128N substitution. In some embodiments, the amino acid substitution at position 129 is a V129X substitution, where X is any amino acid other than the wild type. In some embodiments, the amino acid substitution at position 129 is a V129A substitution. In some embodiments, the amino acid substitution at position 129 is a V129G substitution. In some embodiments, the amino acid substitution at position 196 is a P196X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 196 is a P196S substitution. In certain embodiments, the amino acid substitution at position 196 is a P196T substitution. In certain embodiments, the amino acid substitution at position 196 is a P196F substitution. In some embodiments, the amino acid substitution at position 200 is an N200X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 200 is an N200S substitution. In certain embodiments, the amino acid substitution at position 200 is an N200Y substitution. In some embodiments, the amino acid substitution at position 223 is a V223X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 223 is a V223A substitution. In certain embodiments, the amino acid substitution at position 223 is a V223M substitution. In certain embodiments, the amino acid substitution at position 223 is a V223L substitution. In certain embodiments, the amino acid substitution at position 223 is a V223E substitution.

[0122] In some embodiments, any of the reverse transcriptase variants provided herein further comprises an amino acid substitution from the MMLV reverse transcriptase used in PE2 and PEmax (e.g., any of the amino acid substitutions D200N, T306K, W313F, T330P, and L603W). In certain embodiments, any of the reverse transcriptase variants provided herein comprises a truncation of all or a portion of the C-terminal RNaseH domain of the MMLV reverse transcriptase (e.g., a truncation at amino acid positions 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, or 510 of SEQ ID NO: 30). In certain embodiments, any of the reverse transcriptase variants provided herein comprises a truncation between positions D497 and I498 of SEQ ID NO: 30.

[0123] In some embodiments, the reverse transcriptase variant comprises the amino acid sequence of SEQ ID NO: 25 (the RT domain of "PE6b"), or an amino acid sequence that is 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 amino acid sequence of SEQ ID NO: 25: ISSSKHTLSQMNKVSNIVKEPELPDIYKEFKDITADTNTEKLPKPIKGLEFEVELTQENYRLPIRNYPLTPVKMQAMNDEINQGLKGGIIRESKAINACPVIFVPRKEGTLRMVVDYRPLNKYVKPNV YPLPLIEQLLAKIQGSTIFTKLDLKSAYHQIRVRKGDEHKLAFRCPRGVFEYLVMPYGISTAPAHFQYFINTILGEAKESHVVCYMDDILIHSKSESEHVKHVKDVLQKLKNANLIINQAKCEFHQSQV KFIGYHISEKGLTPCQENIDKVLQWKQPKNRKELRQFLGSVNYLRKFIPKTSQLTHPLNKLLKKDVRWKWTPTQTQAIENIKQCLVSPPVLRHFDFSKKILLETDVSDVAVGAVLSQKHDDDKYYPVGYYSAKMSKAQLNYSVSDKEMLAIIKSLEHWRHYLESTIEPFKILTDHRNLIGRITNESEPENKRLARWQLFLQDFNFEINYRPGSANHIADALSRIVDETEPIPKDNEDNSINFVNQISI (Sequence ID 25).

[0124] In some embodiments, the PE6b prime editor comprises the amino acid sequence: MKRTADGSEFESPKKKRKV[CAS9]SGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSISSSKHTLSQMNKVSNIVKEPELPDIYKEFKDITADTNTEKLPKPIKGLEFEVELTQENYRLPIRNYPLTPVKMQAMNDEINQGLKGGIIRESKAINACPVIFVPRKEGTLRMVVDYRPLNKYVKPNVYPLPLIEQLLAKIQGSTIFTKLDLKSAYHQIRVRKGDEHKLAFRCPRGVFEYLVMPYGISTAPAHFQYFINTILGEAKESHVVCYMDDILIHSKSESEHVKHVKDVLQKLKNANLIINQAKCEFHQSQVKFIGYHISEKGLTPCQENIDKVLQWKQPKNRKELRQFLGSVNYLRKFIPKTSQLTHPLNKLLKKDVRWKWTPTQTQAIENIKQCLVSPPVLRHFDFSKKILLETDVSDVAVGAVLSQKHDDDKYYPVGYYSAKMSKAQLNYSVSDKEMLAIIKSLEHWRHYLESTIEPFKILTDHRNLIGRITNESEPENKRLARWQLFLQDFNFEINYRPGSANHIADALSRIVDETEPIPKDNEDNSINFVNQISIKRTADGSEFESPKKKRKVPAAKRVKLD (SEQ ID NO: 146, 147), wherein [CAS9] comprises any Cas9 protein (e.g., any of the Cas9 variants disclosed herein).

[0125] In some embodiments, the reverse transcriptase variant comprises the amino acid sequence of SEQ ID NO: 26 (the RT domain of "PE6c"), or an amino acid sequence that is 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 amino acid sequence of SEQ ID NO: 26: ISSSKHTLSQMNKVSNIVKEPELPDIYKEFKDITADTNTEKLPKPIKGLEFEVELTQENYRLPIRNYPLTPVKMQAMNDEINQGLKGGIIRESKAINACPVIFVPRKEGTLRMVVDYRPLNKYVKPNV YPLPLIEQLLAKIQGSTIFTKLDLKSAYHQIRVRKGDEHKLAFRCPRGVFEYLVMPYGIKTAPAHFQYFINTILGEAKESHVVCYMDDILIHSKSESEHVKHVKDVLQKLKNANLIINQAKCEFHQSQV KFLGYHISEKGLTPCQENIDKVLQWKQPKNQKELRQFLGQVNYLRKFIPKTSQLTHPLNKLLKKDVRWKWTPTQTQAIENIKQCLVSPPVLRHFDFSKKILLETDVSDVAVGAVLSQKHDDDKYYPVGYYSAKMSKAQLNYSVSDKEMLAIIKSLEHWRHYLESTIEPFKILTDHRNLIGRITNESENKRLARWQLFLQDFNFEINYRPGSANHIADALSRIVDETEPIPKDNEDNSINFVNQISI (Sequence ID 26).

[0126] In some embodiments, the PE6c prime editor comprises the amino acid sequence:MKRTADGSEFESPKKKRKV[CAS9](SEQ ID NOs: 146, 148), where [CAS9] comprises any Cas9 protein (e.g., any of the Cas9 variants disclosed herein).

[0127] In some embodiments, the reverse transcriptase variant includes the amino acid sequence of SEQ ID NO: 27 (the RT domain of "PE6d"), or an amino acid sequence that is 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 amino acid sequence of SEQ ID NO: 27 TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIH PNVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFCEALHRDLADFRIQHPDLILLQYYDDLLLAATSELDCQQGTRALLQTLGNLGYR ASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLD (Sequence ID 27).

[0128] In some aspects, the PE6d Prime Editor includes the amino acid sequence:[any NLS]-[CAS9]-[SEQ ID NO: 27]-[any NLS], Here, CAS9 comprises any Cas9 protein (e.g., any of the Cas9 variants disclosed herein), “any NLS” comprises one or more nuclear localization signals described herein or known in the art, and each of “]-[” independently comprises any peptide linker described herein or known in the art.

[0129] In some embodiments, the N-terminal NLS of the PE6d prime editor includes a binodelf-type SV40 NLS as defined in Sequence ID No. 95.

[0130] In some embodiments, the C-terminal NLS of the PE6d prime editor includes a binocular SV40 NLS as defined by sequence number 97. In some embodiments, the C-myc NLS of the PE6d prime editor includes a binocular SV40 NLS as defined by sequence number 98. In some embodiments, the C-terminal NLS of the PE6d prime editor includes a sequence SGGS KRTADGSEFESPKKKRKV GSG PAAKRVKLD Includes.

[0131] In some embodiments, the C-terminal NLS of the PE6d prime editor includes a binodermous SV40 NLS as defined in Sequence ID No. 96.

[0132] In some embodiments, the peptide linker connecting the Cas9 and MMLV-RT variants of the PE6d fusion protein includes SEQ ID NO: 80.

[0133] In some embodiments, the peptide linker connecting the Cas9 and MMLV-RT variants of the PE6d fusion protein includes SEQ ID NO: 79.

[0134] In some embodiments, the PE6d prime editor comprises the amino acid sequence:MKRTADGSEFESPKKKRKV[CAS9](SEQ ID NOs: 146, 149), where [CAS9] comprises any Cas9 protein (e.g., any of the Cas9 variants disclosed herein).

[0135] In another aspect, this disclosure provides Cas9 variants including various amino acid substitutions to the amino acid sequence of Streptococcus pyogenes Cas9 nickase (H840A) provided below:

[0136] In some embodiments, Cas9 comprises the amino acid sequence defined in SEQ ID NO: 10.

[0137] In some embodiments, Cas9 comprises the amino acid sequence defined in SEQ ID NO: 11.

[0138] In some embodiments, Cas9 comprises the amino acid sequence defined in SEQ ID NO: 133.

[0139] In some embodiments, the disclosure provides Cas9 variants 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% sequence identity with SEQ ID NO: 2, wherein the Cas9 variant includes amino acid substitutions at positions 775 and 918 with respect to SEQ ID NO: 2, or corresponding substitutions in homologous sequences. In some embodiments, the amino acid substitution at position 775 is a K775X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 775 is a K775R substitution. In some embodiments, the amino acid substitution at position 918 is a K918X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 918 is a K918A substitution. In certain embodiments, the Cas9 variant includes K775R and K918A substitutions with respect to SEQ ID NO: 2.

[0140] In some embodiments, the Disclosure provides Cas9 variants 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% sequence identity with SEQ ID NO: 2, wherein the Cas9 variant includes amino acid substitutions at positions 99, 471, 632, 645, and 721 with respect to SEQ ID NO: 2, or corresponding substitutions in homologous sequences. In some embodiments, the amino acid substitution at position 99 is an H99X substitution, where X is any amino acid. In some embodiments, the amino acid substitution at position 99 is an H99R substitution. In some embodiments, the amino acid substitution at position 471 is an E471X substitution, where X is any amino acid. In some embodiments, the amino acid substitution at position 471 is an E471K substitution. In some embodiments, the amino acid substitution at position 632 is an I632X substitution, where X is any amino acid. In some embodiments, the amino acid substitution at position 632 is an I632V substitution. In some embodiments, the amino acid substitution at position 645 is a D645X substitution, where X is any amino acid. In certain embodiments, the amino acid substitution at position 645 is a D645N substitution. In some embodiments, the amino acid substitution at position 721 is an H721X substitution, where X is any amino acid. In certain embodiments, the amino acid substitution at position 721 is an H721Y substitution. In some embodiments, the Cas9 variant further includes an amino acid substitution at position 654 for SEQ ID NO: 2. In some embodiments, the amino acid substitution at position 654 is an R654X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 654 is an R654C substitution. In some embodiments, the Cas9 variant further includes an amino acid substitution at position 918 for SEQ ID NO: 2. In some embodiments, the amino acid substitution at position 918 is a K918X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 918 is a K918A substitution.

[0141] In some embodiments, the Disclosure provides Cas9 variants 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% sequence identity with SEQ ID NO: 2, wherein the Cas9 variant includes amino acid substitutions at positions 99, 471, and 632 with respect to SEQ ID NO: 2, or corresponding substitutions in homologous sequences. In some embodiments, the amino acid substitution at position 99 is an H99X substitution, where X is any amino acid other than the wild type. In some embodiments, the amino acid substitution at position 99 is an H99R substitution. In some embodiments, the amino acid substitution at position 471 is an E471X substitution, where X is any amino acid other than the wild type. In some embodiments, the amino acid substitution at position 471 is an E471K substitution. In some embodiments, the amino acid substitution at position 632 is an I632X substitution, where X is any amino acid other than the wild type. In some embodiments, the amino acid substitution at position 632 is an I632V substitution. In certain embodiments, the Cas9 variant includes amino acid substitutions H99R, E471K, and I632V relative to SEQ ID NO: 2. In some embodiments, the Cas9 variant further includes an amino acid substitution at position 721 relative to SEQ ID NO: 2. In some embodiments, the amino acid substitution at position 721 is an H721X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 721 is an H721K substitution.

[0142] In some embodiments, the present disclosure provides a Cas9 variant 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% sequence identity to SEQ ID NO: 2, wherein the Cas9 variant comprises amino acid substitutions at positions 471 and 918 relative to SEQ ID NO: 2, or corresponding substitutions in a homologous sequence. In some embodiments, the amino acid substitution at position 471 is an E471X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 471 is an E471K substitution. In some embodiments, the amino acid substitution at position 918 is a K918X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 918 is a K918A substitution. In certain embodiments, the Cas9 variant comprises the amino acid substitutions E471K and K918A relative to SEQ ID NO: 2.

[0143] In some embodiments, the present disclosure provides a Cas9 variant 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% sequence identity to SEQ ID NO: 2, wherein the Cas9 variant comprises amino acid substitutions at positions 753 and 1151 relative to SEQ ID NO: 2, or corresponding substitutions in a homologous sequence. In some embodiments, the amino acid substitution at position 753 is an R753X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 753 is an R753G substitution. In some embodiments, the amino acid substitution at position 1151 is a K1151X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 1151 is a K1151E substitution. In certain embodiments, the Cas9 variant comprises the amino acid substitutions R753G and K1151E relative to SEQ ID NO: 2.

[0144] In some embodiments, the Disclosure provides Cas9 variants 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% sequence identity with SEQ ID NO: 2, wherein the Cas9 variants include one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions at positions selected from the group consisting of 260, 298, 395, 769, 778, 1014, 1034, 1100, 1106, 1138, 1152, and 1320, or corresponding substitutions in homologous sequences. In some embodiments, the amino acid substitution at position 260 is an E260X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 260 is an E260K substitution. In some embodiments, the amino acid substitution at position 298 is a D298X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 298 is a D298N substitution. In some embodiments, the amino acid substitution at position 395 is an R395X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 395 is an R395C substitution. In some embodiments, the amino acid substitution at position 769 is a T769X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 769 is a T769P substitution. In some embodiments, the amino acid substitution at position 778 is an R778X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 778 is an R778Q substitution. In some embodiments, the amino acid substitution at position 1014 is a K1014X substitution, where X is any amino acid other than the wild type. In some embodiments, the amino acid substitution at position 1014 is a K1014E substitution. In some embodiments, the amino acid substitution at position 1034 is an A1034X substitution, where X is any amino acid other than the wild type.In certain embodiments, the amino acid substitution at position 1034 is an A1034E substitution. In some embodiments, the amino acid substitution at position 1100 is a V1100X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 1100 is a V1100I substitution. In some embodiments, the amino acid substitution at position 1106 is an S1106X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 1106 is an S1106F substitution. In some embodiments, the amino acid substitution at position 1138 is a T1138X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 1138 is a T1138A substitution. In some embodiments, the amino acid substitution at position 1152 is a G1152X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 1152 is a G1152E substitution. In some embodiments, the amino acid substitution at position 1320 is an A1320X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 1320 is an A1320T substitution. In certain embodiments, the Cas9 variant includes one or more amino acid substitutions E260K, D298N, R395C, T769P, R778Q, K1014E, A1034E, V1100I, S1106F, T1138A, G1152E, and A1320T. In certain embodiments, the Cas9 variant includes the amino acid substitutions E260K, D298N, R395C, T769P, R778Q, K1014E, A1034E, V1100I, S1106F, T1138A, G1152E, and A1320T. In some embodiments, the Cas9 variant further includes one or more additional amino acid substitutions at positions selected from the group consisting of 102, 753, 804, and 1003 relative to SEQ ID NO: 2.In some embodiments, the amino acid substitution at position 102 is an E102X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 102 is an E102K substitution. In some embodiments, the amino acid substitution at position 753 is an R753X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 753 is an R753G substitution. In some embodiments, the amino acid substitution at position 804 is a T804X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 804 is a T804A substitution. In some embodiments, the amino acid substitution at position 1003 is a K1003X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 1003 is a K1003R substitution. In some embodiments, Cas9 variants include amino acid substitutions at positions 102, 395, 753, 778, and 1100; 753, 769, 1034, and 1320; 298, 753, 1034, and 1138; 102, 260, 395, 753, 778, 804, 1003, 1100, 1106, and 1152 with respect to SEQ ID NO: 2. In certain embodiments, Cas9 variants include amino acid substitutions at positions E102K, R395C, R753G, R778Q, and V1100I; R753G, T769P, A1034E, and A1320T; D298N, R753G, A1034E, and T1138A; E102K, E260K, R395C, R753C, R778Q, T804A, K1003R, V1100I, S1106F, and G1152E; or E102K, E260K, R395C, R753G, R778Q, T804A, K1003R, K1014E, V1100I, S1106F, and G1152E with respect to SEQ ID NO: 2.

[0145] In some embodiments, the Disclosure provides Cas9 variants 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% sequence identity with SEQ ID NO: 2, wherein the Cas9 variant includes amino acid substitutions at positions 23 and 754 with respect to SEQ ID NO: 2, or corresponding substitutions in homologous sequences. In some embodiments, the amino acid substitution at position 23 is a D23X substitution, where X is any amino acid other than the wild type (i.e., D). In certain embodiments, the amino acid substitution at position 23 is a D23G substitution. In some embodiments, the amino acid substitution at position 754 is an H754X substitution, where X is any amino acid other than the wild type (i.e., H). In certain embodiments, the amino acid substitution at position 754 is an H754R substitution.

[0146] In certain embodiments, a Cas9 variant includes an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 28 (the Cas9 domain of "PE6e"), or an amino acid sequence that is 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 amino acid sequence of SEQ ID NO: 28

[0147] Here, [reverse transcriptase] includes any reverse transcriptase (e.g., any of the reverse transcriptase variants disclosed herein).

[0148] In certain embodiments, a Cas9 variant includes an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 48 (the Cas9 domain of "PE6f"), or an amino acid sequence that is 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 amino acid sequence of SEQ ID NO: 48:

[0149] Here, [reverse transcriptase] includes any reverse transcriptase (e.g., any of the reverse transcriptase variants disclosed herein).

[0150] In certain embodiments, a Cas9 variant contains an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 49 (the Cas9 domain of "PE6g"), or an amino acid sequence that is 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 amino acid sequence of SEQ ID NO: 49:

[0151] In some embodiments, PE6g Prime Editor has the following amino acid sequence: Here, [reverse transcriptase] includes any reverse transcriptase (e.g., any of the reverse transcriptase variants disclosed herein).

[0152] In certain embodiments, a Cas9 variant contains an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 145, or an amino acid sequence that is 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 amino acid sequence of SEQ ID NO: 145:

[0153] In some embodiments, any of the PE6 prime editors provided herein may include the architecture of PEmax. In some embodiments, any of the PE6 prime editors provided herein may include one or more additional amino acid substitutions relative to the wild-type amino acid sequence (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more additional amino acid substitutions), such as those incorporated into the Cas9 protein of PEmax or the MMLV reverse transcriptase of PEmax.

[0154] In some aspects, this disclosure provides a fusion protein comprising one of the Cas9 variants provided herein and an effector domain. In certain embodiments, the effector domain comprises nuclease activity, nickas activity, recombinase activity, deaminase activity, methyltransferase activity, methylase activity, acetylase activity, acetyltransferase activity, transcriptional activating activity, transcriptional repressing activity, or polymerase activity.

[0155] It should be understood that any amino acid mutation described herein (e.g., E60K), from a first amino acid residue (e.g., E) to a second amino acid residue (e.g., K), may encompass mutations from the first amino acid residue to an amino acid residue similar to the second amino acid residue (e.g., a conserved amino acid). For example, a mutation in an amino acid with a hydrophobic side chain (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan) may be a mutation to a second amino acid with a different hydrophobic side chain (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan). For example, a mutation from alanine to threonine may also be a mutation from alanine to an amino acid similar in size and chemical properties to threonine, such as serine. As another example, a mutation in an amino acid with a positively charged side chain (e.g., arginine, histidine, or lysine) may result in a mutation in a second amino acid with a different positively charged side chain (e.g., arginine, histidine, or lysine). As yet another example, a mutation in an amino acid with a polar side chain (e.g., serine, threonine, asparagine, or glutamine) may result in a mutation in a second amino acid with a different polar side chain (e.g., serine, threonine, asparagine, or glutamine). Additional similar amino acid pairs include, but are not limited to, phenylalanine and tyrosine; asparagine and glutamine; methionine and cysteine; aspartic acid and glutamic acid; and arginine and lysine.

[0156] Those skilled in the art will recognize that such conservative amino acid substitutions are likely to have little effect on protein structure and are likely to be well tolerated without impairing function. In some embodiments, any of the amino acid mutations provided herein from one amino acid to threonine may be amino acid mutations to serine. In some embodiments, any of the amino acid mutations provided herein from one amino acid to arginine may be amino acid mutations to lysine. In some embodiments, any of the amino acid mutations provided herein from one amino acid to isoleucine may be amino acid mutations to alanine, valine, methionine, or leucine. In some embodiments, any of the amino acid mutations provided herein from one amino acid to lysine may be amino acid mutations to arginine. In some embodiments, any of the amino acid mutations provided herein from one amino acid to aspartic acid may be amino acid mutations to glutamic acid or asparagine. In some embodiments, any of the amino acid mutations provided herein from one amino acid to valine may be amino acid mutations to alanine, isoleucine, methionine, or leucine. In some embodiments, any of the amino acid mutations provided herein from one amino acid to glycine may be amino acid mutations to alanine. However, it should be understood that additional conserved amino acid residues will be recognized by those skilled in the art, and any of the amino acid mutations to other conserved amino acid residues are also within the scope of this disclosure.

[0157] In several respects, this disclosure provides reverse transcriptase variants that include mutations at homologous positions on another reverse transcriptase that correspond to any or any combination thereof of the mutations disclosed herein. Examples of additional reverse transcriptases include, but are not limited to, the following: [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11]

[0158] Additional reverse transcriptases are known in this field and will be readily apparent to those skilled in the art.

[0159] In several respects, this disclosure provides Cas9 variants that include mutations at homologous positions on another Cas9 protein that correspond to any or any combination thereof of the mutations disclosed herein. Examples of additional Cas9 proteins include, but are not limited to, the following: [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12]

[0160] The additional Cas9 protein is known in this field and will be readily apparent to those skilled in the art.

[0161] In some aspects, this disclosure provides a prime editor comprising any of the reverse transcriptase variants described herein and / or any of the Cas9 variants. In some embodiments, this disclosure provides a prime editor comprising any of the reverse transcriptase variants provided herein and napDNAbp. In certain embodiments, napDNAbp comprises a Cas9 protein (e.g., Cas9 nickase). In some embodiments, the Cas9 protein comprises an amino acid sequence that is 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 any of the Cas9 proteins of SEQ ID NOs. 2, 6, 8, 9, 12-24, or 133, or any one of SEQ ID NOs. 2, 6, 8, 9, 12-24, or 133. In certain embodiments, the Cas9 protein contains the amino acid sequence of SEQ ID NO: 133, or an amino acid sequence that is 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 SEQ ID NO: 133. In certain embodiments, napDNAbp contains one of the Cas9 variants disclosed herein. In some embodiments, the disclosure provides a prime editor comprising one of the Cas9 variants provided herein and a polymerase. In some embodiments, the polymerase is a reverse transcriptase. In certain embodiments, the reverse transcriptase is one of the reverse transcriptase variants provided herein.

[0162] In some embodiments, the reverse transcriptase variant used in the prime editor includes various amino acid substitutions to the amino acid sequence of Escherichia coli Ec48 reverse transcriptase provided below (SEQ ID NO: 7): GRPYVTLNLNGMFMDKFKPYSKSNAPITTLEKLSKALSISVEELKAIAELSLDEKYTLKEIPKIDGSKRIVYSLHPKMRLLQSRINKRIKRIFKELVVFPSFLFGSVPSKNDVLNSNVKRDYVSCAKAHCGAKTVLKVDISNFFDNIHRDLVRSVFEEILHIKDEALEYLVDICTKDDFVVQGALTSSYIATLCLFAVEGDVVRRAQRKG LVYTRLVDDITVSSKISNYDFSQMQSHIERMLSEHDLPINKHKTKIFHCSSEPIKVHGLRVDYDSPRLPSDEVKRIRASIHNLKLLAAKNNTKTSVAYRKEFNRCMGRVNKLGRVGHEKYESFKKQLQAIKPMPSKRDVAVIDAAIKSLELSYSKGNQNKHWYKRKYDLTRYKMIILTRSESFKEKLECFKSRLASLKPL(Sequence ID 7)

[0163] In some embodiments, the reverse transcriptase variant used in the prime editor comprises a sequence 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% sequence identity with SEQ ID NO: 7, wherein the reverse transcriptase comprises amino acid substitutions at positions 60, 87, 165, 243, 267, 279, 318, and 343 with respect to SEQ ID NO: 7. In some embodiments, the amino acid substitution at position 60 is an E60X substitution, where X is any amino acid other than the wild type. In some embodiments, the amino acid substitution at position 60 is an E60K substitution. In some embodiments, the amino acid substitution at position 87 is a K87X substitution, where X is any amino acid other than the wild type. In some embodiments, the amino acid substitution at position 87 is a K87E substitution. In some embodiments, the amino acid substitution at position 165 is an E165X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 165 is an E165D substitution. In some embodiments, the amino acid substitution at position 243 is a D243X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 243 is a D243N substitution. In some embodiments, the amino acid substitution at position 267 is an R267X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 267 is an R267I substitution. In some embodiments, the amino acid substitution at position 279 is an E279X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 279 is an E279K substitution. In some embodiments, the amino acid substitution at position 318 is a K318X substitution, where X is any amino acid other than the wild type. In certain embodiments, the amino acid substitution at position 318 is a K318E substitution. In some embodiments, the amino acid substitution at position 343 is a K343X substitution, where X is any amino acid other than the wild-type amino acid.In certain embodiments, the amino acid substitution at position 343 is a K343N substitution. In certain embodiments, the reverse transcriptase variant includes amino acid substitutions E60K, K87E, E165D, D243N, R267I, E279K, K318E, and K343N relative to SEQ ID NO: 7.

[0164] In certain embodiments, the reverse transcriptase variant contains an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 50 (the RT domain of "PE6a"), or an amino acid sequence that is 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 amino acid sequence of SEQ ID NO: 50 GRPYVTLNLNGMFMDKFKPYSKSNAPITTLEKLSKALSISVEELKAIAELSLDEKYTLKKIPKIDGSKRIVYSLHPKMRLLQSRINERIFKELVVFPSFLFGSVPSKNDVLNSNVKRDYVSCAKAHCGAKTVLKVDISNFFDNIHRDLVRSVFEEILHIKDEALDYLVDICTKDDFVVQGALTSSYIATLCLFAVEGDVVRRAQRKG LVYTRLVDDITVSSKISNYDFSQMQSHIERMLSEHNLPINKHKTKIFHCSSEPIKVHGLIVDYDSPRLPSDKVKRIRASIHNLKLLAAKNNTKTSVAYRKEFNRCMGRVNELGRVGHEKYESFKKQLQAIKPMPSNRDVAVIDAAIKSLELSYSKGNQNKHWYKRKYDLTRYKMIILTRSESFKEKLECFKSRLASLKPL(Sequence ID 50)

[0165] In some embodiments, the PE6a Prime Editor uses the amino acid sequence: MKRTADGSEFESPKKKRKV[CAS9]SGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSGRPYVTLNLNGMFMDKFKPYSKSNAPITTLEKLSKALSISVEELKAIAELSLDEKYTLKKIPKIDGSKRIVYSLHPKMRLLQ SRINERIFKELVVFPSFLFGSVPSKNDVLNSNVKRDYVSCAKAHCGAKTVLKVDISNFFDNIHRDLVRSVFEEILHIKDEALDYLVDICTKDDFVVQGALTSSYIATLCLFAVEGDVVRRAQRKGLVYTRLVDDITVSSKIS The sequence includes NYDFSQMQSHIERMLSEHNLPINKHKTKIFHCSSEPIKVHGLIVDYDSPRLPSDKVKRIRASIHNLKLLAAKNNTKTSVAYRKEFNRCMGRVNELGRVGHEKYESFKKQLQAIKPMPSNRDVAVIDAAIKSLELSYSKGNQNKHWYKRKYDLTRYKMIILTRSESFKEKLECFKSRLASLKPLKRTADGSEFESPKKKRKVPAAKRVKLD (SEQ ID NOs: 146, 154), where [CAS9] comprises any Cas9 protein (e.g., any of the Cas9 variants described herein).

[0166] In some embodiments, the prime editors provided herein may include both a reverse transcriptase variant and a Cas9 variant provided herein, or both reverse transcriptase variants and Cas9 variants that are 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 either of those provided herein. For example, this disclosure envisions a prime editor including the reverse transcriptase variant of SEQ ID NO: 50 (PE6a) and the Cas9 variant of SEQ ID NO: 28 (PE6e), or reverse transcriptase variants and Cas9 variants that are 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 SEQ ID NO: 50 and SEQ ID NO: 28. In some embodiments, the Prime Editor includes the reverse transcriptase variant of SEQ ID NO: 50 (PE6a) and the Cas9 variant of SEQ ID NO: 48 (PE6f), or a reverse transcriptase variant and Cas9 variant that are 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 SEQ ID NO: 50 and SEQ ID NO: 48. In some embodiments, the Prime Editor includes the reverse transcriptase variant of SEQ ID NO: 50 (PE6a) and the Cas9 variant of SEQ ID NO: 49 (PE6g), or a reverse transcriptase variant and Cas9 variant that are 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 SEQ ID NO: 50 and SEQ ID NO: 49. In some embodiments, the prime editor includes the reverse transcriptase variant of SEQ ID NO: 25 (PE6b) and the Cas9 variant of SEQ ID NO: 28 (PE6e), or a reverse transcriptase variant and Cas9 variant that are 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 SEQ ID NO: 25 and SEQ ID NO: 28.In some embodiments, the Prime Editor includes the reverse transcriptase variant of SEQ ID NO: 25 (PE6b) and the Cas9 variant of SEQ ID NO: 48 (PE6f), or a reverse transcriptase variant and Cas9 variant that are 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 SEQ ID NO: 25 and SEQ ID NO: 48. In some embodiments, the Prime Editor includes the reverse transcriptase variant of SEQ ID NO: 25 (PE6b) and the Cas9 variant of SEQ ID NO: 49 (PE6g), or a reverse transcriptase variant and Cas9 variant that are 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 SEQ ID NO: 25 and SEQ ID NO: 49. In some embodiments, the Prime Editor includes the reverse transcriptase variant of SEQ ID NO: 26 (PE6c) and the Cas9 variant of SEQ ID NO: 28 (PE6e), or a reverse transcriptase variant and Cas9 variant that are 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 SEQ ID NO: 26 and SEQ ID NO: 28. In some embodiments, the Prime Editor includes the reverse transcriptase variant of SEQ ID NO: 26 (PE6c) and the Cas9 variant of SEQ ID NO: 48 (PE6f), or a reverse transcriptase variant and Cas9 variant that are 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 SEQ ID NO: 26 and SEQ ID NO: 48. In some embodiments, the Prime Editor includes the reverse transcriptase variant of SEQ ID NO: 26 (PE6c) and the Cas9 variant of SEQ ID NO: 49 (PE6g), or a reverse transcriptase variant and Cas9 variant that are 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 SEQ ID NO: 26 and SEQ ID NO: 49.In some embodiments, the Prime Editor includes the reverse transcriptase variant of SEQ ID NO: 27 (PE6d) and the Cas9 variant of SEQ ID NO: 28 (PE6e), or a reverse transcriptase variant and Cas9 variant that are 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 SEQ ID NO: 27 and SEQ ID NO: 28. In some embodiments, the Prime Editor includes the reverse transcriptase variant of SEQ ID NO: 27 (PE6d) and the Cas9 variant of SEQ ID NO: 48 (PE6f), or a reverse transcriptase variant and Cas9 variant that are 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 SEQ ID NO: 27 and SEQ ID NO: 48. In some embodiments, the Prime Editor includes the reverse transcriptase variant of SEQ ID NO: 27 (PE6d) and the Cas9 variant of SEQ ID NO: 49 (PE6g), or a reverse transcriptase variant and Cas9 variant that are 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 SEQ ID NO: 27 and SEQ ID NO: 49.

[0167] Nuclear localization sequence (NLS) In various embodiments, the prime editors described herein may include one or more nuclear localization sequences (NLSs) that help facilitate the translocation of proteins into the cell nucleus. Such sequences are well known in the art and may include the following examples: [Table 4-1] [Table 4-2]

[0168] The NLS examples above are not limiting. The prime editors disclosed herein may include any known NLS sequences that encompass either of those described in Cokol et al., “Finding nuclear localization signals,” EMBO Rep., 2000, 1(5):411-415 and Freitas et al., “Mechanisms and Signals for the Nuclear Import of Proteins,” Current Genomics, 2009, 10(8):550-7. Each of these is incorporated herein by reference.

[0169] In various embodiments, the prime editor described herein further comprises one or more (preferably at least two) nuclear localization sequences. In certain embodiments, the prime editor comprises at least two NLSs. In embodiments with at least two NLSs, the NLSs may be the same NLS or they may be different NLSs. In some embodiments, one or more NLSs are binocular NLSs ("bpNLS"). In certain embodiments, the prime editor comprises two binocular NLSs. In some embodiments, the prime editor comprises more than two binocular NLSs.

[0170] The location of the NLS fusion can be at the N-terminus, C-terminus, or within the sequence of the prime editor (for example, inserted between the encoded napDNAbp component (e.g., Cas9) and the polymerase domain (e.g., reverse transcriptase)).

[0171] The NLS could be any known NLS sequence in this field. The NLS could also be any future NLS discovered for nuclear localization. The NLS could also be any naturally occurring NLS or any non-naturally occurring NLS (e.g., an NLS with one or more desired mutations).

[0172] The term “nuclear localization sequence” or “NLS” refers to an amino acid sequence that facilitates the transport of proteins into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and will be obvious to those skilled in the art. For example, NLS sequences are described in Plank et al., International PCT Application PCT / EP2000 / 011690, filed November 23, 2000, published May 31, 2001, as WO / 2001 / 038547, the contents of which are incorporated herein by reference. In some embodiments, the NLS includes the amino acid sequence PKKKRKV (SEQ ID NO: 94), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 99), KRTADGSEFESPKKKRKV (SEQ ID NO: 97), or KRTADGSEFEPKKKRKV (SEQ ID NO: 106). In other embodiments, NLS comprises the amino acid sequences NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 107), PAAKRVKLD (SEQ ID NO: 98), RQRRNELKRSF (SEQ ID NO: 108), or NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 109).

[0173] In one aspect of this disclosure, the prime editors described herein may be modified by one or more nuclear localization sequences (NLSs), preferably at least two NLSs. In certain embodiments, the prime editor is modified by two or more NLSs. This disclosure intends to use any nuclear localization sequence known in the art at the time of this disclosure, or any nuclear localization sequence identified in the state of the art after the time of this application, or otherwise made available. Typical nuclear localization sequences are peptide sequences that direct proteins to the nucleus of the cell in which the sequence is expressed. Nuclear localization signals are overwhelmingly basic, can be located almost anywhere on the amino acid sequence of a protein, and generally consist of short sequences of 4 to 8 amino acids (Autieri & Agrawal, (1998) J.Biol.Chem.273:14731-37, incorporated herein by reference), and are typically rich in lysine and arginine residues (Magin et al., (2000) Virology 274:11-16, incorporated herein by reference). Nuclear localization sequences often contain proline residues. Various nuclear localization sequences have been identified and are used to accomplish the transport of biological molecules from the cytoplasm to the nucleus of a cell. See, for example, Tinland et al., (1992) Proc.Natl.Acad.Sci.USA89:7442-46; Moede et al., (1999) FEBS Lett.461:229-34. This is incorporated herein by reference. Nuclear pore proteins are currently thought to be involved in the transport.

[0174] Most NLSs can be classified into three common groups: (i) monosegmental NLSs, exemplified by the SV40 large T antigen NLS (PKKKRKV (SEQ ID NO: 94)); (ii) bisegmental motifs consisting of two basic domains separated by a variable number of spacer amino acids, exemplified by the Xenopus nucleoplasmin NLS (KRXXXXXXXXXXKKKL (SEQ ID NO: 110)); and (iii) non-classical sequences, such as M9 of the hnRNP Al protein, influenza virus nucleoprotein NLS, and yeast Gal4 protein NLS (Robbins, J. et al., Cell 1991, 64(3), 615-623).

[0175] Nuclear localization sequences appear at various points in the amino acid sequence of a protein. NLSs have been identified at the N-terminus, C-terminus, and central region of a protein. Therefore, this disclosure provides a prime editor that can be modified by one or more NLSs at the C-terminus and / or N-terminus and in the internal region of the prime editor. Longer sequence residues that do not function as component NLS residues should be selected so as not to interfere with the nuclear localization signal itself, for example, tonically or sterically. Thus, there is no strict limitation on the composition of sequences containing NLSs, although in practice, such sequences may be functionally limited in length and composition.

[0176] This disclosure intends to provide any preferred means for modifying a prime editor to include one or more NLSs. In one aspect, a prime editor may be manipulated to express a prime editor that is translationally fused to one or more NLSs at its N-terminus or C-terminus (or both), i.e., to form a prime editor-NLS fusion construct. In another aspect, a nucleotide sequence encoding a prime editor may be genetically modified to incorporate a leading frame encoding one or more NLSs into the internal region of the encoded prime editor. In addition, the NLS may include various amino acid linker or spacer regions encoded between the prime editor and the NLS amino acid sequence attached to the N-terminus, C-terminus, or internally, for example, the central region of the protein. Therefore, this disclosure also provides, among other components, nucleotide constructs, vectors, and host cells for expressing a fusion protein containing a prime editor and one or more NLSs.

[0177] The prime editors described herein may also include one or more linkers, e.g., a nuclear localized sequence linked to the prime editor through a polymer, amino acid, nucleic acid, polysaccharide, chemical, or nucleic acid linker element. Linkers within the scope contemplated of this disclosure are not intended to have any limitations and may be any preferred type of molecule (e.g., a polymer, amino acid, polysaccharide, nucleic acid, lipid, or any synthetic chemical linker domain) and may be linked to the prime editor by any preferred strategy that enables the formation of a bond (e.g., covalent linkage, hydrogen bond) between the prime editor and one or more NLSs.

[0178] In some embodiments, the Prime Editor provided herein includes an NLS containing the amino acid sequence of SEQ ID NO: 95, or an NLS containing an amino acid sequence that is 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%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 95. In some embodiments, the Prime Editor provided herein includes an NLS containing the amino acid sequence of SEQ ID NO: 97, or an NLS containing an amino acid sequence that is 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%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the prime editor provided herein includes an NLS containing the amino acid sequence of SEQ ID NO: 98, or an amino acid sequence that is 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%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 98.In certain embodiments, the Prime Editor provided herein includes a first NLS containing the amino acid sequence of SEQ ID NO: 95, or an amino acid sequence identical to 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%, or at least 99% of the amino acid sequence of SEQ ID NO: 95; a second NLS containing the amino acid sequence of SEQ ID NO: 97, or an amino acid sequence identical to 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%, or at least 99% of the amino acid sequence of SEQ ID NO: 97; and a third NLS containing the amino acid sequence of SEQ ID NO: 98, or an amino acid sequence identical to 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%, or at least 99% of the amino acid sequence of SEQ ID NO: 98.

[0179] Linker In various embodiments, the napDNAbp and reverse transcriptase of the prime editor provided herein may be provided in trans or otherwise unfused to one another. In other embodiments, the prime editor provided herein comprises napDNAbp and reverse transcriptase fused to one another, for example, via one or more linkers. As defined above, the term “linker” as used herein means a chemical group or molecule that links two molecules or parts, e.g., a binding domain and a cleavage domain of a nuclease. In some embodiments, a linker links the gRNA binding domain of an RNA-programmed nuclease and a polymerase (e.g., reverse transcriptase). In some embodiments, a linker links a Cas9 protein and a reverse transcriptase (e.g., any of the Cas9 variants provided herein and / or any of the reverse transcriptase variants provided herein). Typically, a linker is located between or flanked by two groups, molecules, or other parts, and is linked to one each via a covalent bond, thus linking the two. In some embodiments, the linker is an amino acid or a group of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical part. In some embodiments, the linker is 5 to 100 amino acids long, 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 long. Longer or shorter linkers are also conceived.

[0180] The linker can be as simple as a covalent bond, or it can be a polymer linker with a length of many atoms. In certain embodiments, the linker is a polypeptide or amino acid-based. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is covalent (e.g., carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is an amide-linked carbon-nitrogen bond. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises monomers, dimers, or polymers of aminoalkanoic acids. In certain embodiments, the linker contains aminoalkanoic acids (e.g., glycine, ethaneic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutyric acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises an amino acid. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include a functionalized moiety to facilitate the attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any of the electrophiles may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0181] In some other embodiments, the linker is an amino acid sequence (GGGGS) n (Sequence ID 84), (G) n(Sequence ID 85), (EAAAK) n (Sequence 86), (GGS) n (Sequence No. 87), (SGGS) n (Sequence ID 81), (XP) n (Sequence ID 88), or any combination thereof, where n is an integer between 1 and 30 independently, and X is any amino acid. In some embodiments, the linker is the amino acid sequence (GGS) n The linker includes (SEQ ID NO: 87), where n is 1, 3, or 7. In some embodiments, the linker includes the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 89). In some embodiments, the linker includes the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 90). In some embodiments, the linker includes the amino acid sequence SGGSGGSGGS (SEQ ID NO: 91). In some embodiments, the linker includes the amino acid sequence SGGS (SEQ ID NO: 82). In other embodiments, the linker includes the amino acid sequence SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSSGGS (SEQ ID NO: 83, 60AA). In some embodiments, the linker comprises the amino acid sequence GGS, GGSGGS (SEQ ID NO: 92), GGSGGSGGS (SEQ ID NO: 93), SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 80), SGSETPGTSESATPES (SEQ ID NO: 89), or SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSSGGS (SEQ ID NO: 83).

[0182] In certain embodiments, a linker may be used to link any of the peptides or peptide domains or portions of the present invention (e.g., napDNAbp linked to or fused to a reverse transcriptase domain, and / or napDNAbp linked to one or more NLSs). Furthermore, any of the domains of the prime editors described herein may be linked to one another through any of the linkers described herein.

[0183] PegRNA The prime editing systems and methods described herein intend to use any suitable pegRNA to introduce a recombinase recognition site onto a target DNA sequence, such as a genome, using prime editing.

[0184] PEgRNA architecture In some embodiments, the extended guide RNA or pegRNA used in the prime editing systems and methods disclosed herein comprises a spacer sequence (e.g., a spacer sequence of approximately 20 nt) and a gRNA core region that binds to napDNAbp. In some embodiments, the pegRNA comprises an extended RNA fragment, i.e., an extended arm, i.e., a 5' extension, at its 5' end. In some embodiments, the 5' extension comprises a DNA synthesis template sequence, a primer binding site, and an optional 5-20 nucleotide linker sequence. The RT primer binding site hybridizes to the free 3' end, which is formed after a nick is formed on the non-target strand of the R loop, thereby priming the reverse transcriptase for DNA polymerization in the 5'-3' direction.

[0185] In another embodiment, the extended guide RNA (i.e., pegRNA) used in the prime editing systems and methods provided herein comprises a spacer sequence (e.g., a spacer sequence of approximately 20 nt) and a gRNA core that binds to napDNAbp. In some embodiments, the pegRNA comprises an extended RNA fragment, i.e., an extended arm, i.e., a 3' extension, at its 3' end. In some embodiments, the 3' extension comprises a DNA synthesis template sequence and a reverse transcription primer binding site. The RT primer binding site hybridizes to the free 3' end formed after a nick is formed on the non-target strand of the R loop, thereby priming the reverse transcriptase for DNA polymerization in the 5'-3' direction.

[0186] In another embodiment, the extended guide RNA (i.e., pegRNA) used in the prime editing systems and methods provided herein comprises a spacer sequence (e.g., a spacer sequence of approximately 20 nt) and a gRNA core that binds to napDNAbp. In some embodiments, the pegRNA comprises an extended RNA fragment, i.e., an extended arm, i.e., an intramolecular extension, at an intermolecular position within the gRNA core. In some embodiments, the intramolecular extension comprises a DNA synthesis template sequence and a reverse transcription primer binding site. The RT primer binding site hybridizes to the free 3' end formed after a nick is formed on the non-target strand of the R loop, thereby priming the reverse transcriptase for DNA polymerization in the 5'-3' direction.

[0187] In one embodiment, the location of the intermolecular RNA extension is not on the spacer sequence of the guide RNA. In another embodiment, the location of the intermolecular RNA extension is on the gRNA core. In yet another embodiment, the location of the intermolecular RNA extension is anywhere within the guide RNA molecule, with the exception of within the spacer sequence, or at a location that blocks the spacer sequence. In one embodiment, the intermolecular RNA extension is inserted downstream from the 3' end of the spacer sequence. In another embodiment, the intermolecular RNA extension is inserted at least 1 nucleotide, at least 2 nucleotides, 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 nucleotides, 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 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides downstream from the 3' end of the spacer sequence.

[0188] In another embodiment, the intermolecular RNA extension is inserted onto a gRNA core, which refers to a portion of a conventional guide RNA containing a tracrRNA that binds to and / or interacts with a napDNAbp, such as the Cas9 protein or its equivalent (i.e., a different napDNAbp). Preferably, the insertion of the intermolecular RNA extension does not block or minimizes the interaction between the tracrRNA portion and the napDNAbp.

[0189] The length of the RNA extension (which includes at least the RT template and primer binding site) can be any useful length. In various embodiments, the RNA extension is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, 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 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides.

[0190] The RT template sequence may also be of any preferred length. For example, the RT template sequence may have a length of 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 nucleotides, 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, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides.

[0191] In yet another embodiment, the reverse transcription primer binding site 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 nucleotides, 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, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides.

[0192] In another embodiment, any linker or spacer 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 nucleotides, 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, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides.

[0193] The RT template sequence, in certain embodiments, encodes a single-stranded DNA molecule that is homologous to the non-target strand (and therefore complementary to the corresponding site on the target strand), but includes, for example, one or more nucleotide changes for introducing a recombinase recognition sequence onto the target DNA molecule. These one or more nucleotide changes may include one or more single-nucleotide changes, one or more deletions, and / or one or more insertions.

[0194] The single-stranded DNA product synthesized from the RT template sequence is homologous to the non-target strand, with the exception that it contains one or more nucleotide changes. The single-stranded DNA product of the RT template sequence hybridizes with the complementary target strand sequence in equilibrium, thereby displacing the homologous endogenous target strand sequence. The displaced endogenous strand can, in some embodiments, be called a 5' endogenous DNA flap species. This 5' endogenous DNA flap species can be removed by a 5' flap endonuclease (e.g., FEN1), and the single-stranded DNA product now hybridized with the endogenous target strand can be ligated, thereby creating a mismatch between the endogenous sequence and the newly synthesized strand. This mismatch can be resolved by the cell's innate DNA repair and / or replication processes.

[0195] In various embodiments, the nucleotide sequence of the RT template sequence corresponds to a nucleotide sequence of the non-target strand that is substituted as a 5' flap species and overlaps with the site to be edited.

[0196] In various embodiments of the extended guide RNA, the DNA synthesis template sequence may encode a single-stranded DNA flap complementary to an endogenous DNA sequence adjacent to the nick site, where the single-stranded DNA flap contains the desired nucleotide alteration. The single-stranded DNA flap may strand-displace the endogenous single-stranded DNA at the nick site. The strand-displaced endogenous single-stranded DNA at the nick site may have a 5' end and form an endogenous flap, which can be excised by the cell. In various embodiments, excision of the 5' end endogenous flap may help drive product formation because removal of the 5' end endogenous flap promotes hybridization of the single-stranded 3' DNA flap to the corresponding complementary DNA strand, and the incorporation or assimilation of the desired nucleotide alteration carried by the single-stranded 3' DNA flap onto the target DNA.

[0197] In the context of prime editing, the terms “cleavage site,” “nick site,” and “cut site,” as used interchangeably herein, refer to a specific location between two nucleotides or two base pairs on a double-stranded target DNA sequence. In some embodiments, the location of the nick site is determined relative to a specific PAM sequence. In some embodiments, the nick site is the specific location where a nick will occur when the double-stranded target DNA is brought into contact with a nickase, such as Cas nickase, that recognizes a particular PAM sequence. For each PEgRNA described herein, the nick site (e.g., PE3, PE5, and “first nick site” as referred to in the context of similar approaches) is characteristic of a specific napDNAbp to which the gRNA core of the PEgRNA binds, and is characteristic of a specific PAM required for the recognition and function of the napDNAbp. For example, in PEgRNA containing a gRNA core that binds to SpCas9, the phosphodiester bond nick site is between base 3 (position -3 relative to position 1 in the PAM sequence) and base 4 (position -4 relative to position 1 in the PAM sequence).

[0198] In some embodiments, the nick site is on the target strand of the double-stranded target DNA sequence. In some embodiments, the nick site is on the non-target strand of the double-stranded target DNA sequence. In some embodiments, the nick site is on a protospacer sequence. In some embodiments, the nick site is adjacent to a protospacer sequence. In some embodiments, the nick site may be downstream of a region, for example, on the non-target strand, that is complementary to the primer-binding site of PEgRNA. In some embodiments, the nick site is downstream of a region, for example, on the non-target strand, that binds to the primer-binding site of PEgRNA. In some embodiments, the nick site is immediately downstream of a region, for example, on the non-target strand, that is complementary to the primer-binding site of PEgRNA. In some embodiments, the nick site is upstream of a specific PAM sequence on the non-target strand of the double-stranded target DNA, where the PAM sequence is specific to recognition by napDNAbp that binds to the gRNA core of PEgRNA. In some embodiments, the nick site is located downstream of a specific PAM sequence on the non-target strand of a double-stranded target DNA, where the PAM sequence is specific to recognition by napDNAbp that binds to the gRNA core of PEgRNA. In some embodiments, the nick site is located 3 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by Streptococcus pyogenes Cas9 nickase, P. lavamentivorans Cas9 nickase, C. diphtheriae Cas9 nickase, N. cinerea Cas9, S. aureus Cas9, or N. lari Cas9 nickase. In some embodiments, the nick site is located 3 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by Cas9 nickase, where Cas9 nickase comprises a nuclease-active HNH domain and a nuclease-inactive RuvC domain. In some embodiments, the nick site is located 2 base pairs upstream of the PAM sequence, and the PAM sequence is recognized by S. thermophilus Cas9 nickase.

[0199] In various embodiments of the extended guide RNA, cellular repair of a single-stranded DNA flap results in the placement of a desired nucleotide change, thereby forming the desired product.

[0200] In yet another embodiment, the desired nucleotide change is placed on an editing window located between approximately -5 and +5 of the nick site, or between approximately -10 and +10, or between approximately -20 and +20, or between approximately -30 and +30, or between approximately -40 and +40, or between approximately -50 and +50, or between approximately -60 and +60, or between approximately -70 and +70, or between approximately -80 and +80, or between approximately -90 and +90, or between approximately -100 and +100, or between approximately -200 and +200.

[0201] In another embodiment, the desired nucleotide change is approximately +1 to +2 from the nick site, or approximately +1 to +3, +1 to +4, +1 to +5, +1 to +6, +1 to +7, +1 to +8, +1 to +9, +1 to +10, +1 to +11, +1 to +12, +1 to +13, +1 to +14, +1 to +15, +1 to +16, +1 to +17, +1 to +18, +1 to +19, +1 to +20, +1 to +21, +1 to +22, +1 to +23, +1 to +24, +1 to +25, +1 to +26, +1 to +27, +1 to +28, +1 to +29, +1 to +30, +1 to +31, +1~+32, +1~+33, +1~+34, +1~+35, +1~+36, +1~+37, +1~+38, +1~+39, +1~+40, +1~+41, +1~+42, +1~+43, +1~+44, +1~+45, +1~+46, +1~+47, +1~+48, +1~+49, +1~+50, +1~+51, +1~+52, +1~+53, +1~+54, +1~+55, +1~+56, +1~+57, +1~+58, +1~+59, +1~+60, +1~+61, +1~+62, +1~+63, +1~+64, +1~+ 65, +1~+66, +1~+67, +1~+68, +1~+69, +1~+70, +1~+71, +1~+72, +1~+73, +1~+74, +1~+75, +1~+76, +1~+77, +1~+78, +1~+79, +1~+80, +1~+81, +1~+82, +1~+83, +1~+84, +1~+85, +1~+86, +1~+87, +1~+88, +1~+89, +1~+90, +1~+90, +1~+91, +1~+92, +1~+93, +1~+94, +1~+95, +1~+96, +1~+97, +1~+9 It is placed on the editing window between 8, +1~+99, +1~+100, +1~+101, +1~+102, +1~+103, +1~+104, +1~+105, +1~+106, +1~+107, +1~+108, +1~+109, +1~+110, +1~+111, +1~+112, +1~+113, +1~+114, +1~+115, +1~+116, +1~+117, +1~+118, +1~+119, +1~+120, +1~+121, +1~+122, +1~+123, +1~+124, or +1~+125.

[0202] In yet another embodiment, the desired nucleotide change is approximately +1 to +2 from the nick site, or approximately +1 to +5, +1 to +10, +1 to +15, +1 to +20, +1 to +25, +1 to +30, +1 to +35, +1 to +40, +1 to +45, +1 to +50, +1 to +55, +1 to +100, +1 to +105, +1 to +110, +1 to +115, + It is placed on the editing window if the value is between 1 and +120, +1 and +125, +1 and +130, +1 and +135, +1 and +140, +1 and +145, +1 and +155, +1 and +160, +1 and +165, +1 and +170, +1 and +175, +1 and +180, +1 and +185, +1 and +190, +1 and +195, or between +1 and +200.

[0203] In various respects, extended guide RNA is a modified form of extended guide RNA. PegRNA (i.e., extended guide RNA) and ngRNA can be expressed from coding nucleic acids or chemically synthesized. Methods for obtaining or otherwise synthesizing guide RNA, and for determining suitable sequences of pegRNA containing protospacer sequences that interact with and hybridize with target strands at desired genomic target sites, are well known in this field.

[0204] In various embodiments, the specific design aspects of pegRNA and ngRNA sequences will depend, among other factors, on the nucleotide sequence of the target genome site (i.e., the desired site to be edited), as well as the type of napDNAbp (e.g., Cas9 protein) present in the prime editing system used in the methods and compositions described herein, such as the location of the PAM sequence, the G / C content (%) on the target sequence, the degree of microhomology region, and the secondary structure.

[0205] Generally, a spacer sequence (i.e., guide sequence) of pegRNA or ngRNA can be any polynucleotide sequence that hybridizes with the target sequence and has sufficient complementarity to the target polynucleotide sequence to lead to sequence-specific binding of napDNAbp (e.g., Cas9, Cas9 homolog, or Cas9 variant) to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more, or about more, when optimally aligned using a preferred alignment algorithm. Optimal alignment can be determined by the use of any preferred algorithm for aligning the sequences. Examples of this include, but are not limited to, the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (e.g., 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). In some embodiments, the guide sequence is approximately 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides in length, or more, or approximately more.

[0206] In some embodiments, the guide sequence is less than or equal to approximately 75, 50, 45, 40, 35, 30, 25, 20, 15, or 12 nucleotides in length. The ability of the guide sequence to guide sequence-specific binding of the prime editor to the target sequence can be evaluated by any suitable assay. For example, a prime editor component containing the guide sequence to be tested may be provided to a host cell having the corresponding target sequence by transfection with a vector encoding the prime editor component disclosed herein, for example, and then the preferred cleavage within the target sequence may be evaluated by a Surveyor assay described herein, for example. Similarly, cleavage of a target polynucleotide sequence may be evaluated in vitro by providing the target sequence, a prime editor component containing the guide sequence to be tested, and a control guide sequence different from the test guide sequence, and by comparing the binding or cleavage rate at the target sequence between the test and control guide sequence reactions. Other assays are possible and will be recalled by those skilled in the art.

[0207] A guide sequence may be selected to target any of the target sequences. In some embodiments, the target sequence is a sequence within the cell's genome. Exemplary target sequences include those unique on the target genome. For example, for S. pyogenes Cas9, a unique target sequence on the genome may include the Cas9 target site of morphology MMMMMMMNNNNNNNNNNNNXGG, where NNNNNNNNNNNNXGG (where N is A, G, T, or C; X can be any). A unique target sequence on the genome may include the S. pyogenes Cas9 target site of morphology MMMMMMMMMNNNNNNNNNNNXGG, where NNNNNNNNNNNXGG (where N is A, G, T, or C; X can be any). For S. thermophilus CRISPR1Cas9, the genomically specific target sequence may encompass the Cas9 target site of morphology MMMMMMMMNNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be any; W is A or T). The genomically specific target sequence may encompass the S. thermophilus CRISPR 1 Cas9 target site of morphology MMMMMMMMMNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be any; W is A or T). For S. pyogenes Cas9, the genomically specific target sequence may encompass the Cas9 target site of morphology MMMMMMMMNNNNNNNNNNNNXGGXG, where NNNNNNNNNNNNXGGXG (N is A, G, T, or C; X can be any). Unique target sequences on the genome may encompass the S. pyogenes Cas9 target site of morphology MMMMMMMMMNNNNNNNNNNNXGGXG, where NNNNNNNNNNNXGGXG (N is A, G, T, or C; X can be any of these). In each of these sequences, "M" can be A, G, T, or C, and does not require any consideration in identifying the sequence uniquely.

[0208] In some embodiments, the guide sequence is selected to reduce the degree of secondary structure within the guide sequence. The secondary structure can be determined by any suitable polynucleotide folding algorithm. Several programs are based on calculating the minimum Gibbs free energy. An example of one such algorithm is mFold, described by Zuker and Stiegler (Nucleic Acids Res.9 (1981), 133-148). Another example of a folding algorithm is the online web server RNAfold, developed at the Institute for Theoretical Chemistry, University of Vienna, which uses a centroid structure prediction algorithm (see, e.g., AR Gruber et al., 2008, Cell 106(1):23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12):1151-62). Further algorithms can be found in US application Ser. No. 61 / 836,080, incorporated herein by reference. In some embodiments, silent mutations are introduced onto the guide sequence to modulate its secondary structure and increase the efficiency of prime editing.

[0209] In some embodiments, the pegRNA backbone or gRNA core portion contains sequences corresponding to the tracr and tracr mate sequences of a conventional guide RNA. Generally, the tracr mate sequence contains any sequence that has sufficient complementarity with the tracr sequence to facilitate one or more of the following: (1) excision of the guide sequence by flanking by the tracr mate sequence in a cell containing the corresponding tracr sequence; and (2) formation of a complex in the target sequence, where the complex contains the tracr mate sequence hybridized to the tracr sequence. Generally, the degree of complementarity refers to the optimal alignment of the tracr mate sequence and the tracr sequence along the shorter of the two sequences. The optimal alignment may be determined by any preferred alignment algorithm, and may further take into account secondary structures such as self-complementarity within either the tracr sequence or the tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and the tracr mate sequence along the shorter of the two optimally aligned lengths is approximately 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher, or approximately more. In some embodiments, the tracr sequence is approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 nucleotides in length, or more, or approximately more. In some embodiments, the tracr sequence and the tracr mate sequence are contained within a single transcript such that hybridization between the two produces a transcript having a secondary structure such as a hairpin. A preferred loop-forming sequence for use in a hairpin structure is 4 nucleotides in length and most preferably has the sequence GAAA. However, longer or shorter loop sequences may be used as alternative sequences. The sequences preferably include a nucleotide triplet (e.g., AAA) and an additional nucleotide (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG.In one embodiment of the present invention, the transcript or the polynucleotide sequence to be transcribed has at least two hairpins. In a preferred embodiment, the transcript has two, three, four, or five hairpins. In a further embodiment of the present invention, the transcript has at most five hairpins. In some embodiments, the single transcript further includes a transcription termination sequence; preferably, this is a poly-T sequence, e.g., six T nucleotides. Further, non-limiting examples of single polynucleotides comprising a guide sequence, a tracr mate sequence, and a tracr sequence are listed below (listed from 5' to 3'), where "N" represents a base of the guide sequence and the last poly-T sequence represents a transcription terminator:

[0210] (1)NNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT(Sequence code 113); (2)NNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT(Sequence code 114); (3)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtTTTTT(Sequence code 115); (4)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTT(Sequence No. 116); (5)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTTT(Sequence ID 117); and (6)NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT(Sequence ID 118).

[0211] In some embodiments, sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) to (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from the transcript containing the tracr mate sequence.

[0212] It will be apparent to those skilled in the art that, in order to target any of the fusion proteins containing a Cas9 domain and a single-stranded DNA-binding protein disclosed herein to a target site, for example, the site where the recombinase recognition sequence is to be introduced, it is typically necessary to co-express the fusion protein together with a guide RNA, such as sgRNA. As will be described in more detail elsewhere in this specification, the guide RNA typically includes a tracrRNA framework that allows Cas9 binding and a guide sequence that confers sequence specificity to the Cas9: nucleic acid editing enzyme / domain fusion protein.

[0213] In some embodiments, the pegRNA comprises the structure 5'-[guide sequence]-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUU(SEQ ID NO: 119)-extension arm-3', where the guide sequence comprises a sequence complementary to the target sequence. The guide sequence, also referred herein as a spacer sequence, is typically 20 nucleotides long. Suitable guide RNA sequences for targeting Cas9: nucleic acid editing enzyme / domain fusion proteins to specific genomic target sites will be apparent to those skilled in the art based on this disclosure. Such suitable guide RNA sequences typically comprise a guide sequence complementary to a nucleic acid sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Several exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to specific target sequences are provided herein. Additional guide sequences are well known in the art and may be used in prime editors utilized in the methods and compositions described herein.

[0214] In some embodiments, PEgRNA includes three main constituent elements in a 5'-to-3' order: a spacer, a gRNA core, and an extension arm at the 3' end. In some embodiments, the extension arm may be further divided into the following structural elements in the 5'-to-3' order: an editing template, a homology arm, and a primer binding site. In some embodiments, the extension arm may be further divided into the following structural elements in the 5'-to-3' order: a homology arm, an editing template, and a primer binding site. In some embodiments, the extension arm may be further divided into the following structural elements in the 5'-to-3' order: a DNA synthesis template (e.g., an RT template) and a primer binding site. In addition, PEgRNA may include an optional 3' end modification region and an optional 5' end modification region. Furthermore, PEgRNA may include a transcription termination signal at the 3' end of PEgRNA. These structural elements are further defined herein. The description of the structure of PEgRNA is not intended to be limiting and encompasses variations in the arrangement of elements. For example, any sequence modification may be located within or between any of the other regions shown, and is not limited to being located at the 3' and 5' ends.

[0215] PEgRNA modification PEgRNA may also encompass additional design modifications that modulate the properties and / or characteristics of PEgRNA, thereby improving the effectiveness of prime editing. In various embodiments, these modifications may belong to one or more of several different categories, including but not limited to: (1) designs that enable efficient expression of functional PEgRNA from a non-polymerase III (pol III) promoter, which would allow expression of longer PEgRNA without cumbersome sequence requirements; (2) modifications to the core, Cas9-binding PEgRNA backbone, which may improve effectiveness; (3) modifications to PEgRNA to improve RT processing, allowing insertion of longer sequences at targeted genomic loci; and (4) additions of RNA motifs to the 5' or 3' end of PEgRNA, which improve PEgRNA stability, enhance RT processing, prevent PEgRNA misfolding, or recruit additional factors important for genome editing. Such modifications are further described, for example, in PCT Publication WO 2022 / 067130, which is incorporated herein by reference.

[0216] Pharmaceutical composition Other aspects of this disclosure relate to pharmaceutical compositions comprising any of the reverse transcriptase variants, Cas9 variants, prime editors, fusion proteins, or complexes provided herein, or any of the polynucleotides or vectors encoding such reverse transcriptase variants, Cas9 variants, prime editors, fusion proteins, or complexes provided herein. The term “pharmaceutical composition” as used herein means a composition formulated for pharmaceutically acceptable use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g., compounds for specific delivery, for increasing half-life, or other therapeutic agents). In some embodiments, the pharmaceutical composition further comprises pegRNA, or a polynucleotide encoding pegRNA.

[0217] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or base, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or steric acid), or solvent encapsulation material, involved in transporting or delivering a protein, fusion protein, polynucleotide, or vector from one site in the body (e.g., a delivery site) to another site (e.g., an organ, tissue, or other part of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense that it is compatible with the other components of the formulation and is non-harmful to the target tissue (e.g., physiologically compatible, sterile, physiological pH, etc.). Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, e.g., lactose, glucose, and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, e.g., magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, e.g., cocoa butter and suppository waxes; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (1 0) Glycols, e.g., propylene glycol; (11) Polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) Esters, e.g., ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, e.g., magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Pyrogen-free water; (17) Isotonic saline; (18) Ringer's solution; (19) Ethyl alcohol; (20) pH buffer solution; (21) Polyesters, polycarbonates, and / or polyanhydrides; (22) Expanders, e.g., polypeptides and amino acids; (23) Serum components, e.g., serum albumin, HDL, and LDL; (22) C2-C 12Alcohols, such as ethanol; and (23) other non-toxic, suitable substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulations. Terms such as “excipient,” “carrier,” “pharmaceutically acceptable carrier,” or similar terms are used interchangeably herein.

[0218] In some embodiments, the pharmaceutical compositions are formulated for delivery to a target, for example, for gene editing. Preferred routes for administering the pharmaceutical compositions described herein include, without limitation, the following: topical, subcutaneous, transdermal, intradermal, intrafocal, intraarticular, intraperitoneal, intrabladderal, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periophthalmosal, intratumoral, intracerebral, and intraventricular administration.

[0219] In some embodiments, the pharmaceutical compositions described herein are administered topically to a diseased site (e.g., a tumor site). In some embodiments, the pharmaceutical compositions described herein are administered to a target by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being made of a porous, non-porous, or gelatinous material and comprising a membrane or fiber such as a sialastic membrane.

[0220] In some embodiments, pharmaceutical compositions are formulated according to customary procedures as compositions suitable for intravenous or subcutaneous administration to a subject, e.g., a human. In some embodiments, pharmaceutical compositions for administration by injection are aqueous solutions in sterile isotonic buffer. Where necessary, the pharmaceutical composition may also include a solubilizer and a local anesthetic, such as a lignocaine, for pain relief at the injection site. Generally, the components are supplied either separately or mixed together in a combination, for example, as dry lyophilized powder or water-free concentrate in airtight containers such as ampoules or pouches indicating the quantity of the active drug. Where the pharmaceutical composition is to be administered by drip infusion, it may be administered by drip bottle containing sterile pharmaceutical-grade water or saline. Where the pharmaceutical composition is to be administered by injection, ampoules of sterile water or saline for injection may be provided so that the components can be mixed prior to administration.

[0221] Pharmaceutical compositions for systemic administration may be liquids, such as sterile saline, Ringer's lactate, or Hanks' solution. In addition, pharmaceutical compositions may be in solid form and may be redissolved or suspended immediately prior to use. Lyophilized forms are also intended.

[0222] Pharmaceutical compositions may be contained within lipid particles or vesicles, such as liposomes or microcrystals, which are also suitable for parenteral administration. The particles may have any preferred structure, such as monolayer or multilayer, as long as the composition is contained within them. Proteins, fusion proteins, polynucleotides, or vectors may be captured in “stabilized plasmid-lipid particles” (SPLPs) containing the fusion lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipids, and stabilized by polyethylene glycol (PEG) coating (Zhang YP et al., Gene Ther. 1999, 6:1438-47). Positively charged lipids such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate or “DOTAP” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, for example, U.S. Patent Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of these is incorporated herein by reference.

[0223] The pharmaceutical compositions described herein may be administered or packaged, for example, as unit doses. When used in reference to the pharmaceutical compositions of this disclosure, the term “unit dose” means a physically individual unit suitable as a unit dose for a subject, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect when combined with a required diluent, i.e., a carrier or base.

[0224] Furthermore, the pharmaceutical composition may be provided as a pharmaceutical kit comprising (a) a container containing the protein, fusion protein, complex (e.g., ribonucleoprotein complex), polynucleotide, or vector of the present invention in lyophilized form, and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent may be used for the reconstitution or dilution of the lyophilized protein, fusion protein, complex (e.g., ribonucleoprotein complex), polynucleotide, or vector of the present invention. Optionally, a notice in the form prescribed by the government agency regulating the manufacture, use, or sale of a pharmaceutical or biological product may be attached to such container(s). This notice reflects the approval by the agency for manufacture, use, or sale for human administration.

[0225] In another aspect, the invention encompasses molded articles containing materials useful for treating the diseases described above. In some embodiments, the molded article includes a container and a label. Preferred containers include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from a variety of materials, such as glass or plastic. In some embodiments, the container may hold a composition effective for treating a disease and have a sterile access port. For example, the container may be a vial or intravenous solution bag with a stopper that can be punctured by a subcutaneous injection needle. The active agent in the composition is a protein, fusion protein, polynucleotide, or vector of the invention. In some embodiments, a label on or associated with the container indicates that the composition is used to treat a selected disease. The molded article may further include a second container containing 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 perspective, including other buffers, diluents, filters, needles, syringes, and a package insert having accompanying documentation.

[0226] Polynucleotides, vectors, AAVs, kits, and cells In some aspects, this disclosure provides polynucleotides and vectors encoding any of the reverse transcriptase variants, Cas9 variants, fusion proteins, or prime editors provided herein. In some aspects, this disclosure provides one or more polynucleotides and vectors encoding any of the complexes provided herein. In some embodiments, the polynucleotides and vectors provided herein include DNA. In some embodiments, the polynucleotides and vectors provided herein include RNA. In some aspects, any of the polynucleotides described herein may be provided on a vector.

[0227] In some embodiments, one or more polynucleotides encoding any of the complexes provided herein are delivered to cells, for example, using AAV. In certain embodiments, two polynucleotides encoding any of the complexes provided herein are delivered to cells, for example, using AAV. In certain embodiments, the two polynucleotides include a split intein which two halves of the Prime Editor described herein can be reassembled into a Prime Editor molecule. In some embodiments, one or more polynucleotides encoding any of the complexes provided herein are delivered to cells in one or more adeno-associated virus (AAV) particles. Delivery of the Prime Editor complexes is described, for example, in US Provisional Application USSN 63 / 426,336 filed November 17, 2022, US Provisional Application USSN 63 / 491,013 filed March 17, 2023, and in Davis, JR, et al. Nat. Biotechnol. 2023. Each of these is incorporated herein by reference. In certain embodiments, one or more polynucleotides encoding the complex are delivered to the cell in two AAV particles. In some embodiments, one or both AAV particles contain AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. In certain embodiments, one or both AAV particles contain AAV9.

[0228] In some embodiments, first and second AAV particles are delivered to a cell. In certain embodiments, the first AAV particle contains a polynucleotide comprising the structure 5'-[inverted terminal repeat (ITR) sequence]-[promoter]-[napDNAbp N-terminal fragment]-[N-intane]-[terminator sequence]-[ITR sequence]-3'. In certain embodiments, the second AAV particle contains a polynucleotide comprising the structure 5'-[ITR sequence]-[promoter]-[C-intane]-[napDNAbp C-terminal fragment]-[reverse transcriptase]-[terminator sequence]-[optional nicking gRNA]-[pegRNA]-[ITR]-3'.

[0229] The reverse transcriptase variants, Cas9 variants, prime editors, fusion proteins, or complexes provided herein may also be assembled into kits. In some embodiments, the kit comprises a polynucleotide for the expression of any of the reverse transcriptase variants, Cas9 variants, prime editors, or complexes provided herein. In other embodiments, the kit further comprises a suitable pegRNA or a nucleic acid vector for the expression of such pegRNA.

[0230] The kits described herein may comprise one or more containers containing components for carrying out the methods described herein, and optionally instructions for use. In some embodiments, the kits may comprise instructions for editing or treating a particular disease by prime editing. In some embodiments, the kits may comprise instructions for editing a particular gene in a cell. Any of the kits described herein may further comprise components required to carry out any of the methods described herein. Each component of the kit may be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder) where applicable. In certain cases, some of the components may be reconstituted or otherwise processed (e.g., into an active form) by adding a suitable solvent or other kind (e.g., water) which may or may not be provided by the kit.

[0231] In some embodiments, the kit may optionally include instructions and / or promotions for the use of the components provided. As used herein, “Instructions” defines the components of the instructions and / or promotions and typically involves written instructions on or associated with the packaging of this disclosure. Instructions may also include any oral or electronic instructions provided in any format that clearly recognizes to the user that the instructions should be associated with the kit, such as audiovisual (e.g., videotape, DVD, etc.), internet, and / or web-based communications. Written instructions may be in any form specified by government agencies that regulate the manufacture, use, or sale of pharmaceutical or biological products. These may also reflect approval by authorities for manufacture, use, or sale for animal administration. As used herein, “promoted” includes all methods of doing business that includes pharmaceutical industry activities relating to this disclosure, including education, hospital and other clinical instructions, scientific research, drug discovery or drug development, academic research, drug sales, and any advertising or other promotional activities that include any form of written, oral, and electronic communications. In addition, the kit may include other components depending on the specific application, as described herein.

[0232] The kit may contain one or more of the components described herein in one or more containers. The components may be aseptically prepared, packaged in syringes, and shipped refrigerated. Alternatively, they may be contained in vials or other containers for storage. A second container may contain other aseptically prepared components. Alternatively, the kit may contain active drugs that are pre-mixed and shipped in vials, tubes, or other containers.

[0233] The kit may have various forms, such as blister pouches, shrink-packed pouches, vacuum-sealable pouches, sealable thermoformed trays, or similar pouches or trays, in which the accessories are loosely packed in pouches, one or more tubes, containers, boxes, or bags. The kit may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to not be packaged differently. The kit may be sterilized using any appropriate sterilization technique, such as radiation sterilization, thermal sterilization, or other sterilization methods known in the art. Depending on the specific application, the kit may also include other components, such as containers, cell culture media, salts, buffers, reagents, syringes, needles, fabrics such as gauze for applying or removing disinfectants, disposable gloves, supports for drugs preceding administration, etc. Some aspects of this disclosure provide kits comprising nucleic acid constructs containing nucleotide sequences encoding the Prime Editor System described herein or various components thereof (e.g., reverse transcriptase variants, Cas9 variants, Prime Editors, complexes, polynucleotides, and / or vectors provided herein). In some embodiments, a nucleotide sequence(s) comprises heterologous promoters (or more than a single promoter) that drive the expression of one or more prime editor system components.

[0234] Cells that may contain any of the reverse transcriptase variants, Cas9 variants, prime editors, fusion proteins, complexes, polynucleotides, and / or vectors described herein include prokaryotic and eukaryotic cells. The methods described herein may be used to deliver pegRNA and prime editors to eukaryotic cells (e.g., mammalian cells such as human cells). In some embodiments, the cells are in vitro (e.g., cultured cells). In some embodiments, the cells are in vivo (e.g., in a subject such as a human subject). In some embodiments, the cells are ex vivo (e.g., isolated from a subject and administered again to the same or a different subject).

[0235] Several aspects of this disclosure provide cells comprising any of the vectors or other constructs disclosed herein. In some embodiments, host cells are transiently or nontransiently transfected or electroporated with one or more vectors described herein. In some embodiments, cells are transfected or electroporated as they are naturally occurring in the subject. In some embodiments, the cells to be transfected or electroporated are taken from the subject. In some embodiments, the cells are derived from cells taken from the subject, such as a cell line.

[0236] How to use In several respects, this disclosure provides a method for editing nucleic acid molecules by prime editing, comprising contacting the nucleic acid molecule with a prime editor or complex (or a polynucleotide or vector encoding it) to thereby install one or more modifications to the nucleic acid molecule at a target site. Prime editing refers to an approach for gene editing that uses napDNAbp, a polymerase (e.g., reverse transcriptase), and a specialized guide RNA containing a DNA synthesis template and primer binding site for encoding (or deleting) desired new genetic information to be incorporated onto a target DNA sequence. For example, prime editing may be used to incorporate one or more recombinase recognition sequences onto a target DNA sequence, such as a genome, as described herein. Prime editing is described in Anzalone, AV 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 on 19 March 2020, published as WO 2020 / 191239, which is incorporated herein by reference.

[0237] Prime Editing represents a platform for genome editing, a versatile and precise method for directly writing new genetic information to a designated DNA site using a nucleic acid-programmable DNA-binding protein ("napDNAbp") that works in conjunction with polymerase (i.e., provided in the form of a fusion protein, or otherwise in trans with napDNAbp), where the Prime Editing system is programmed by a Prime Editing (PE) guide RNA ("pegRNA"), which both designates the target site and serves as a template for the synthesis of the desired edit (e.g., a recombinase-recognizing sequence to be inserted onto the target DNA) in the form of a replacement DNA strand as an manipulated extension (either DNA or RNA) on the guide RNA (e.g., at the 5' or 3' end or in the interior of the guide RNA). The replacement strand containing the desired edit (e.g., a single nucleic acid substitution) shares (or is homologous to) the same sequence as (or is homologous to) the endogenous strand immediately downstream of the nic site of the target site to be edited (with the exception that it contains the desired edit). Through DNA repair and / or replication mechanisms, the endogenous strand downstream of a nick site is replaced by a newly synthesized replacement strand containing the desired edit. In some cases, prime editing can be considered a “search and replace” genome editing technology, because the prime editors described herein not only search and locate the desired target site to be edited, but also encode a replacement strand containing the desired edit that is placed in place of the corresponding endogenous DNA strand at the target site. The prime editors of this disclosure relate in part to the discovery that the mechanism of reverse transcription (TPRT) or “prime editing” primed by a target can be utilized or adapted to perform precise CRISPR / Cas-based genome editing with high efficiency and gene flexibility. TPRT is naturally used by mobile DNA factors such as mammalian non-LTR retrotransposons and bacterial group II introns.A Cas protein-reverse transcriptase fusion or related system is used to target a specific DNA sequence by guide RNA, generate a single-strand nick at the target site, and use the nicked DNA as a primer for reverse transcription of the manipulated DNA synthesis template incorporated into the guide RNA. However, while the concept begins with a prime editor that uses reverse transcriptase as a DNA polymerase component, the prime editors described herein are not limited to reverse transcriptase and may substantially encompass the use of any DNA polymerase. In fact, this application may consistently refer to a prime editor having “reverse transcriptase,” but it is hereby defined that reverse transcriptase is only one type of DNA polymerase that can act in prime editing. Therefore, wherever this specification refers to “reverse transcriptase,” those skilled in the art should understand that any suitable DNA polymerase may be used instead of reverse transcriptase. Therefore, in one aspect, a prime editor may include a Cas9 (or equivalent napDNAbp) programmed to target a DNA sequence by binding to a specialized guide RNA (i.e., PEgRNA) containing a spacer sequence that anneals to a complementary sequence on the target DNA (a sequence complementary to an endogenous protospacer sequence). The PEgRNA also contains novel genetic information in the form of an extension encoding a replacement strand of DNA containing the desired nucleotide change, which is used to replace the corresponding endogenous DNA strand at the target site. To transfer the information from the PEgRNA to the target DNA, the mechanism of prime editing involves nicking the target site on one strand of DNA to expose a 3' hydroxyl group. The exposed 3' hydroxyl group can then be used to prime DNA polymerization directly from the edit-encoding extension on the PEgRNA to the target site. In various embodiments, the extension providing a template for polymerization of the replacement strand containing the edit may be formed from RNA or DNA. In the case of RNA extension, the polymerase of the prime editor may be an RNA-dependent DNA polymerase (e.g., reverse transcriptase).In the case of DNA extension, the polymerase of the prime editor may be a DNA-dependent DNA polymerase. The newly synthesized strand formed by the prime editor (i.e., the replacement DNA strand containing the desired nucleotide edit) will be homologous to the genomic target sequence (i.e., have the same sequence) with respect to the genomic target sequence, with the exception of the inclusion of one or more desired nucleotide changes (e.g., a single nucleotide substitution, deletion, or insertion, or a combination thereof). The newly synthesized (or replacement) strand of DNA may also be called a single-stranded DNA flap. This will compete for hybridization with a complementary homologous endogenous DNA strand, thereby displacing the corresponding endogenous strand. The resolution of a hybridized intermediate (also called a heteroduplex, including a single-stranded DNA flap synthesized by a reverse transcriptase hybridized to an endogenous DNA strand, with the exception of a mismatch at the site where the desired nucleotide edit is placed on the edited strand) may include removal of the strand-substituted flap resulting from the endogenous DNA (e.g., by the 5' end DNA flap endonuclease FEN1), ligation of the synthesized single-stranded DNA flap onto the target DNA, and assimilation of the desired nucleotide alteration as a result of cellular DNA repair and / or replication processes. Since template-based DNA synthesis provides one-nucleotide precision for any nucleotide modification, including insertions and deletions, the scope of this approach is extremely broad and predictably applicable to countless applications in basic science and therapeutics. In certain embodiments, the system may be combined with the use of error-prone reverse transcriptase (e.g., provided as a fusion protein with a Cas9 domain or trans to a Cas9 domain). Error-prone reverse transcriptase may introduce modulation during the synthesis of the single-stranded DNA flap. Therefore, in certain embodiments, error-prone reverse transcriptase can be used to introduce nucleotide changes into target DNA. Depending on the error-prone reverse transcriptase used in the system, the changes may be random or non-random.

[0238] In various embodiments, prime editing operates by contacting a target DNA molecule (in which a nucleotide sequence change is desired) with a nucleic acid-programmed DNA-binding protein (napDNAbp) complexed with a prime editing guide RNA (PEgRNA). In various embodiments, the prime editing guide RNA (PEgRNA) contains an extension at the 3' or 5' end of the guide RNA or at an intramolecular location on the guide RNA, encoding the desired nucleotide change (e.g., single base substitution, insertion, or deletion). First, the napDNAbp / extended gRNA complex contacts the DNA molecule, and the extended gRNA guides the napDNAbp to bind to the target locus. Next, a nick is introduced on one of the DNA strands of the target locus (e.g., by a nuclease or chemical agent), thereby creating an available 3' end on one of the strands of the target locus. In certain embodiments, the nick is created on the DNA strand corresponding to the R loop strand, i.e., the strand that is not hybridized to the guide RNA sequence, i.e., the "non-target strand". However, the nick can be introduced on either strand. In other words, the nick can be introduced onto 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 that forms the single-stranded portion of the R-loop and is complementary to the target strand). In the next step, the 3' end of the DNA strand (formed by the nick) interacts with the extended portion of the guide RNA to prime the 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., a “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 from the 3' end of the primed site toward the 5' end of the prime-editing guide RNA to synthesize a single strand of DNA. The DNA polymerase (e.g., reverse transcriptase) can be fused to napDNAbp or, alternatively, provided trans toward napDNAbp.This involves forming a single-stranded DNA flap containing the desired nucleotide alteration (e.g., a single base change, insertion, or deletion, or a combination thereof; e.g., a recombinase recognition sequence to be inserted onto a target DNA sequence such as the genome) and otherwise homologous to the endogenous DNA at or adjacent to the nicking site. In the next step, napDNAbp and guide RNA are released. The last two steps involve the dissolution of the single-stranded DNA flap so that the desired nucleotide alteration is incorporated into the target locus. This process can be driven toward the formation of the desired product by removing the corresponding 5' endogenous DNA flap that forms once the 3' single-stranded DNA flap invades and hybridizes with the endogenous DNA sequence. Although not constrained by theory, the cell's endogenous DNA repair and replication processes resolve mismatched DNA and incorporate nucleotide alterations (one or more) to form the desired modulated product. The process can also be driven toward product formation via "second-strand nicking." This process can introduce at least one of the following genetic changes: transversion, transition, deletion, and insertion (e.g., insertion of a recombinase-recognition sequence). In some embodiments, one or more recombinase-recognition sequences are inserted onto a target DNA sequence using prime editing, and these recombinase-recognition sequences are then brought into contact with a recombinase (e.g., one of the evolved recombinases provided herein) and optionally a donor DNA sequence into which the recombinase-recognition sequences are to be inserted onto the target DNA sequence.

[0239] The terms “prime editor (PE) system” or “prime editor (PE)” or “PE system” or “PE editing system” refer to compositions relating to methods of genome editing using reverse transcription (TPRT) primed with a target as described herein, and include, but are not limited to, napDNAbp, reverse transcriptase, fusion protein (e.g., napDNAbp and reverse transcriptase), prime editing guide RNA, and complexes comprising the fusion protein and prime editing guide RNA, as well as auxiliary elements such as second-strand nickeling components (e.g., second-strand nickeling sgRNA) and 5' endogenous DNA flap removal endonuclease (e.g., FEN1) to help drive the prime editing process toward editing product formation.

[0240] In the embodiments described so far, PEgRNA constitutes a single molecule comprising a guide RNA (which itself contains a spacer sequence and a gRNA core or backbone) and a 5' or 3' extension arm containing a primer binding site and a DNA synthesis template; however, PEgRNA can also take the form of two individual molecules. For example, in some embodiments, PEgRNA may comprise a guide RNA and a transprime editor RNA template (tPERT). This essentially houses an extension arm (specifically containing the primer binding site and DNA synthesis domain) and an RNA-protein recruitment domain (e.g., an MS2 aptamer or hairpin) on the same molecule, which colocalizes or recruits to a modified prime editor complex containing a tPERT recruiting protein (e.g., an MS2cp protein that binds to an MS2 aptamer).

[0241] A prime editor system may comprise one or more prime editing guide RNAs (PEgRNAs). In some embodiments, a prime editor system has one PEgRNA that targets one strand of double-stranded DNA, e.g., a target genomic site ("single-flap prime editing system"). For example, a single-flap prime editing system may comprise a spacer sequence comprising complementarity to the target strand of the double-stranded target DNA, a primer binding site comprising complementarity to the non-target strand of the double-stranded target DNA, and a DNA synthesis template comprising (encoding) a nucleotide editing site, e.g., a recombinase recognition site, compared to the double-stranded target DNA sequence. In some embodiments, a prime editor system ("dual-flap prime editing system," "twin prime editing," or "twinPE") comprises at least two different PEgRNAs capable of targeting opposing strands of double-stranded target DNA, e.g., a target genomic site. For example, a twin-prime editing system may include two PEgRNAs, each containing a DNA synthesis template with complementary regions to the other, leading to the synthesis of two 3' flaps with complementary regions to the other, containing nucleotide edits (e.g., recombinase recognition sequences) compared to a double-stranded target DNA sequence. Unlike single-flap prime editing, there is no requirement for a pair of edited DNA strands (3' flaps) to directly compete with the 5' flap on the endogenous genomic DNA (i.e., no requirement for homology arms on extension arms that would generate regions complementary to the endogenous DNA). This is because, instead, complementary edited strands are available for hybridization. Since both strands of the double helix are synthesized as edited DNA, the dual-flap prime editing system eliminates the need for replacing the unedited complementary DNA strand required by classical prime editing. Instead, cellular DNA repair mechanisms only need to excise the paired 5' flap (original genomic DNA) and ligate the paired 3' flap (edited DNA) to the gene locus.Therefore, it is not necessary to include sequences homologous to the genomic DNA on the newly synthesized DNA strand, allowing for selective hybridization of the new strand and facilitating editing with minimal genomic homology. Nuclease-active versions of prime editors that cut both strands of DNA can also be used to accelerate the removal of the original DNA sequence.

[0242] Therefore, in several respects, this disclosure provides a method for simultaneously editing both strands of a double-stranded nucleic acid molecule by twin-prime editing at a target site to be edited, which includes contacting the double-stranded nucleic acid molecule with: (a) any of the prime editors disclosed herein (or one or more polynucleotides encoding it); (b) (i) a first spacer sequence that binds to a first binding site on the first strand of a double-stranded DNA sequence upstream of the target site to the second strand; (ii) a first gRNA core that can be complexed with the prime editor; and (iii) a first single strand A polynucleotide encoding a first prime-edit guide RNA (first pegRNA) or first pegRNA, comprising a first DNA synthesis template encoding a DNA sequence; and (c)(i) a second spacer sequence that binds to a second binding site on the second strand of a double-stranded DNA sequence downstream of the target site relative to the second strand; (ii) a second gRNA core that can complex with a prime editor; and (iii) a polynucleotide encoding a second prime-edit guide RNA (second pegRNA) or second pegRNA, comprising a second DNA synthesis template encoding a second single-stranded DNA sequence.

[0243] A variant of twin-prime editing encompasses quadruple-flap prime editing. This uses two sets of twin-prime editors to introduce genetic alterations at two different loci, for example, two different recombinase recognition sequences located at the 5' and 3' ends of the gene.

[0244] Like classical prime editing, twin-prime editing (including dual-flap and quadruple-flap prime editing) is a versatile and precise genome editing method that uses a nucleic acid-programmable DNA-binding protein ("napDNAbp") that works in conjunction with polymerase (i.e., provided in the form of a fusion protein or otherwise in trans with napDNAbp) to directly write new genetic information to a designated DNA site, where the prime editing system is programmed by prime editing (PE) guide RNA ("PEgRNA"), which both designates the target site and serves as a template for the synthesis of the desired edit in the form of a replacement DNA strand, as an manipulated extension (either DNA or RNA) on the guide RNA (e.g., at the 5' or 3' end or in the interior of the guide RNA). The replacement strand containing the desired edit (e.g., a recombinase-recognition sequence for insertion into the target DNA sequence) shares the same sequence as the intrinsic strand of the target site to be edited (with the exception that it contains the desired edit). Through DNA repair and / or replication mechanisms, the endogenous strand at the target site is replaced by a newly synthesized replacement strand containing the desired edit.

[0245] In some embodiments, the methods provided herein include placing a recombinase recognition site on a target nucleic acid molecule. In some embodiments, the methods provided herein combine site-directed recombination with the use of prime editing (e.g., using one of the prime editors described herein). In some embodiments, such methods facilitate the introduction of large DNA (e.g., full-length genes) into the genome of an organism (e.g., in the CNS).

[0246] The term "site-directed recombination" refers to a type of genetic modification also known as "conserved site-directed recombination." Site-directed recombination is a type of genetic modification in which DNA strand exchange occurs between fragments that possess at least a certain degree of sequence homology. Enzymes known as site-directed recombinases ("SSRs"), such as Bxb1, recognize and bind to short, specific DNA sequences ("recombinase recognition sites"), where they carry out rearrangement of DNA fragments by cleaving the DNA backbone, exchanging the two DNA helices involved, and rejoining the DNA strands. In some cases, the presence of a recombinase enzyme and a recombination site is sufficient for the reaction to proceed; in other systems, several accessory proteins and / or accessory sites are required. Many different genome modification strategies, including advanced approaches for the targeted introduction of transcription units to a given genomic locus, and cassette exchanges (RMCEs) mediated by these recombinases, rely on SSRs. Site-directed recombination systems are highly specific, rapid, and efficient, even when faced with complex eukaryotic genomes. These are naturally used in a variety of cellular processes, including bacterial genome replication, differentiation, and disease development, as well as the movement of mobile genetic elements. Recombination sites are typically between 30 and 200 nucleotides in length and generally consist of two motifs with partial inverted repeat symmetry to which recombinases bind, and which flank the central cross-sequence where recombination occurs. The pair of sites where recombination occurs between them is usually identical, but there are exceptions (e.g., attP and attB).

[0247] Once a recombinase recognition site is established on the genome, the corresponding recombinase that recognizes the established recombinase recognition site can be used to catalyze precise cleavage, strand exchange, and recombination of DNA fragments at the defined recombinase recognition site. This is achieved without relying on the cell's endogenous repair mechanisms for repairing double-strand breaks that could otherwise induce indels and other undesirable DNA rearrangements. Reactions catalyzed by recombinases and recombinase recognition sites result in large-scale genomic changes such as insertions, deletions, inversions, substitutions, and chromosomal translocations of one or more chromosomal regions encompassing one or more loci, one or more genes, or one or more parts of a gene (e.g., gene exons, introns, and gene regulatory regions).

[0248] In certain embodiments, one or more recombinase recognition sites can be inserted or introduced anywhere within the genome. In some organisms, the genome is organized as a single chromosome (e.g., bacteria), and the recombinase recognition site can be inserted into any locus within the chromosome. The insertion site can be within a gene or intergeneric region of the chromosome. The insertion can occur within an exon, an intron, or between them, or within a regulatory sequence such as a promoter, enhancer, or transcription-binding sequence. In other organisms, such as humans, the genome is organized into more than one chromosome, and the recombinase recognition site can be inserted into any locus within the chromosome. For example, in humans, the genome contains 23 pairs of chromosomes. In addition, the genome can also be mitochondrial DNA. The insertion site can be within a gene or intergeneric region of the chromosome. The insertion can occur within an exon, an intron, or between them, or within a regulatory sequence such as a promoter, enhancer, or transcription-binding sequence.

[0249] As used herein, “inserting into the genome” in any organism may include inserting one or more SSR recognition sites into one or more chromosomes (depending on the number of chromosomes making up the genome) and one or more chromosomal loci of a given genome. Where the genome contains more than one chromosome, “inserting into the genome” may include inserting one or more SSRs into one or more chromosomes of the genome. For example, in humans, who have 23 pairs of chromosomes, "inserting into the genome" means inserting one or more SSR recognition sites into any one of the following chromosomes: chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10, chromosome 11, chromosome 12, chromosome 13, chromosome 14, chromosome 15, chromosome 16, chromosome 17, chromosome 18, chromosome 19, chromosome 20, chromosome 21, chromosome 22, or chromosome 23 (also known as XX chromosomes or XY chromosomes), or inserting into any combination of the aforementioned chromosomes or into the mitochondrial genome.

[0250] In various embodiments, the Disclosure provides compositions and methods for installing one or more recombinase recognition sites using single-flap prime editing ("classical PE"), twin-prim editing (or twinPE), or multi-flap PE.

[0251] In some embodiments, classical PEs may be used to insert one or more recombinase recognition ...

Claims

1. A reverse transcriptase variant 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% sequence identity with SEQ ID NO: 1, comprising amino acid substitutions at positions 70, 72, 87, 102, 106, 118, 128, 158, 269, 363, 413, and 492 with respect to SEQ ID NO: 1, or corresponding substitutions in homologous sequences.

2. The reverse transcriptase variant according to claim 1, wherein the amino acid substitutions include P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, and S492N relative to SEQ ID NO:

1.

3. The reverse transcriptase variant according to claim 1 or 2, further comprising amino acid substitutions at positions 188, 260, 297, and 288 relative to SEQ ID NO:

1.

4. The reverse transcriptase variant according to claim 3, wherein the amino acid substitutions include S188K, I260L, S297Q, and R288Q relative to SEQ ID NO:

1.

5. A reverse transcriptase variant 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% sequence identity with SEQ ID NO: 30, wherein the reverse transcriptase variant comprises amino acid substitutions at positions 128 and 200, or corresponding substitutions in homologous sequences with respect to SEQ ID NO:

30.

6. The reverse transcriptase variant according to claim 5, wherein the amino acid substitutions include T128N and D200C relative to SEQ ID NO:

30.

7. The reverse transcriptase variant according to claim 5 or 6, further comprising amino acid substitutions at positions 223, 306, 313, and 330 relative to SEQ ID NO:

30.

8. The reverse transcriptase variant according to claim 7, wherein the amino acid substitutions include V223Y, T306K, W313F, and T330P relative to SEQ ID NO:

30.

9. A reverse transcriptase variant 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% sequence identity with SEQ ID NO: 30, comprising amino acid substitutions T128N and V223M; T128N and V223Y; T128F and V223M; or D200C and V223M, or corresponding substitutions in homologous sequences, optionally further comprising one or more amino acid substitutions D200N, T306K, W313F, T330P, and L603W with respect to SEQ ID NO:

30.

10. A reverse transcriptase variant 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% sequence identity with SEQ ID NO: 30, comprising amino acid substitutions at positions 128, 129, 196, 200, and 223 with respect to SEQ ID NO: 30, or corresponding substitutions in homologous sequences.

11. The reverse transcriptase variant according to claim 10, wherein the amino acid substitutions include T128N; V129A or V129G; P196S, P196T, or P196F; N200S or N200Y; and V223A, V223M, V223L, or V223E relative to SEQ ID NO: 30, and optionally further comprising one or more of the amino acid substitutions D200N, T306K, W313F, T330P, and L603W relative to SEQ ID NO:

30.

12. A reverse transcriptase variant according to any one of claims 5 to 11, wherein the reverse transcriptase variant comprises a C-terminal shortening of part or all of the RNaseH domain of SEQ ID NO:

30.

13. The reverse transcriptase variant according to claim 12, wherein the C-terminal shortening is located between amino acid positions D497 and I498 of SEQ ID NO:

30.

14. A Cas9 variant 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% sequence identity with SEQ ID NO: 2, wherein the Cas9 variant comprises amino acid substitutions at positions 775 and 918, or corresponding substitutions in homologous sequences with respect to SEQ ID NO:

2.

15. The Cas9 variant according to claim 14, wherein the amino acid substitutions include K775R and K918A relative to SEQ ID NO:

2.

16. A Cas9 variant 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% sequence identity with SEQ ID NO: 2, comprising amino acid substitutions at positions 99, 471, 632, 645, and 721 with respect to SEQ ID NO: 2, or corresponding substitutions in homologous sequences.

17. The Cas9 variant according to claim 16, wherein the amino acid substitutions include H99R, E471K, I632V, D645N, and H721Y relative to SEQ ID NO:

2.

18. The Cas9 variant according to claim 16 or 17, further comprising an amino acid substitution at position 654 relative to SEQ ID NO:

2.

19. The Cas9 variant according to claim 18, wherein the amino acid substitution comprises R654C relative to SEQ ID NO:

2.

20. A Cas9 variant according to any one of claims 16 to 19, further comprising an amino acid substitution at position 918 relative to SEQ ID NO:

2.

21. The Cas9 variant according to claim 20, wherein the amino acid substitution comprises K918A relative to SEQ ID NO:

2.

22. A Cas9 variant 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% sequence identity with SEQ ID NO: 2, comprising amino acid substitutions at positions 99, 471, and 632 with respect to SEQ ID NO: 2, or corresponding substitutions in homologous sequences.

23. The Cas9 variant according to claim 22, wherein the amino acid substitutions include H99R, E471K, and I632V relative to SEQ ID NO:

2.

24. The Cas9 variant according to claim 22 or 23, further comprising an amino acid substitution at position 721 relative to SEQ ID NO:

2.

25. The Cas9 variant according to claim 24, wherein the amino acid substitution is H721Y relative to SEQ ID NO:

2.

26. A Cas9 variant 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% sequence identity with SEQ ID NO: 2, wherein the Cas9 variant includes amino acid substitutions at positions 471 and 918, or corresponding substitutions in homologous sequences with respect to SEQ ID NO:

2.

27. The Cas9 variant according to claim 26, wherein the amino acid substitutions include E471K and K918A relative to SEQ ID NO:

2.

28. A Cas9 variant 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% sequence identity with SEQ ID NO: 2, wherein the Cas9 variant includes amino acid substitutions at positions 753 and 1151, or corresponding substitutions in homologous sequences with respect to SEQ ID NO:

2.

29. The Cas9 variant according to claim 28, wherein the amino acid substitutions include R753G and K1151E relative to SEQ ID NO:

2.

30. A Cas9 variant 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% sequence identity with SEQ ID NO: 2, comprising one or more amino acid substitutions at positions selected from the group consisting of 260, 298, 395, 769, 778, 1014, 1034, 1100, 1106, 1138, 1152, and 1320, or corresponding substitutions in homologous sequences with respect to SEQ ID NO:

2.

31. The Cas9 variant according to claim 30, wherein one or more amino acid substitutions are selected from the group consisting of E260K, D298N, R395C, T769P, R778Q, K1014E, A1034E, V1100I, S1106F, T1138A, G1152E, and A1320T.

32. A Cas9 variant according to claim 30 or 31, further comprising one or more additional amino acid substitutions at positions selected from the group consisting of 102, 753, 804, and 1003 relative to SEQ ID NO:

2.

33. The Cas9 variant according to claim 32, wherein one or more additional amino acid substitutions are selected from the group consisting of E102K, R753G, T804A, and K1003R.

34. A Cas9 variant according to any one of claims 30 to 33, comprising an amino acid substitution in any one of the following positions: For sequence number 2, 102, 395, 753, 778, and 1100; 753, 769, 1034, and 1320; 298, 753, 1034, and 1138; 102, 260, 395, 753, 778, 804, 1003, 1100, 1106, and 1152; or 102, 260, 395, 753, 778, 804, 1003, 1014, 1100, 1106, and 1152.

35. A Cas9 variant according to any one of claims 30 to 34, comprising an amino acid substitution in any one of the following positions: For sequence number 2, E102K, R395C, R753G, R778Q, and V1100I; R753G, T769P, A1034E, and A1320T; D298N, R753G, A1034E, and T1138A; E102K, E260K, R395C, R753C, R778Q, T804A, K1003R, V1100I, S1106F, and G1152E; or E102K, E260K, R395C, R753G, R778Q, T804A, K1003R, K1014E, V1100I, S1106F, and G1152E.

36. A Cas9 variant 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% sequence identity with SEQ ID NO: 2, wherein the Cas9 variant includes amino acid substitutions at positions 23 and 754, or corresponding substitutions in homologous sequences with respect to SEQ ID NO:

2.

37. The Cas9 variant according to claim 36, wherein the amino acid substitutions are D23G and H754R.

38. A prime editor comprising a reverse transcriptase variant according to any one of claims 1 to 13 and a nucleic acid programmed DNA-binding protein (napDNAbp).

39. The prime editor according to claim 38, wherein napDNAbp contains the Cas9 protein.

40. The Prime Editor according to claim 39, wherein the Cas9 protein is Cas9 nickase.

41. The prime editor according to any one of claims 38 to 40, wherein napDNAbp comprises the Cas9 variant according to any one of claims 14 to 37.

42. The Prime Editor according to any one of claims 38 to 40, wherein napDNAbp contains an amino acid sequence that is 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 any one of the Cas9 proteins of sequence numbers 2, 6, 8, 9, 12-24, or 133.

43. The Prime Editor according to any one of claims 38 to 40, wherein napDNAbp comprises an amino acid sequence that is 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 Cas9 protein of SEQ ID NO: 133, or SEQ ID NO:

133.

44. A prime editor comprising a Cas9 variant and polymerase according to any one of claims 14 to 37.

45. The Prime Editor according to claim 44, wherein the polymerase is a reverse transcriptase.

46. The Prime Editor according to claim 45, wherein the reverse transcriptase is a reverse transcriptase variant according to any one of claims 1 to 13.

47. Prime Editor according to claim 45, wherein the reverse transcriptase comprises a sequence 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% sequence identity with SEQ ID NO: 7, wherein the reverse transcriptase comprises amino acid substitutions at positions 60, 87, 165, 243, 267, 279, 318, and 343, or at corresponding positions in homologous sequences with respect to SEQ ID NO:

7.

48. Prime Editor according to claim 47, wherein the amino acid substitutions include E60K, K87E, E165D, D243N, R267I, E279K, K318E, and K343N relative to SEQ ID NO:

7.

49. The Prime Editor according to any one of claims 38 to 48, wherein the napDNAbp and reverse transcriptase are provided in trans or not fused to each other.

50. The prime editor according to any one of claims 38 to 48, wherein napDNAbp and reverse transcriptase are provided as a fusion protein or are fused to each other.

51. The prime editor according to claim 50, wherein napDNAbp and reverse transcriptase are fused via a linker.

52. The prime editor according to claim 51, wherein the linker includes one of sequence numbers 80 to 93.

53. A prime editor according to any one of claims 38 to 52, further comprising a nuclear localization sequence (NLS).

54. A prime editor comprising a Cas9 protein and a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 1, wherein the reverse transcriptase variant comprises amino acid substitutions P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, and S492N relative to SEQ ID NO:

1.

55. A prime editor comprising a Cas9 protein and a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 1, wherein the reverse transcriptase variant comprises amino acid substitutions P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, S188K, I260L, F269L, R288Q, S297Q, A363V, K413E, and S492N relative to SEQ ID NO:

1.

56. A prime editor comprising a Cas9 protein and a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 30, wherein the reverse transcriptase variant comprises the amino acid substitutions T128N, D200C, and V223Y relative to SEQ ID NO:

30.

57. A prime editor according to any one of claims 54 to 56, wherein the Cas9 protein comprises a Cas9 variant 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% sequence identity with SEQ ID NO: 2, wherein the Cas9 variant comprises amino acid substitutions K775R and K918A; H99R, E471K, I632V, D645N, H721Y, and K918A; or H99R, E471K, I632V, D645N, R654C, and H721Y with respect to SEQ ID NO:

2.

58. A reverse transcriptase, and a prime editor comprising a Cas9 variant 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% sequence identity with SEQ ID NO: 2, wherein the Cas9 variant comprises amino acid substitutions K775R and K918A relative to SEQ ID NO:

2.

59. A reverse transcriptase, and a prime editor comprising a Cas9 variant 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% sequence identity with SEQ ID NO: 2, wherein the Cas9 variant comprises amino acid substitutions H99R, E471K, I632V, D645N, H721Y, and K918A relative to SEQ ID NO:

2.

60. A reverse transcriptase, and a prime editor comprising a Cas9 variant 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% sequence identity with SEQ ID NO: 2, wherein the Cas9 variant comprises amino acid substitutions H99R, E471K, I632V, D645N, R654C, and H721Y relative to SEQ ID NO:

2.

61. The reverse transcriptase comprises a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 1, wherein the reverse transcriptase variant is an amino acid substitution P70T, G72V, S87G, M102I, K106R, K11 A prime editor according to any one of claims 58 to 60, comprising 8R, I128V, L158Q, F269L, A363V, K413E, and S492N; or P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, S188K, I260L, F269L, R288Q, S297Q, A363V, K413E, and S492N.

62. A prime editor according to any one of claims 58 to 60, wherein the reverse transcriptase comprises a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 30, wherein the reverse transcriptase variant comprises amino acid substitutions T128N, D200C, and V223Y relative to SEQ ID NO:

30.

63. A prime editor according to any one of claims 58 to 60, wherein the reverse transcriptase comprises a reverse transcriptase variant 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% sequence identity with SEQ ID NO: 7, wherein the reverse transcriptase comprises amino acid substitutions E60K, K87E, E165D, D243N, R267I, E279K, K318E, and K343N relative to SEQ ID NO:

7.

64. A prime editor comprising one Cas9 variant of SEQ ID NO: 28, 48, or 49 and one reverse transcriptase variant of SEQ ID NO: 25–27 or 50, or 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% of Cas9 variants and 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% of reverse transcriptase variants of one of SEQ ID NO: 25–27 or 50.

65. Prime Editor, Structure NH 2 A prime editor according to any one of claims 38 to 64, comprising -[binodelux NLS]-[Cas9]-[linker]-[reverse transcriptase]-[binodelux NLS]-[NLS].

66. The prime editor according to any one of claims 38 to 65, wherein the prime editor comprises a fusion protein architecture of PEmax.

67. A prime editor according to any one of claims 38 to 66, wherein the prime editor is smaller in size than PE2 and has comparable editing efficiency to that of PE2.

68. The prime editor according to any one of claims 38 to 67, wherein the prime editor has increased editing efficiency compared to PEmax for editing that requires a structured pegRNA reverse transcriptase template (RTT).

69. A fusion protein comprising a Cas9 variant and an effector domain as described in any one of claims 14 to 37.

70. The fusion protein according to claim 69, wherein the effector domain comprises nuclease activity, nickas activity, recombinase activity, deaminase activity, methyltransferase activity, methylase activity, acetylase activity, acetyltransferase activity, transcriptional activating activity, transcriptional repressing activity, or polymerase activity.

71. A reverse transcriptase variant comprising 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% of the sequence of any one of sequence numbers 25, 27, or 50.

72. A Cas9 variant containing 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% of any one of sequence numbers 28, 48, 49, or 145.

73. A prime editor containing 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% of any one of sequence numbers 155–158.

74. A complex comprising a pegRNA and a prime editor according to any one of claims 38 to 68 or a fusion protein according to claim 69 or 70, wherein the pegRNA is optionally epegRNA.

75. A polynucleotide encoding a reverse transcriptase variant according to any one of claims 1 to 13 or 71.

76. A polynucleotide encoding a Cas9 variant according to any one of claims 14 to 37 or 72.

77. One or more polynucleotides encoding a prime editor according to any one of claims 38 to 68 or a fusion protein according to claim 69 or 70.

78. A vector comprising the polynucleotide described in claim 75 and / or the polynucleotide described in claim 76.

79. The vector according to claim 78, further comprising a polynucleotide encoding pegRNA.

80. One or more vectors comprising one or more polynucleotides as described in claim 77.

81. The vector according to claim 80, further comprising a polynucleotide encoding pegRNA.

82. One or more AAV particles comprising one or more polynucleotides according to any one of claims 75 to 77 or one or more vectors according to any one of claims 78 to 81.

83. The AAV particle according to claim 82, wherein the AAV particle comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.

84. One or more AAV particles according to claim 82 or 83, wherein the AAV particles include AAV9.

85. One or more AAV particles according to any one of claims 82 to 84, wherein the first AAV particle comprises a polynucleotide having the structure 5'-[inverted terminal repeat (ITR) sequence]-[promoter]-[Cas9 N-terminal fragment]-[N-intane]-[terminator sequence]-[ITR sequence]-3', and the second AAV particle comprises a polynucleotide having the structure 5'-[ITR sequence]-[promoter]-[C-intane]-[Cas9 C-terminal fragment]-[reverse transcriptase]-[terminator sequence]-[any nickeling gRNA]-[pegRNA]-[ITR]-3', and the first AAV particle comprises a polynucleotide comprising a first AAV particle and a second AAV particle.

86. A cell comprising a reverse transcriptase variant according to any one of claims 1 to 13 or 71, a Cas9 variant according to any one of claims 14 to 37 or 72, a prime editor according to any one of claims 38 to 68 or 73, a fusion protein according to claim 69 or 70, a complex according to claim 74, one or more polynucleotides according to any one of claims 75 to 77, one or more vectors according to any one of claims 78 to 81, or one or more AAV particles according to any one of claims 82 to 85.

87. A pharmaceutical composition comprising a reverse transcriptase variant according to any one of claims 1 to 13 or 71, a Cas9 variant according to any one of claims 14 to 37 or 72, a prime editor according to any one of claims 38 to 68 or 73, a fusion protein according to claim 69 or 70, a complex according to claim 74, one or more polynucleotides according to any one of claims 75 to 77, one or more vectors according to any one of claims 78 to 81, one or more AAV particles according to any one of claims 82 to 85, or a cell according to claim 86.

88. A method for editing a nucleic acid molecule by prime editing, comprising contacting the nucleic acid molecule with a prime editor according to any one of claims 38 to 68 or 73, a complex according to claim 74, one or more polynucleotides according to any one of claims 75 to 77, or one or more vectors according to any one of claims 78 to 81, thereby installing one or more modifications to the nucleic acid molecule at a target site.

89. A method for simultaneously editing both strands of a double-stranded nucleic acid molecule at a target site to be edited, comprising contacting the double-stranded nucleic acid molecule with the following: (a) A prime editor according to any one of claims 38 to 68, or a polynucleotide encoding a prime editor; (b) A first prime editing guide RNA (first pegRNA) or a polynucleotide encoding the first pegRNA, comprising: (i) A first spacer sequence that binds to a first binding site on the first strand of a double-stranded DNA sequence upstream of the target site relative to the second strand, (ii) A first gRNA core that can be complexed with a prime editor, and (iii) A first DNA synthesis template that encodes a first single-stranded DNA sequence, Furthermore (c) A second prime editing guide RNA (second pegRNA) or a polynucleotide encoding the second pegRNA, including the following: (i) A second spacer sequence that binds to a second binding site on the second strand of a double-stranded DNA sequence downstream of the target site relative to the second strand; (ii) A second gRNA core that can be complexed with a prime editor, and (iii) A second DNA synthesis template that encodes a second single-stranded DNA sequence.

90. The method according to claim 88 or 89, further comprising contacting a nucleic acid molecule with one or more second-strand nickeling gRNAs.

91. The method according to any one of claims 88 to 90, wherein the method comprises placing a recombinase recognition site on a nucleic acid molecule.

92. A kit comprising a reverse transcriptase variant according to any one of claims 1 to 13 or 71, a Cas9 variant according to any one of claims 14 to 37 or 72, a prime editor according to any one of claims 38 to 68 or 73, a fusion protein according to claim 69 or 70, a complex according to claim 74, one or more polynucleotides according to any one of claims 75 to 77, one or more vectors according to any one of claims 78 to 81, one or more AAV particles according to any one of claims 82 to 85, or a cell according to claim 86.

93. Use in the manufacture of a pharmaceutical product of a reverse transcriptase variant according to any one of claims 1 to 13 or 71, a Cas9 variant according to any one of claims 14 to 37 or 72, a prime editor according to any one of claims 38 to 68 or 73, a fusion protein according to claim 69 or 70, a complex according to claim 74, one or more polynucleotides according to any one of claims 75 to 77, one or more vectors according to any one of claims 78 to 81, one or more AAV particles according to any one of claims 82 to 85, or a cell according to claim 86.

94. A reverse transcriptase variant according to any one of claims 1 to 13 or 71, a Cas9 variant according to any one of claims 14 to 37 or 72, a prime editor according to any one of claims 38 to 68 or 73, a fusion protein according to claim 69 or 70, a complex according to claim 74, one or more polynucleotides according to any one of claims 75 to 77, one or more vectors according to any one of claims 78 to 81, one or more AAV particles according to any one of claims 82 to 85, or a cell according to claim 86, for use in medicine.

95. The following are systems of polynucleotides for phage-assisted continuous and discontinuous evolution in Prime Editor: i) The first polynucleotide encoding the pegRNA and gIII gene; ii) A second polynucleotide encoding the Cas9 protein fused to the N-intene; iii) A third polynucleotide encoding RNA polymerase; iv) A fourth polynucleotide, optionally, which encodes a protein that can be mutated into a phage, where the fourth polynucleotide includes the MP6 plasmid; and v) A fifth polynucleotide encoding reverse transcriptase fused to C-intene.

96. The following are systems of polynucleotides for phage-assisted continuous and discontinuous evolution in Prime Editor: i) The first polynucleotide encoding the pegRNA and gIII gene; ii) A second polynucleotide encoding the prime editor; iii) A third polynucleotide encoding RNA polymerase; and iv) A fourth polynucleotide, optionally, that encodes a protein that can be mutated into a phage, where the fourth polynucleotide is the MP6 plasmid.

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