Systems, methods, and compositions for cystic fibrosis gene editing

Abstract

Provided herein are systems, methods, and compositions for cystic fibrosis gene correction using programmable genomic editing. The programmable genomic editing can efficiently edit cystic fibrosis genes, such as the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Description

SYSTEMS, METHODS, AND COMPOSITIONS FOR CYSTIC FIBROSIS GENE EDITINGCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 496,596, filed on April 17, 2023, the entire disclosure of which is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with support from the Cystic Fibrosis Foundation under Grant No. 032542-00002 and government support under Grant No. EB031957 awarded by the National Institute of Health. The Government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing, which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 15, 2024, is named 752971_083474-039PC_SL.xml and is 213,354 bytes in size.BACKGROUND

[0004] Cystic fibrosis (CF) is a genetic disease, more specifically an autosomal recessive genetic disease, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Mutations in this gene can result in total or partial loss of CFTR protein production. The CFTR gene is primarily expressed in cells lining the respiratory tract, the gastrointestinal tract, the pancreas, sweat glands, and the genitourinary system. This gene is involved in the production of a protein that regulates the transfer of sodium and chloride ions across cell membranes. Affected individuals experience multi-glandular malfunction, as secretions from the glands become very thick and sticky causing widespread inflammation, obstruction, and infection. Many patients experience lung disease, heart disease, gastrointestinal obstruction syndromes, liver dysfunction, exocrine and endocrine pancreatic dysfunction, and infertility. Current treatment of patients with CF is limited to symptomatic management of the disease, rather than permanent correction of the causative genetic defect.

[0005] Investigational approaches for treating CF include gene editing. One approach is to edit the CF gene via short fragment homologous recombination using DNA fragments containing the correct CFTR sequence, which can recombine with F508del CFTR genomic DNA (Goncz, et al., Hum Mol Genet., 7:1913-1919 (1998); Goncz, et al., Gene Then, 8:961-965 (2001); Bruscia, et al., Gene Then, 9:683-685 (2002)). Another approach involves the introduction of the F508del mutation into normal mouse lung tissue (Goncz, et al., Gene Then, 8:961-965 (2001)). Yet another approach includes the use of Zinc finger nucleases (ZFNs (Beumer, et al., Genetics, 172:2391-2403 (2006)) to insert a CFTR transgene at the CCR5 locus 21 and for modification of F508del at levels <1 % in vitro (Lee Ciaran, et al., BioResearch Open Access, 1 :99-108 (2012)). Still another approach includes using a CRISPR / Cas-9 enzyme to correct CF genes in intestinal organoids from CF patients in culture (Schwank, et al., Cell Stem Cell., 13:653-658 (2013)). Unfortunately, these approaches suffer significant drawbacks, such as high off-target effects, low efficiency, and high cost.

[0006] Therefore, there is a need for effective systems, methods, and compositions for the treatment and / or prevention of cystic fibrosis.SUMMARY

[0007] Provided herein are systems, methods, and compositions for cystic fibrosis treatment and / or prevention via programmable genomic editing. The programmable genomic editing can efficiently edit cystic fibrosis genes, such as the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

[0008] In a first aspect, disclosed is an editing system capable of site-specifically integrating an exogenous nucleic acid into a cystic fibrosis transmembrane conductance regulator (CFTR) gene. The system comprises (a) an editing polypeptide, wherein the editing polypeptide comprises a DNA binding nuclease domain linked via a linker to a reverse transcriptase domain, wherein the DNA binding nuclease domain comprises a nickase activity that nicks a strand of the CFTR gene to form a nicked strand; (b) a guide RNA (gRNA) comprising a primer binding sequence, a sequence complementary to an integration sequence; and a targeting sequence that can recognize a target site of the CFTR gene and can hybridize to a strand from the CFTR gene complementary to the nicked strand, wherein the gRNA comprises a nucleic acid sequence 85%-100% identical to any one of the nucleic acid sequences of Table 11 , and wherein the exogenous nucleic acid is linked to a sequence that is an integration cognate of the integration sequence; and (c) an integration enzyme.

[0009] In one embodiment, the DNA binding nuclease comprises a nickase activity selected from the group consisting of Cas9-D10A, Cas9-H840A, and Cas12a / b / c / d / e / f / g / h / i / j.

[0010] In another embodiment, the primer binding site can hybridize to the 3' end of the nicked DNA strand.

[0011] In another embodiment, the integration sequence for the integration enzyme is selected from an attB sequence, an attP sequence, an attL sequence, an attR sequence, a Iox71 sequence, and a FRT sequence.

[0012] In another embodiment, the recognition site for the integration enzyme is a Bxb1 sequence.

[0013] In another embodiment, the linker is cleavable or non-cleavable.

[0014] In another embodiment, wherein the linker can be replaced by two associating binding domains of the DNA binding nuclease linked to a reverse transcriptase.

[0015] In another embodiment, the integration enzyme is selected from the group consisting of Cre, Dre, Vika, Bxb1 , q>C31 , RDF, FLP, q>BT1, R1 , R2, R3, R4, R5, TP901-1, A118, q)FC1, cpC1 , MR11 , TG1 , cp370.1 , Wp, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, Conceptll, Museum, Severus, Airmid, Benedict, Hinder, ICIeared, Sheen, Mundrea, BxZ2, cpRV, retrotransposases encoded by R2, L1 , Tol2 Tc1 , Tc3, Mariner (Himar 1), Mariner (mos 1), and Minos, and any mutants thereof.

[0016] In another embodiment, the integration enzyme is Bxb1 or a mutant thereof.

[0017] In another embodiment, wherein the reverse transcriptase domain comprises a mutation relative to a wild-type sequence.

[0018] In another embodiment, the reverse transcriptase domain is selected from the group consisting of Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain, transcription xenopolymerase (RTX), avian myeloblastosis virus reverse transcriptase (AMV- RT), and Eubacterium rectale maturase RT.

[0019] In another embodiment, the M-MLV reverse transcriptase domain comprises one or more mutations selected from the group consisting of D200N, T306K, W313F, T330P, and L603W.

[0020] In a second aspect, disclosed is a method of site-specifically integrating an exogenous nucleic acid sequence into a cystic fibrosis transmembrane conductance regulator (CFTR) gene.

[0021] In one embodiment, the method comprises (a) incorporating at least one integration sequence at a specific target site in the CFTR gene by introducing ex vivo into a mammalian cell: (i) an editing polypeptide, wherein the editing polypeptide comprises a DNA binding nuclease domain linked via a linker to a reverse transcriptase domain, wherein the DNAbinding nuclease domain comprises a nickase activity; and (ii) a guide RNA (gRNA) comprising a targeting sequence, a primer binding sequence, a sequence complementary to the at least one integration sequence, and a nucleic acid sequence 85%-100% identical to any one of the nucleic acid sequences of T able 11 , wherein the gRNA interacts with the editing polypeptide to direct the editing polypeptide to the target site of the CFTR gene, wherein the DNA binding nuclease domain nicks a strand of the CFTR gene to form a nicked site, and wherein the reverse transcriptase domain reverse transcribes the complement of the at least one integration sequence and thereby incorporates the at least one integration sequence into the nicked site, thereby incorporating the at least one integration sequence at the specific target site of the CFTR gene; and (b) integrating the exogenous nucleic acid sequence into the CFTR gene target nucleic acid by introducing into the cell: (i) the exogenous nucleic acid sequence linked to a sequence that is an integration cognate of the at least one integration sequence; and (ii) an integration enzyme that integrates the exogenous nucleic acid sequence into the CFTR gene at the at least one integration sequence, thereby site-specifically integrating the exogenous nucleic acid sequence into the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

[0022] In another embodiment, the editing polypeptide, the gRNA, the integration enzyme, and the exogenous nucleic acid sequence are introduced into the cell concurrently or not concurrently.

[0023] In another embodiment, the gRNA, an expressible polynucleotide construct encoding the editing polypeptide, and an expressible polynucleotide construct encoding the integration enzyme are introduced to the cell using a virus, a RNP, an mRNA, a lipid, or a polymeric nanoparticle.

[0024] In another embodiment, the gRNA hybridizes to a strand of the CFTR gene.

[0025] In another embodiment, the exogenous nucleic acid is introduced into the cell as an adeno-associated virus (AAV) or an adenovirus (AdV).

[0026] In another embodiment, the exogenous nucleic acid is introduced into the cell as a minicircle, a plasmid, mRNA, or a linear DNA.

[0027] In another embodiment, the minicircle does not comprise a sequence of a bacterial origin.

[0028] In another embodiment, the integration sequence is an attB sequence, an attP sequence, an attL sequence, an attR sequence, a Iox71 sequence, a Vox sequence, or a FRT sequence.

[0029] In another embodiment, the method further comprises introducing a nicking guide RNA (ngRNA).

[0030] In another embodiment, the exogenous nucleic acid is a reporter gene; the exogenous nucleic acid is a degradation tag for programmable knockdown of proteins in the presence of small molecules; the exogenous nucleic acid is a T-cell receptor (TCR), a chimeric antigen receptor (CAR), an interleukin, a cytokine, or an immune checkpoint gene for integration into a T-cell or natural killer (NK) cell; the exogenous nucleic acid is a beta hemoglobin (HBB) gene and the cell is a hematopoietic stem cell (HSC); or the exogenous nucleic acid is a metabolic gene.

[0031] In another embodiment, the reporter gene is a fluorescent protein.

[0032] In another embodiment, the cell is a dividing cell or a non-dividing cell.

[0033] In another embodiment, the exogenous nucleic acid is CFTR exon 1-27.

[0034] In a third aspect disclosed is a genome editing system for a cystic fibrosis transmembrane conductance regulator (CFTR) gene.

[0035] In one embodiment, the system comprises (a) an R2 element enzyme; and (b) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology region, a 3’ homology region, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the CFTR gene, and wherein the R2 element enzyme comprises a reverse transcriptase domain, and a nickase domain.

[0036] In another embodiment, the R2 element enzyme further comprises a targeting domain.

[0037] In another embodiment, the targeting domain is a natural targeting domain or an engineered targeting domain.

[0038] In another embodiment, the nucleic acid insertion into the genome is a DNA or RNA insertion template.

[0039] In another embodiment, the R2 element enzyme is a modified R2 element enzyme.

[0040] In another embodiment, the coding sequence of the R2 element enzyme is modified.

[0041] In another embodiment, the modified R2 element enzyme is modified by an N-terminal or C-terminal truncation of the R2 element enzyme sequence.

[0042] In another embodiment, the modified R2 element enzyme comprises a linker.

[0043] In another embodiment, the linker is an XTEN linker.

[0044] In another embodiment, the modified R2 element enzyme is a fusion protein.

[0045] In another embodiment, the modified R2 element is fused to a Cas9 protein that is fully active, catalytically dead (H840A / D10A for SpCas9), or functioning as a nickase (H840A or D10A for SpCas9).

[0046] In another embodiment, the modified R2 element is fused to a Cas12 protein that is fully active, catalytically dead, or functioning as a nickase.

[0047] In another embodiment, the modified R2 element is fused to a TALEN protein, zinc finger protein, argonaute, or meganuclease protein.

[0048] In another embodiment, the 5’ homology region, the 3’ homology region, or both the 5’ and 3’ homology region target an exogenously introduced landing sequence.

[0049] In another embodiment, the genome editing system further comprises a guide RNA.

[0050] In a fourth aspect, disclosed is a method of inserting a nucleic acid into a cystic fibrosis transmembrane conductance regulator (CFTR) gene in a cell, wherein the method comprises supplying a Cas9 or Cas12 fusion protein to the cell, wherein the Cas9 or Cas12 fusion protein is supplied with a payload RNA template, wherein the RNA template is reverse transcribed by the Cas9 or Cas12 fusion protein prior to being inserted into the gene of the cell; and wherein the nucleic acid is inserted into the gene.

[0051] In one embodiment, the Cas9 fusion protein comprises a Cas9 portion and an R2 element portion, optionally wherein the Cas9 fusion protein comprises a targeting domain, a reverse transcriptase domain, and a nickase domain; or optionally wherein the Cas12 fusion protein comprises a Cas12 portion and an R2 element portion.BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Aspects, features, benefits, and advantages of the embodiments described herein will be apparent with regard to the following description, appended claims, and accompanying drawings where:

[0053] FIG. 1 is a schematic representation of the Programmable Addition via Site-specific Targeting Elements (PASTE) correction of cystic fibrosis according to embodiments of the present teaching. A payload encoding for CFTR exons 2-27 and a splice acceptor and theinsertion of the payload into the intron 1 of the CFTR gene to introduce functional CF protein are illustrated;

[0054] FIG. 2A is a schematic representation of the CFTR intron 1 (24,105 bp) and the intron 1 Region 3 (5,837bp from exon 1);

[0055] FIG. 2B is a histogram of the percent of AttB insertion for Forward Guides 1-8 paired with Reverse Guides 1-5 at intron 1 region 3 of the CFTR gene;

[0056] FIG. 3A is a schematic representation of the CFTR intron 1 (24,105 bp) and the intron 1 Region 4 (11 ,295bp from exon 1);

[0057] FIG. 3B is a histogram of the percent of AttB insertion for Forward Guides 1-8 paired with Reverse Guides 1-5 at intron 1 region 4 of the CFTR;

[0058] FIG. 4 is a histogram of the percent integration of EGFP Insert 800 bp and EGFP Insert 2981 bp;

[0059] FIG. 5 is a histogram of the percent integration of EGFP Insert 800 bp;

[0060] FIG. 6 is a histogram of the percent integration of EGFP Insert 800 bp;

[0061] FIG. 7 is a schematic representation of the Site-specific Target-primed Insertion via Targeted CRISPR Homing of Retroelements (STITCHR) correction of cystic fibrosis according to embodiments of the present teaching. A STITCHR CFTR template and a genomic target CFTR intron 1 are illustrated;

[0062] FIG. 8 is a histogram of the percent payload insertion at CFTR intron 1 region 1 using STITCHR according to embodiments of the present teaching. About 1% integration rates were achieved at the CFTR locus. Five forward facing guides were co-transfected with three reverse guides at intron 1 region 1 using STITCHR (R2Toc-SpCas9H840A);

[0063] FIG. 9 is a histogram of the percent payload insertion at the CFTR intron 1 region 2 using STITCHR according to embodiments of the present teaching. Five forward facing guides were co-transfected with six reverse guides at intron 1 region 2 using STITCHR (R2Toc- SpCas9H840A);

[0064] FIG. 10 is a histogram of the percent payload insertion at the CFTR intron 1 region 3 using STITCHR according to embodiments of the present teaching. Five forward facing guides were co-transfected with seven reverse guides at intron 1 region 3 using STITCHR (R2Toc- SpCas9H840A);

[0065] FIG. 11 is a histogram of the percent payload insertion at the CFTR intron 1 region 4 using STITCH R according to embodiments of the present teaching. Seven forward facing guides were co-transfected with six reverse guides at intron 1 region 4 using STITCH R) (R2Toc-SpCas9H840A);

[0066] FIG. 12 is a histogram of the percent payload insertion at the CFTR intron 1 region 5 using STITCH R according to embodiments of the present teaching. Seven forward facing guides were co-transfected with nine reverse guides at intron 1 region 5 using STITCH R (R2Toc-SpCas9H840A);

[0067] FIG. 13 are histograms of the percent payload insertion at the CFTR intron 1 , regions 1 , 2, 4, and 5, and intron 2, region 1 using PASTE;

[0068] Fig. 14 are schematic representations of locations of CFTR intron 1 , regions 1 , 2, 4 and 5, and intron 2, region 2;

[0069] FIG. 15 are histograms of the percent payload insertion at the CFTR intron 1 , region 2 using PASTE;

[0070] FIG. 16 are histograms of the percent payload insertion at the CFTR intron 1 , region 2 of -2000 bp EGFP payload using PASTE;

[0071] FIG. 17 are histograms of the percent payload insertion at the CFTR intron 1 , region 2 of -5000 bp EGFP payload using PASTE;

[0072] FIG. 18 is a histogram of the optimization of pegRNAs;

[0073] FIG. 19 is a histogram of the optimization of the linker;

[0074] FIG. 20 is a histogram of the percent payload insertion at the CFTR intron 1 in HEK293FT cells; and

[0075] FIG. 21 is a histogram of the percent payload insertion at the CFTR intron 1 in 16Hbe cells.DETAILED DESCRIPTIONDefinitions

[0076] The use of the singular forms herein includes the plural unless specifically stated otherwise. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0077] Regarding proteins contemplated herein, it should be understood that polynucleotides encoding the proteins are also provided, as are vectors comprising the polynucleotides encoding the proteins.

[0078] As used herein, the term “Cas9” refers to an RNA-guided nuclease comprising a Cas9 domain, or a functional fragment or variant thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and / or the gRNA binding domain of Cas9).

[0079] As used herein, the term “DNA binding nickase” such as a Cas9 or Cas12 nickase refers to a variant of DNA binding nuclease which is capable of cleaving only one strand of a target double stranded polynucleotide, thereby introducing a single-strand break in the target double strand polynucleotide. Similar terminology is used herein in reference to other Cas nucleases that exhibit nickase activity. For example, a “Cas12e nickase” would be used similarly herein to refer to a Cas12e which is capable of cleaving only one strand of a target double stranded polynucleotide, thereby introducing a single-strand break in the target double strand polynucleotide.

[0080] As used herein, the term “derived from,” with reference to a polynucleotide sequence refers to a polynucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a reference naturally occurring nucleic acid sequence from which it is derived. The term “derived from,” with reference to an amino acid sequence refers to an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a reference naturally occurring amino acid sequence from which it is derived. The term “derived from” as used herein does not denote any specific process or method for obtaining the polynucleotide or amino acid sequence. For example, the polynucleotide or amino acid sequence can be chemically synthesized.

[0081] As used herein, the term “DNA” or “DNA polynucleotides” refers to macromolecules that include multiple deoxyribonucleotides that are polymerized via phosphodiester bonds. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose.

[0082] As used herein, the term “functional fragment” in reference to a nucleic acid sequence, an amino acid sequence, or the like refers to a fragment of a reference nucleic acid sequence, an amino acid sequence, or the like that retains at least one particular function. For example, a functional fragment of an aptamer binding protein can refer to a fragment of the protein that retains the ability to bind the cognate aptamer. Not all functions of the reference protein need be retained by a functional fragment of the protein. In some instances, one or more functions are selectively reduced or eliminated.

[0083] As used herein, the term “functional variant” in reference to a nucleic acid sequence, an amino acid sequence, or the like refers to a nucleic acid sequence, an amino acid sequence, or the like that comprises at least one nucleic acid or amino acid modification (e.g., a substitution, deletion, addition) compared to the nucleic acid or amino acid sequence of a reference nucleic acid sequence, an amino acid sequence, or the like, that retains at least one particular function. For example, a functional variant of an aptamer binding protein refers to a protein that binds an aptamer comprising an amino acid substitution as compared to a wildtype reference protein that retains the ability to bind the cognate aptamer. Not all functions of the reference wild type protein need be retained by the functional variant of the protein. In some instances, one or more functions are selectively reduced or eliminated.

[0084] As used herein, the term “fusion protein” and grammatical equivalents thereof refer to a protein that comprises an amino acid sequence derived from at least two separate proteins. The amino acid sequence of the at least two separate proteins can be directly connected through a peptide bond; or can be operably connected through an amino acid linker. Therefore, the term fusion protein encompasses embodiments, wherein the amino acid sequence of e.g., Protein A is directly connected to the amino acid sequence of Protein B through a peptide bond (Protein A - Protein B), and embodiments, wherein the amino acid sequence of e.g., Protein A is operably connected to the amino acid sequence of Protein B through an amino acid linker (Protein A - linker - Protein B).

[0085] A used herein, the term “fuse” and grammatical equivalents thereof refer to the operable connection of an amino acid sequence derived from one protein to the amino acid sequence derived from different protein. The term fuse encompasses both a direct connection of the two amino acid sequences through a peptide bond, and the indirect connection through an amino acid linker.

[0086] As used herein, the term “guide RNA” or “gRNA” refers to an RNA polynucleotide that guides the insertion or deletion of one or more polynucleotides of interest (e.g., a gene of interest) into a target polynucleotide (e.g., genome) via a nuclease, nickase, or functional fraction or variant thereof (e.g., a Cas protein, e.g., Cas9).

[0087] As used herein, the term “integrase” refers to a protein capable of integrating a polynucleotide of interest (e.g., a gene) into a desired location (e.g., at an integration site) in a target polynucleotide (e.g., the genome of a cell). The integration can occur in a single reaction or multiple reactions.

[0088] As used herein, the term “integration sequence” refers to a polynucleotide sequence that encodes an integration site.

[0089] As used herein, the term “integration site” refers to a polynucleotide sequence capable of being recognized by an integrase.

[0090] As used herein, the term “modification,” with reference to a polynucleotide sequence, refers to a polynucleotide sequence that comprises at least one substitution, alteration, inversion, addition, or deletion of nucleotide compared to a reference polynucleotide sequence. Modifications can include the inclusion of non-naturally occurring nucleotide residues. As used herein, the term “modification,” with reference to an amino acid sequence refers to an amino acid sequence that comprises at least one substitution, alteration, inversion, addition, or deletion of an amino acid residue compared to a reference amino acid sequence. Modifications can include the inclusion of non-naturally occurring amino acid residues. Naturally occurring amino acid derivatives are not considered modified amino acids for purposes of determining percent identity of two amino acid sequences. For example, a naturally occurring modification of a glutamate amino acid residue to a pyroglutamate amino acid residue would not be considered an amino acid modification for purposes of determining percent identity of two amino acid sequences. Further, for example, a naturally occurring modification of a glutamate amino acid residue to a pyroglutamate amino acid residue would not be considered an amino acid “modification” as defined herein.

[0091] As used herein, the term “nickase” refers to a protein (e.g., a nuclease) that has the ability to cleave only one strand of a target double stranded polynucleotide, thereby introducing a single-strand break in the target double strand polynucleotide. In some embodiments, for example, an editing polypeptide described herein comprises a Cas9 nuclease with one of the two nuclease domains inactivated, e.g., by amino acid substitution of H840A, wherein the Cas9 has nickase activity but is not able to make a double strand break in a target double stranded polynucleotide.

[0092] As used herein, the terms “operably connected” and “operably linked” are used interchangeably and refer to a linkage of polynucleotide sequence elements or polypeptide sequence elements in a functional relationship. For example, a polynucleotide sequence is operably connected when it is placed into a functional relationship with another polynucleotide sequence. In some embodiments, a transcription regulatory polynucleotide sequence e.g., a promoter, enhancer, or other expression control element is operably-linked to a polynucleotide sequence that encodes a protein if it affects the transcription of the polynucleotide sequence that encodes the protein.

[0093] As used herein, the term “orthogonal integration sites” refers to integrations sites that do not significantly recognize the recognition site or nucleotide sequence of the integrase (e.g., recombinase) recognized by the other.

[0094] The determination of “percent identity” between two sequences (e.g., polypeptide or polynucleotides) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is herein incorporated by reference in its entirety. Such an algorithm is incorporated into the N BLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which is herein incorporated by reference in its entirety. BLAST nucleotide searches can be performed with the N BLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc. Acids Res. 25: 3389-3402, which is herein incorporated by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (See, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is herein incorporated by reference in its entirety. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0095] As used herein the term “pharmaceutical composition” means a composition that is suitable for administration to an animal, e.g., a human subject, and comprises a therapeutic agent and a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable carrier or diluent” means a substance for use in contact with the tissues of human beingsand / or non-human animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable therapeutic benefit / risk ratio.

[0096] The terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of DNA or RNA. The nucleic acid molecule can be single-stranded or double-stranded; contain natural, non-natural, or altered nucleotides; and contain a natural, non-natural, or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified nucleic acid molecule. Nucleic acid molecules include, but are not limited to, all nucleic acid molecules which are obtained by any means available in the art, including, without limitation, recombinant means, e.g., the cloning of nucleic acid molecules from a recombinant library or a cell genome, using ordinary cloning technology and polymerase chain reaction, and the like, and by synthetic means. The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application will recite thymidine (T) in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the thymidines (Ts) would be substituted for uracils (Us). Thus, any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each thymidine (T) of the DNA sequence is substituted with uracil (U).

[0097] As used herein, the term “primer binding site” or “PBS” refers to the portion of a gRNA that binds to the polynucleotides sequence at the 3' end of the flap that is formed after the DNA binding nickase nicks the target polynucleotide sequence.

[0098] The terms “protein” and “polypeptide” are used interchangeably herein and refer to a polymer of at least two amino acids linked by a peptide bond.

[0099] As used herein, the term “protospacer” refers to the DNA sequence that has the same (or similar) nucleotide sequence as the spacer sequence of a gRNA. The gRNA anneals to the complement of the protospacer sequence on the opposite strand of the DNA.

[0100] As used herein, the term “protospacer adjacent motif” or “PAM” refers to a short DNA sequence, typically 2-6 base pairs, that functions to aid a Cas nickase in recognizing the target DNA.

[0101] As used herein, the term “recognition site” refers to a polynucleotide sequence that pairs with an integration site to mediate integration by an integrase (e.g., a recombinase).

[0102] As used herein, the term “RNA” or “RNA polynucleotide” refers to macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds. Ribonucleotides are nucleotides in which the sugar is ribose. RNA may contain modified nucleotides; and contain natural, non-natural, or altered internucleotide linkages, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified nucleic acid molecule.

[0103] As used herein, the term “hairpin loop” in reference to an RNA polynucleotide (e.g., an aptamer) refers to an RNA sequence that under physiological conditions is able to basepair to form a double helix that ends in an unpaired loop.

[0104] As used herein, the term “reverse transcriptase” refers to a protein (e.g., a polymerase) that is capable of RNA-dependent DNA synthesis. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. An exemplary reverse transcriptase commonly used in the art is derived from the moloney murine leukemia virus (M-MLV). See, e.g., Gerard, G. R., DNA 5:271-279 (1986) and Kotewicz, M. L., et al., Gene 35:249-258 (1985).

[0105] As used herein, the term “reverse transcriptase template sequence” refers to the portion of a gRNA that encodes the polynucleotide desired to be integrated into the target polynucleotide (e.g., genome) that is synthesized by the reverse transcriptase. The reverse transcriptase template sequence is used as a template during DNA synthesis by the reverse transcriptase.

[0106] As used herein, the term “scaffold” in reference to a gRNA refers to a polynucleotide in a gRNA that mediates binding to a nuclease (e.g., nickase) or a functional fragment or variant thereof (e.g., Cas9 (e.g., Cas9 nickases)).

[0107] As used herein, the term “spacer” in reference to a gRNA refers to a polynucleotide in a gRNA that mediates binding to a polynucleotide comprising a sequence complementary to the protospacer.

[0108] As used herein, the term “therapeutic nucleotide modification” refers to a polynucleotide of interest that encodes at least one nucleotide modification (e.g., substitution, deletion, or insertion) relative to the endogenous target polynucleotide (e.g., gene) sequence that is intended to have or does have a therapeutic effect in a subject.

[0109] A “therapeutically effective amount” of a therapeutic agent (e.g., a composition or system described herein) refers to any amount of the therapeutic agent that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of adisease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0110] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disease and / or symptom(s) associated therewith or obtaining a desired pharmacologic and / or physiologic effect. It will be appreciated that, although not precluded, treating a disease does not require that the disease, or symptom(s) associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, i.e. , without limitation, the effect partially or completely reduces, diminishes, abrogates, abates, alleviates, decreases the intensity of, or cures a disease and / or adverse symptom attributable to the disease. In some embodiments, the effect is preventative, i.e., the effect protects or prevents an occurrence or reoccurrence of a disease. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of a composition as described herein.PASTE and Prime Gene Editing

[0111] The present disclosure relates in part to gene editing using Programmable Addition via Site-specific Targeting Elements (PASTE) and prime editing. PASTE editing utilizes a modified PRIME gene editing technique to site-specifically insert an integration site within a target polynucleotide (e.g., genome) and subsequently utilizing the site to integrate a polynucleotide of interest. PASTE-REPLACE editing utilizes PASTE but with a paired set of gRNAs that enable the simultaneous deletion of a polynucleotide sequence (e.g., a gene) and replacement of the polynucleotide with an exogenous polynucleotide of interest (e.g., a variant gene) (See, e.g., US Patent Appl. No. 17 / 451 ,734). The first step in PASTE and PASTEREPLACE editing generally comprises the use of a nickase (e.g., a Cas9 nickase) fused to a reverse transcriptase and an extended gRNA (pegRNA). The pegRNA comprises at least three functional polynucleotides (i) a targeting sequence (targeting the nickase to the target polynucleotide site), (ii) a primer binding site (PBS), and (iii) a reverse transcriptase template sequence containing the integration site. (See, e.g., Scholefield, J., Harrison, P.T. Prime editing - an update on the field. Gene Ther 28, 396-401 (2021), the entire contents of which are incorporated by reference herein).

[0112] In some embodiments, the compositions and systems described herein are useful in the method of PASTE editing. PASTE editing utilizes a modified prime technique to site- specifically insert an integration site within a target polynucleotide and subsequently utilizing the site to integrate a polynucleotide sequence of interest.DNA Binding Nickases

[0113] In some embodiments, the compositions, systems, and methods described herein utilize a DNA binding nickase (or a functional fragment or variant thereof). In some embodiments, a functional fragment, or functional variants of a DNA binding nickase is used, wherein the fragment or variant maintains nickase activity.

[0114] In some embodiments, the DNA binding nickase is a naturally occurring nickase (or functional fragment or variant thereof). In some embodiments, the DNA binding nickase (or a functional fragment or variant thereof) is a nickase that has been modified (e.g., incorporates one or more amino acid modifications compared to a reference sequence) to impart nickase activity. For example, the DNA binding nickase (or a functional fragment or variant thereof) may be a Cas9 nuclease (or functional fragment or variant thereof) with one of the two nuclease domains inactivated, e.g., by amino acid substitution of H840A, wherein the Cas9 has nickase activity but is not able to make a double strand break in a target double stranded polynucleotide.

[0115] In some embodiments, the DNA binding nickase comprises a Cas9 nickase, Cas12e (CasX) nickase, Casl2d (CasY) nickase, Cas12a (Cpfl) nickase, Cas12bl (C2cl) nickase, Cas13a (C2c2) nickase, Cas12c (C2c3) nickase (or a functional fragment or variant of any of the foregoing).

[0116] In some embodiments, the DNA binding nickase is a Cas9 nickase (or a functional fragment or variant thereof). The wild type Cas9 comprises two separate nuclease domains, the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand). In some embodiments, the Cas9 nickase comprises only a single functioning nuclease domain.

[0117] In some embodiments, the Cas9 nickase comprises a mutation in the RuvC domain which inactivates the RuvC nuclease activity. Suitable mutations include, but are not limited to, e.g., in aspartate (D) 10, histidine (H) 983, aspartate (D) 986, or glutamate (E) 762, (See, e.g., Nishimasu et al., “Crystal structure of Cas9 in complex with guide RNA and target DNA,” Cell / 156(5), 935-949, which is incorporated herein by reference). In some embodiments, the Cas9 nickase (or a functional fragment or variant thereof) comprises at least one of thefollowing amino acid substitutions D10X, H983X, D986X, or E762X, wherein X is any amino acid other than the wild-type amino acid. In some embodiments, the Cas9 nickase (or a functional fragment or variant thereof) comprises at least one of the following amino acid substitutions D10A, H983A, D986A, or E762A, or a combination thereof. A Cas9 nickase (or a functional fragment or variant thereof) comprising a D10A amino acid substitution is also referred to herein as Cas9-D10A. Likewise, a Cas9 nickase (or a functional fragment or variant thereof) comprising a H983A amino acid substitution is also referred to herein as Cas9-H983A. A Cas9 nickase (or a functional fragment or variant thereof) comprising a D986A amino acid substitution is also referred to herein as Cas9-D986A. A Cas9 nickase (or a functional fragment or variant thereof) comprising a E762A amino acid substitution is also referred to herein as Cas9-E762A.

[0118] In some embodiments, the Cas9 nickase (or a functional fragment or variant thereof) comprises a mutation in the HNH domain which inactivates the HNH nuclease activity. Suitable mutations include, but are not limited to, a mutation in histidine (H) 840 or asparagine (R) 863 (amino acid numbering relative to SEQ ID NO: 1) (See supra). In some embodiments, the Cas9 nickase (or a functional fragment or variant thereof) comprises at least one of the following amino acid substitutions H840X or R863X, wherein X is any amino acid other than the wild-type amino acid. In some embodiments, the Cas9 nickase (or a functional fragment or variant thereof) comprises at least one of the following amino acid substitutions H840A or R863A, or a combination thereof. A Cas9 nickase (or a functional fragment or variant thereof) comprising an H840A amino acid substitution is also referred to herein as Cas9-H840A. Likewise, a Cas9 nickase (or a functional fragment or variant thereof) comprising an R863A amino acid substitution is also referred to herein as a Cas9-R863A.

[0119] In some embodiments, the DNA binding nickase (or a functional fragment or variant thereof) comprises Cas9-D10A, Cas9-H983A, Cas9-D986A, Cas9-E762A, Ca9s-H840A, or Cas9-R863A (or a functional fragment or variant of any of the foregoing). In some embodiments, the DNA binding nickase (or a functional fragment or variant thereof) comprises Cas9-D10A, Cas9-H983A, Cas9-D986A, or Cas9-E762A (or a functional fragment or variant of any of the foregoing). In some embodiments, the DNA binding nickase comprises Cas9- H840A or Cas9-R863A (or a functional fragment or variant of any of the foregoing). In some embodiments, the DNA binding nickase (or a functional fragment or variant thereof) comprises Cas9-H840A (or a functional fragment or variant of any of the foregoing).Reverse Transcriptases

[0120] In some embodiments, the compositions, systems, and methods described herein utilize a reverse transcriptase (or a functional fragment or variant thereof). In some embodiments, a functional fragment or functional variants of a reverse transcriptase is used, wherein the fragment or variant maintains reverse transcriptase activity.

[0121] In some embodiments, the reverse transcriptase is a naturally occurring reverse transcriptase (or functional fragment or variant thereof). In some embodiments, the reverse transcriptase is derived from a naturally occurring reverse transcriptase (or functional fragment or variant thereof). In some embodiments, the reverse transcriptase (or a functional fragment or variant thereof) is a reverse transcriptase that has been modified (e.g., incorporates one or more amino acid modifications compared to a reference sequence). In some embodiments, the modified reverse transcriptase comprises one or more improved properties as compared to the corresponding reference sequence (e.g., thermostability, fidelity, reverse transcriptase activity).

[0122] Exemplary reverse transcriptases include, but are not limited to, moloney murine leukemia virus (M-MLV) reverse transcriptase; human immunodeficiency virus (HIV) reverse transcriptase and avian sarcoma-leukosis virus (ASLV) reverse transcriptase, which includes but is not limited to rous sarcoma virus (RSV) reverse transcriptase, avian myeloblastosis virus (AMY) reverse transcriptase, avian erythroblastosis virus (AEV) helper virus MCAV reverse transcriptase, avian myelocytomatosis virus MC29 helper virus MCAV reverse transcriptase, avian reticuloendotheliosis virus (REV-T) helper virus REV-A reverse transcriptase, avian sarcoma virus UR2 helper virus LIR2AV reverse transcriptase, avian sarcoma virus Y73 helper virus YAV reverse transcriptase, rous associated virus (RAV) reverse transcriptase, and myeloblastosis associated virus (MAV) reverse transcriptase.

[0123] Any of the aforementioned exemplary reverse transcriptases can be modified, e.g., comprises at least one amino acid substitution, deletion, or addition.

[0124] In some embodiments, the reverse transcriptase is derived from the M-MLV reverse transcriptase. In some embodiments, the M-MLV reverse transcriptase is naturally occurring. In some embodiments, the M-MLV reverse transcriptase is non-naturally occurring.Integrases

[0125] In some embodiments, the compositions, systems, and methods described herein utilize an integrase (or a functional fragment or variant thereof) and a cognate integration sequence. Integrases, integration sequences, and integration sites are particularly useful inmethods of PASTE editing (e.g., as described herein). It is understood by the person of ordinary skill in the art that integration sites and integrases for use in the compositions, systems, and methods described herein will be selected in pairs, wherein the selected integrase will specifically recognize the selected integration site.

[0126] The integrase (or functional fragment or variant thereof) can be provided as part of the editing polypeptide (e.g., as described herein, e.g., as a fusion protein) or as a separate polypeptide. In some embodiments, the integrase (or functional fragment or variant thereof) is part of the editing polypeptide (e.g., a fusion protein). In some embodiments, the integrase (or functional fragment or variant thereof) is polypeptide separate from the editing polypeptide.

[0127] Exemplary integrases include recombinases, reverse transcriptases, and retrotransposases. Exemplary integrases include, but are not limited to, Cre, Dre, Vika, Bxb1 , <pC31 , RDF, FLP, q)BT1, R1 , R2, R3, R4, R5, TP901-1 , A118, q)FC1, <pC1 , MR11 , TG1 , cp370.1 , Wp, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, Conceptll, Museum, Severus, Airmid, Benedict, Hinder, ICIeared, Sheen, Mundrea, BxZ2, cpRV, and retrotransposases encoded by R2, L1 , Tol2 Tc1 , Tc3, Mariner (Himar 1), Mariner (mos 1), and Minos. In some embodiments, the integrase is Bxb1.

[0128] The integrases (e.g., recombinases) explicitly provided herein are not meant to be exclusive examples of integrases (e.g., recombinases) that can be used in embodiments of the disclosure. The methods and compositions of the disclosure can be expanded by mining databases for new orthogonal integrases (e.g., recombinases) or designing synthetic integrases (e.g., recombinases) with defined DNA specificities (See, e.g., Groth et al., “Phage integrases: biology and applications.” J. Mol. Biol. 2004; 335, 667-678; Gordley et al., “Synthesis of programmable integrases.” Proc. Natl. Acad. Sci. USA. 2009; 106, 5053-5058; the entire contents of each of which is hereby incorporated by reference in their entirety for all purposes).

[0129] In some embodiments, the integrase (or functional fragment or variant thereof) is a recombinase that incorporates the polynucleotide of interest into the target polynucleotide (e.g., a genome of a cell) at an integration site by recombination. Exemplary recombinases include serine recombinases and tyrosine recombinases. In some embodiments, the integrase is a serine recombinase. In some embodiments, the integrase is a tyrosine recombinase. Exemplary serine recombinases include, but are not limited to, Hin, Gin, Tn3, p-six, CinH, ParA, yb, Bxb1 , q)C31, TP901 , TG1 , q)BT1 , R1 , R2, R3, R4, R5, q>RV1, q)FC1, MR11 , A118, U153, gp29. Examples of serine recombinases also include, without limitation, recombinasesPeaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, Conceptll, Museum, Severus, Airmid, Benedict, Hinder, ICIeared, Sheen, Mundrea, and BxZ2 from Mycobacterial phages. In some embodiments, the integrase is Hin, Gin, Tn3, p-six, CinH, ParA, yb, Bxb1 , cpC31 , TP901 , TG1 , q)BT1, R1 , R2, R3, R4, R5, q)RV1 , q)FC1 , MR11 , A118, U153, or gp29. In some embodiments, the integrase is a tyrosine recombinase. Exemplary, tyrosine recombinases include, but are not limited to, Cre, FLP, R, Lambda, HK101 , HK022, and pSAM2.

[0130] In some embodiments, the integrase is a reverse transcriptase that incorporates the polynucleotide of interest into the target polynucleotide (e.g., a genome of a cell) at an integration site by reverse transcription.

[0131] In some embodiments, the integrase (or functional fragment or variant thereof) is a retrotransposase that incorporates the polynucleotide of interest into the target polynucleotide (e.g., a genome of a cell) at an integration site by retrotransposition. Exemplary retrotransposases include, but are not limited to, retrotransposases encoded by elements such as R2, L1 , Tol2 Tc1 , Tc3, Mariner (Himar 1), Mariner (mos 1), Minos, and any functional variants thereof.Reverse Transcriptase Template Sequence

[0132] In some embodiments, the compositions, systems, and methods described herein utilize a reverse transcriptase template sequence. The reverse transcriptase template sequence serves as a template (i.e., encodes) the polynucleotide of interest (e.g., polynucleotide comprising, e.g., therapeutic nucleotide modification, diagnostic nucleotide modification; or e.g., a polynucleotide comprising an integration sequence encoding an integration site) for incorporation into a target polynucleotide (e.g., a gene or genome of a cell). In some embodiments, the reverse transcriptase template sequence comprises a therapeutic or diagnostic target nucleotide modification (e.g., in some embodiments a single nucleotide substitution, e.g., for use in prime editing methods). In some embodiments, the reverse transcriptase template sequence comprises an integration sequence comprising an integration site.Integration Sequences and Integration Sites

[0133] In some embodiments, the compositions, systems, and methods described herein utilize an integration sequence (e.g., comprising an integration site) and a cognate integrase (e.g., as described herein). Integration sequences, integration sites, and integrases are particularly useful in methods of PASTE editing (e.g., as described herein). In someembodiments, the gRNA comprises an integration sequence encoding an integration site. Inclusion of the integration sequence encoding an integration site in the gRNA allows for the incorporation of the integration site into a desired (site-specific) location in the polynucleotide (e.g., gene or genome) being edited.

[0134] Integration sites and integrases for use in the compositions, systems, and methods described herein will be selected in pairs, wherein the selected integrase will specifically recognize the selected integration site. Exemplary integration sites include, but are not limited to, Iox71 sites, attB sites, attP sites, attL sites, attR sites, Vox sites, FRT sites, or pseudo attP sites.

[0135] Integration typically requires (e.g., as with serine integrases) an integration site (encoded by the gRNA) and a recognition site (e.g., linked to a polynucleotide of interest for insertion) both of which are recognized by the integrase. The integration site can be inserted into the target polynucleotide (e.g., of a cell) using a nuclease (e.g., a nickase), a gRNA, and / or an integrase. A single or a plurality of integration sites can be added to a target polynucleotide (e.g., a genome). In some embodiments, one integration site is added to a target polynucleotide (e.g., a genome). In some embodiments, more than one integration site is added to a target polynucleotide (e.g., a genome). The recognition site may be operably linked to a target polynucleotide (e.g., gene of interest) in an exogenous DNA or RNA (e.g., as described herein).

[0136] To insert more than one unique polynucleotide (e.g., gene) of interest, each at a specific site, multiple orthogonal integrations sites can be added to the specific desired locations within the polynucleotide (e.g., genome) to mediate site-specific integration of the multiple polynucleotides. A first integration site is “orthogonal” to a second integration site when it does not significantly recognize the recognition site or the integrase (e.g., recombinase) recognized by the second integration site. Thus, for example, one attB site of an integrase (e.g., a recombinase) can be orthogonal to an attB site of a different recombinase (e.g., integrase). In addition, one pair of attB and attP sites of an integrase (e.g., a recombinase) can be orthogonal to another pair of attB and attP sites recognized by the same integrase (e.g., recombinase). A pair of recombinases are considered orthogonal to each other, as defined herein, when there is recognition of each other' s attB or attP site sequences. In some embodiments, the same integrase (e.g., recombinase) or two different recombinases (e.g., integrases) recognize the same integration site less than 30%, 28%, 26%, 24%, 22%, 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, or 1 %, or any range that is formed from any two of those values as endpoints of the time.

[0137] A single or a plurality of integration sites can be added to a target polynucleotide (e.g., a genome). In some embodiments, one integration site is added to a target polynucleotide (e.g., a genome). In some embodiments, more than one integration site is added to a target polynucleotide (e.g., a genome).

[0138] The central dinucleotide of some integrases is involved in the association of the two paired integration sites. For example, the central dinucleotide of BxbINT is involved in the association of the AttB integration site with the AttP recognition site. Therefore, changing the matched central dinucleotide can modify the integrase activity and provide orthogonality for the insertion of multiple genes. Therefore, expanding the set of AttB / AttP dinucleotides can enable multiplex gene insertion using gRNAs.

[0139] In some embodiments, the attB and / or attP site sequences comprise a central dinucleotide sequence. It has been shown that, for example, the central dinucleotide can be changed to GA from GT and that only GA containing attB / attP sites interact and will not cross react with GT containing sequences. In some embodiments, the central dinucleotide is selected from the group consisting of AG, AC, TG, TC, CA, CT, GA, AA, TT, CC, GG, AT, TA, GC, CG and GT. In some embodiments, the central dinucleotide is nonpalindromic. In some embodiments, the central dinucleotide is palindromic. In some embodiments, the integration site and the recognition site of a pair share the same central dinucleotide and can mediate recombination in the presence of the cognate integrase.Guide RNAs (gRNA)s

[0140] In some embodiments, the compositions, systems, and methods described herein comprise or utilize a gRNA. A gRNA typically functions to guide the insertion or deletion of one or more polynucleotides of interest (e.g., a gene of interest) into a target polynucleotide (e.g., genome). In some embodiments, the gRNA molecule is naturally occurring. In some embodiments, a gRNA molecule is non-naturally occurring. In some embodiments, a gRNA molecule is a synthetic gRNA molecule. In some embodiments, the gRNA comprises one or nucleotide modifications (e.g., to improve stability and / or half-life after being introduced into a cell).

[0141] In some embodiments, the compositions, systems, and methods described herein comprise or utilize one or more set of paired gRNAs that allow for the simultaneous deletion of an endogenous polynucleotide (e.g., gene) and insertion of a polynucleotide of interest (e.g., modified gene). The target double-string DNA (dsDNA) comprises two protospacers each on opposite strands of the target dsDNA. One gRNA (e.g., targeting gRNA) is targeted to one strand, while the other gRNA (e.g., targeting gRNA) of the pairs is targeted to the oppositestrand. The targeting gRNA:editing polypeptide complex generates a single strand nick at each target site.

[0142] In some embodiments, the gRNA comprises one or nucleotide modifications (e.g., to improve stability and / or half-life after being introduced into a cell). In some embodiments, chemical modifications on the ribose rings and phosphate backbone of gRNAs are incorporated. Ribose modifications are typically placed at the 2'OH as it is readily available for manipulation. Simple modifications at the 2'OH include 2'-O-methyl, 2'-fluoro, and 2'-deoxy-2 - fluoro-beta-D-arabinonucleic acid (2'fluoro-ANA). More extensive ribose modifications such as 2'F-4'-Ca-OMe and 2',4'-di-Ca-OMe combine modification at both the 2' and 4' carbons. Exemplary phosphodiester modifications include sulfide-based phosphorothioate (PS) or acetate-based phosphonoacetate alterations. Combinations of the ribose and phosphodiester modifications can also be utilized such as 2'-O-methyl 3'phosphorothioate (MS), or 2'-O- methyl-3'-thioPACE (MSP), and 2'-O-methyl-3'-phosphonoacetate (MP) RNAs. Locked and unlocked nucleotides such as locked nucleic acid (LNA), bridged nucleic acids (BNA), S- constrained ethyl (cEt), and unlocked nucleic acid (UNA) are examples of sterically hindered nucleotide modifications that can also be utilized.Delivery of gRNAs

[0143] In some embodiments, the compositions, systems, and methods described herein comprise or utilize a gRNA (e.g., targeting gRNAs, ngRNAs) delivered to a cell or a population of cells by any suitable method known in the art. For example, and without limitation, via an RNA polynucleotide; via a vector (e.g., a plasmid or viral vector) comprising an RNA polynucleotide; via a particle (e.g., a viral particle, lipid particle, nanoparticle (e.g., a lipid nanoparticle)) encapsulating the polynucleotide or vector. Methods of delivering each of the aforementioned are known to the person of ordinary skill in the art. Also provided herein are pharmaceutical compositions comprising a gRNA described herein (e.g., targeting gRNA, ngRNA) polynucleotide; a vector (e.g., a plasmid or viral vector) comprising the polynucleotide; a particle (e.g., a viral particle, lipid particle, nanoparticle (e.g., a lipid nanoparticle)) encapsulating the polynucleotide; and a pharmaceutically acceptable excipient.

[0144] Exemplary viral vectors include, but are not limited to, adenovirus vectors, adeno- associated virus vectors, lentivirus vectors, retrovirus vectors, poxvirus vectors, parapoxivirus vectors, vaccinia virus vectors, fowlpox virus vectors, herpes virus vectors, adeno-associated virus vectors, alphavirus vectors, lentivirus vectors, rhabdovirus vectors, measles virus, Newcastle disease virus vectors, picornaviruses vectors, or lymphocytic choriomeningitis virus vectors.Genome Editing with Site-specific Retrotransposons and Site-specific Target-primed Insertion via Targeted CRISPR Homing of Retroelements (STITCHR)

[0145] The present disclosure relates, in part, to genome editing with site-specific retrotransposons and by Site-specific Target-primed Insertion via Targeted CRISPR Homing of Retroelements (STITCHR). Disclosed are site specific non-Long Terminal Repeat (LTR) retrotransposons and systems incorporating these non-LTR retrotransposons for inserting large nucleic acids at targeted locations within a genome. Disclosed are also site-specific non- LTR retrotransposons and related systems for performing small nucleotide changes in a genome. In some embodiments, a small nucleotide change comprises a point mutation. In some embodiments, a small nucleotide change comprises a small nucleotide insertion.

[0146] The present disclosure relates in part to modified R2 fusion proteins for inserting large nucleic acids at targeted locations within a genome. The present disclosure also relates in part to Cas9 fusion proteins for inserting large nucleic acids at targeted locations within a genome, which includes Cas9-R2 fusion proteins. In some embodiments, the genome is a human genome.

[0147] The present disclosure relates in part to the insertion of exogenous R2 landing sites within a genome, such that a R2 protein, modified R2 protein, or R2 fusion protein that may target a non-28S locus for insertion of a large genetic element. In some embodiments, the R2 fusion protein is an R2-Cas9 fusion protein. In some embodiments, the R2 fusion protein is a Cas12-R2 fusion protein. In some embodiments, the R2 fusion protein is a TALEN-R2 fusion protein.RLE domain containing non-LTR retrotransposon Families

[0148] In addition to canonical single-ORF RLE domain containing non-LTR retrotransposons, such as R2, R4, R5, R8, R9, Dong, and Cre families, retrotransposons for use in or as part of the genome editing system described herein may also be characterized as part of a larger phylogenetic family. The retrotransposons in these larger phylogenetic families contemplated for use in or as a part of the genome editing systems described herein include the 8,248 RLE-domain containing retrotransposon. These 8,248 retrotransposon-like orthologs are divided into 9 families, termed RLED1-RLED9. In some embodiments, the non-LTR retrotransposon is a member of the RLED1 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED2 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED3 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED4 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED5 family. In some embodiments, the non-LTRretrotransposon is a member of the RLED6 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED7 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED8 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED9 family. In some embodiments, the non-LTR retrotransposon is a member of the R1 family. In some embodiments, the non-LTR retrotransposon is a member of the R2 family. In some embodiments, the non-LTR retrotransposon is a member of the R4 family. In some embodiments, the non-LTR retrotransposon is a member of the R5 family. In some embodiments, the non-LTR retrotransposon is a member of the R6 family. In some embodiments, the non-LTR retrotransposon is a member of the R7 family. In some embodiments, the non-LTR retrotransposon is a member of the R8 family. In some embodiments, the non-LTR retrotransposon is a member of the R9 family. In some embodiments, the non-LTR retrotransposon is a member of the Cre family. In some embodiments, the non-LTR retrotransposon is a member of the NeSL family. In some embodiments, the non-LTR retrotransposon is a member of the HERO family. In some embodiments, the non-LTR retrotransposon is a member of the Utopia family.R2 Element Enzymes

[0149] Without limiting the instant disclosure to any one particular theory, R2 retrotransposons are thought to work via a mechanism known as target-primed reverse transcription, or “TPRT.” TPRT is a mechanism by which an endonuclease creates a nick in a first DNA strand at a specific location, creating a “primed” 3’ hydroxyl end for reverse transcription. After the initial DNA nick, an mRNA molecule is reverse transcribed by the reverse transcriptase.

[0150] In some embodiments, the R2 element enzyme is modified. In some embodiments, the R2 element enzyme is modified by an N-terminal truncation of the R2 element enzyme sequence, a C-terminal truncation of the R2 element enzyme sequence, or both an N-terminal and a C-terminal truncation of the R2 element enzyme sequence.

[0151] In some embodiments, the R2 element enzyme is a fusion protein. In some embodiments, the R2 element enzyme comprises a fusion of an R2 protein with a Cas9 protein. In some embodiments, the R2 element enzyme comprises a fusion of an R2 protein with a Cas12 protein. In some embodiments, the R2 element enzyme comprises a fusion of an R2 protein with a Cas9 protein, wherein the Cas9 portion and the R2 protein portion are connected by a linker. In some embodiments, the R2 element enzyme comprises a fusion ofan R2 protein with a Cas12 protein, wherein the Cas12 portion and the R2 protein portion are connected by a linker.Protein Binding Elements

[0152] Protein binding elements of the disclosure can come in a multitude of forms such as the forms described above. The protein binding element may be an endogenous nucleic acid sequence. The protein binding element may be an exogenous or introduced nucleic acid sequence. The protein binding element may be a synthesized nucleic acid sequence.Guide Elements

[0153] Guide elements of the disclosure can come in a multitude of forms such as the forms described above. The genome editing systems disclosed herein can comprise a guide RNA. The genome editing systems disclosed herein can comprise multiple guide RNAs. The genome editing systems disclosed herein can comprise paired guide RNAs.Genomic Insertion Sites and Targets

[0154] The R2 element naturally targets the 28S rRNA locus. The instant disclosure contemplates the insertion of payloads into either the 28S rRNA locus or into other genomic loci. In some embodiments, the insertion site is a targeted genomic insertion site. In some embodiments, the insertion site is targeted by a targeting domain in a fusion protein. In some embodiments, the insertion site has been exogenously introduced to the genome. In some embodiments, the insertion site has been exogenously introduced by a site-directed genome editing system that is not capable of delivering large genetic insertions. In some embodiments, the targeted genomic site is targeted for a point mutation. In some embodiments, the targeted genomic site is targeted for a small nucleotide insertion.

[0155] The instant disclosure also contemplates additional non-LTR site-specific retrotransposons for use in or as part of the genome editing system described herein that do not target the 28S rRNA locus. In some embodiments, the genome is targeted for a large genetic insertion. In some embodiments, the insertion site is a targeted genomic insertion site. In some embodiments, the insertion site is targeted by a targeting domain in a fusion protein. In some embodiments, the insertion site has been exogenously introduced to the genome. In some embodiments, the insertion site has been exogenously introduced by a site-directed genome editing system that is not capable of delivering large genetic insertions. In some embodiments, the targeted genomic site is targeted for a point mutation. In some embodiments, the targeted genomic site is targeted for a small nucleotide insertion.Payloads

[0156] Payloads for the genome editing systems disclosed herein may encode proteins, such as enzymes. In some embodiments, the payload may act as a regulatory element. Thus, if an embodiment of the disclosure states, by way of example, that “the payload comprises a therapeutic protein,” it is generally understood that the payload comprises a template that, upon insertion, will lead to expression of a therapeutic protein encoded by the template.

[0157] In some embodiments, the insertion region comprises a template for a reporter gene. In some embodiments, the reporter gene encodes a fluorescent protein. In some embodiments, the reporter gene encodes a green fluorescent protein. In some embodiments, the reporter gene encodes eGFP.

[0158] In some embodiments, the insertion region comprises a template for a transcription factor gene.

[0159] In some embodiments, the insertion region comprises a template for a transgene.

[0160] In some embodiments, the insertion region comprises a template for an enzyme gene, or a therapeutic gene. In some embodiments, the therapeutic protein can be used in conjunction with another therapeutic.

[0161] In some embodiments, the payload comprises a protein that is capable of converting one cell type to another.

[0162] In some embodiments, the payload comprises a protein that is capable of killing a specific cell type. In some embodiments, the payload comprises a protein that is capable of killing a tumor cell. In some embodiments, the payload comprises an immune modulating protein.

[0163] In some embodiments, the payload comprises a 5’IITR. In some embodiments, the payload comprises a 3’IITR. In some embodiments, the payload comprises a 5’IITR and a 3’ UTR. In some embodiments, the payload consists of a 5’IITR. In some embodiments, the payload consists of a 3’IITR. In some embodiments, the payload comprises a 5’IITR and a 5’ homology region. In some embodiments, the payload comprises a 3’IITR and a 3’ homology region. In some embodiments, the payload comprises a 5’IITR, a 5’ homology region, a 3’IITR and a 3’ homology region. In some embodiments, the payload comprises a 5’ homology region, a 3’IITR and a 3’ homology region. In some embodiments, the payload comprises a 5’IITR, a 5’ homology region, and a 3’ homology region. In some embodiments, the payload comprises a 5’ homology region and a 3’ homology region. In some embodiments, the 3’ homology region comprises less than 30 base pairs. In some embodiments the 3’ homologyregion comprises less than 20 base pairs. In some embodiments, the 3’ homology region comprises less than 10 base pairs. In some embodiments, the 3’ homology region comprises less than 5 base pairs.Programmable Nucleases, Nickases, and DNA Binding Proteins

[0164] The instant disclosure contemplates programmable nucleases or nickases for use in or as a part of the genome editing systems as described above. In some embodiments, the programmable nuclease or nickase is a Cas9 protein. In some embodiments, the programmable nuclease or nickase is a Cas12 protein. In some embodiments the programmable nuclease or nickase is IscB. In some embodiments, the programmable nuclease or nickase is IsrB. In some embodiments, the programmable nuclease or nickase is TnpB. In some embodiments, the programmable nuclease or nickase is a TALEN nuclease. In some embodiments, the programmable nuclease or nickase is fused to the non-LTR sitespecific retrotransposon element. In some embodiments, the programmable nuclease or nickase is non-covalently linked to the non-LTR site-specific retrotransposon element. In some embodiment, the programmable nuclease or nickase acts in cis with the non-LTR site-specific retrotransposon element. In some embodiments, the programmable nuclease or nickase acts in trans with the non-LTR site-specific retrotransposon element.Linkers

[0165] In some embodiments, the compositions, systems, and methods described herein utilize a linker (e.g., a peptide linker) (e.g., one or more different linkers). Common linkers (e.g., glycine and glycine / serine linkers) are known in the art. Any suitable linker(s) can be utilized as long as each component can mediate the desired function.

[0166] In some embodiments, at least two components of an editing polypeptide (e.g., described herein) are operably connected via a linker. In some embodiments, each component of an editing polypeptide (e.g., described herein) is operably connected to the preceding and / or subsequent component of the editing polypeptide via a linker. In some embodiments, each component of an editing polypeptide (e.g., described herein) is operably connected to the preceding and / or subsequent component of the editing polypeptide via a different linker.

[0167] In some embodiments, the linker is from about 2-100, 2-50, 2-25, 2-10, 4-100, 4-50, 4-25, 4-10, 5-100, 5-50, 5-25, 5-10, 10-100, 10-50, or 10-25 amino acids in length. In some embodiments, the linker is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

[0168] In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker comprises a polypeptide portion and a non-peptide portion. In some embodiments, the linker comprises an extended recombinant polypeptide (XTEN). In some embodiments, the linker comprises the amino acid sequence (Gly4Ser)n (SEQ ID NO: 1), where n is an integer. In some embodiments, the linker comprises the amino acid sequence (Gly4Ser)n, wherein n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 2). In some embodiments, the linker comprises the amino acid sequence (Gly4Ser)n, wherein n is greater than 10 (SEQ ID NO: 3). In some embodiments, the linker comprises a synthetic portion. In some embodiments, the linker comprises polyethylene glycol (PEG). In some embodiments, the linker is a synthetic linker. In some embodiments (Gly2Ser)n, wherein n is an integer. In some embodiments, the linker comprises the amino acid sequence (Gly2Ser)n, wherein n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 4). In some embodiments, the linker comprises the amino acid sequence (Gly2Ser)n, wherein n is greater than 10 (SEQ ID NO: 5). In some embodiments, the linker comprises the amino acid sequence (Ser-Gly-Gly-Ser)n (SEQ ID NO: 6), where n is an integer. In some embodiments, the linker comprises the amino acid sequence (Ser-Gly-Gly-Ser)n, wherein n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 7). In some embodiments, the linker comprises the amino acid sequence (Ser-Gly-Gly-Ser)n, wherein n is greater than 10 (SEQ ID NO: 8). In some embodiments the linker comprises the amino acid sequence (Glu-Ala-Ala-Ala-Lys)n (SEQ ID NO: 9), wherein n is an integer. In some embodiments, the linker comprises the amino acid sequence (Glu-Ala- Ala-Ala-Lys)n, wherein n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 10). In some embodiments, the linker comprises the amino acid sequence (Glu-Ala-Ala-Ala-Lys)n, wherein n is greater than 10 (SEQ ID NO: 11). In some embodiments, the linker comprises a proline linker.Compositions, Pharmaceutical Compositions, Systems, and Kits

[0169] Provided herein are compositions (including pharmaceutical compositions), systems, and kits comprising any one or more (e.g., all) of the components described herein (e.g., an editing polypeptide, one of more gRNAs, polynucleotide inserts). In one aspect, provided herein is a system comprising at least two components of an editing system described herein (e.g., a DNA binding nickase, a reverse transcriptase, an integration enzyme, a gRNA pair). In one aspect, provided herein are compositions comprising at least one components of an editing system described herein (e.g., a DNA binding nickase, a reverse transcriptase, an integration enzyme, a gRNA pair).Pharmaceutical Compositions

[0170] Pharmaceutical compositions descried herein comprise at least one component of an editing system described herein (e.g., a DNA binding nickase) and a pharmaceutically acceptable excipient (see, e.g., Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA, the entire contents of which is incorporated by reference herein for all purposes).

[0171] In one aspect, also provided herein are methods of making pharmaceutical compositions described herein comprising providing at least one component of an editing system described herein (e.g., a DNA binding nickase) and formulating it into a pharmaceutically acceptable composition by the addition of one or more pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a single component described herein (e.g., a DNA binding nickase). In some embodiments, the pharmaceutical composition comprises a plurality of the components described herein (e.g., a DNA binding nickase, a reverse transcriptase, an integration enzyme, a gRNA pair, etc.).

[0172] Acceptable excipients (e.g., carriers and stabilizers) are preferably nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants including ascorbic acid or methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol;or m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, or other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn- protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0173] A pharmaceutical composition may be formulated for any route of administration to a subject. The skilled person knows the various possibilities to administer a pharmaceutical composition described herein a in order to deliver the editing system or composition to a target cell. Non-limiting embodiments include parenteral administration, such as intramuscular, intradermal, subcutaneous, transcutaneous, or mucosal administration. In one embodiment, the pharmaceutical composition is formulated for intravenous administration. In oneembodiment, the pharmaceutical composition is formulated for administration by intramuscular, intradermal, or subcutaneous injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions. The injectables can contain one or more excipients. Exemplary excipients include, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical compositions to be administered can also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins. In some embodiments, the pharmaceutical composition is formulated in a single dose. In some embodiments, the pharmaceutical compositions if formulated as a multi-dose.

[0174] Pharmaceutically acceptable excipients (e.g., carriers) used in the parenteral preparations described herein include for example, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents or other pharmaceutically acceptable substances. Examples of aqueous vehicles, which can be incorporated in one or more of the formulations described herein, include sodium chloride injection, Ringer’s injection, isotonic dextrose injection, sterile water injection, dextrose, or lactated Ringer’s injection. Nonaqueous parenteral vehicles, which can be incorporated in one or more of the formulations described herein, include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to the parenteral preparations described herein and packaged in multiple-dose containers, which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride or benzethonium chloride. Isotonic agents, which can be incorporated in one or more of the formulations described herein, include sodium chloride or dextrose. Buffers, which can be incorporated in one or more of the formulations described herein, include phosphate or citrate. Antioxidants, which can be incorporated in one or more of the formulations described herein, include sodium bisulfate. Local anesthetics, which can be incorporated in one or more of the formulations described herein, include procaine hydrochloride. Suspending and dispersing agents, which can be incorporated in one or more of the formulations described herein, include sodium carboxymethylcelluose, hydroxypropyl methylcellulose or polyvinylpyrrolidone. Emulsifying agents, which can be incorporated in one or more of the formulations described herein, include Polysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metal ions, which can be incorporated in one or more ofthe formulations described herein, is EDTA. Pharmaceutical carriers, which can be incorporated in one or more of the formulations described herein, also include ethyl alcohol, polyethylene glycol or propylene glycol for water miscible vehicles, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0175] The precise dose to be employed in a pharmaceutical composition will also depend on the route of administration, and the seriousness of the condition caused by it, and should be decided according to the judgment of the practitioner and each subject’s circumstances. For example, effective doses may also vary depending upon means of administration, target site, physiological state of the subject (including age, body weight, and health), other medications administered, or whether therapy is prophylactic or therapeutic. Therapeutic dosages are titrated to optimize safety and efficacy.Kits

[0176] Also provided herein are kits comprising at least one pharmaceutical composition described herein. In addition, the kit may comprise a liquid vehicle for solubilizing or diluting, and / or technical instructions. The technical instructions of the kit may contain information about administration and dosage and subject groups. In some embodiments, the kit contains a single container comprising a single pharmaceutical composition described herein. In some embodiments, the kit at least two separate containers, each comprising a different pharmaceutical composition described herein (e.g., a first container comprising a pharmaceutical composition comprising one component of an editing system described herein).

[0177] The following examples are included solely for purposes of illustration and are not considered limiting embodiments. All patents and publications referred to herein are expressly incorporated by reference.EXAMPLESExample 1Insertion at Intron 1 Region 3 of CFTR Gene Using PASTE Gene Editing

[0178] Insertions at intron 1 region 3 of CFTR gene using PASTE gene editing were assessed (FIGS. 1, 2A, and 2B).

[0179] HEK293FT cells were plated at 5-15K the day prior to transfection in a 96-well plate. HEK293FT were transfected with Lipofectamine 3000 (Thermo Fisher Scientific) according tomanufacturer’s specifications. For PASTE insertions, 18 ng of each atgRNA guide-encoding plasmid (36ng total for one forward and one reverse guide encoding plasmid), 64.5 ng cargo plasmid, and 97 ng SpCas9-RT-P2A-Bxb1 complex-encoding plasmid were delivered to each well unless otherwise specified. Cells were harvested for genomic DNA 3 days later and subjected to NGS analysis for efficiency of AttB insertion.

[0180] The 8 forward facing guides were co-transfected with 5 reverse guides at intron 1 region 3 using PASTEvI (Cas9-RT-P2A-Bxb1). The guide commination with the most efficient rate of AttB insertion in CFTR intron 1 region 3 was forward guide 8 paired with reverse guide 3, which deleted 61 DNA base pairs between the two guides and inserted the 46bp AttB sequence with an average efficiency of 10%.

[0181] Table 1 below shows examples of Cas9 guide and full atgRNA sequences.Table 1.Example 2Insertion at Intron 1 Region 4 of CFTR Gene Using PASTE Gene Editing

[0182] Insertions at intron 1 region 4 of CFTR gene using PASTE gene editing were assessed (FIGS. 1, 3A and 3B).

[0183] HEK293FT cells were plated at 5-15K the day prior to transfection in a 96-well plate.HEK293FT were transfected with Lipofectamine 3000 (Thermo Fisher Scientific) according to manufacturer’s specifications. For PASTE insertions, 18 ng of each atgRNA guide-encoding plasmid (36ng total for one forward guide and one reverse guide encoding plasmid), 64.5 ng cargo plasmid, and 97 ng SpCas9-RT-P2A-Bxb1 complex-encoding plasmid were delivered to each well unless otherwise specified. Cells were harvested for genomic DNA 3 days later and subjected to NGS analysis for efficiency of AttB insertion.

[0184] The 8 forward facing guides were co-transfected with 8 reverse guides at intron 1 region 4 using PASTEvI (Cas9-RT-P2A-Bxb1). Forward Guide 4 with Reverse Guide 6 resulted in 62 pb deletion 146 bp insertion for a total of 16 bp deletion. The guide commination with the most efficient rate of AttB insertion in CFTR intron 1 region 4 was forward guide 4 paired with reverse guide 3, which deleted 55 DNA base pairs between the two guides and inserted the 46bp AttB sequence with an average efficiency of 10%.

[0185] Table 2 below shows examples of Cas9 guide and full atgRNA sequences.Table 2.Example 3Integrations of EGFP Insert 800 bp and EGFP Insert 2981 bp Using PASTE Gene Editing

[0186] The integrations of EGFP Insert 800 bp and EGFP Insert 2981 bp using PASTE gene editing were assessed (FIGS. 1 and 4).

[0187] HEK293FT cells were plated at 5-15K the day prior to transfection in a 96-well plate.HEK293FT were transfected with Lipofectamine 3000 (Thermo Fisher Scientific) according to manufacturer’s specifications. For PASTE insertions at CFTR, 18 ng of each atgRNA guideencoding plasmid (36ng total for one forward guide and one reverse guide encoding plasmid), 64.5 ng cargo plasmid, and 97 ng SpCas9-RT-P2A-Bxb1 complex-encoding plasmid were delivered to each well. For PASTE insertion at ACTB, 25.5ng of pegRNA guide-encoding plasmid and 12.8ng of nicking guide encoding plasmid were delivered to each well along with the 64.5ng cargo plasmid and 97 ng SpCas9-RT-P2A-Bxb1 complex-encoding plasmid. Cells were harvested for genomic DNA 3 days later and subjected to digital droplet PCR analysis to determine the insertion efficiency of the full payload template.

[0188] Best pair from region 3 and region 4 in intron 1 were analyzed by ddPCR for full insertion of the payload template. Two different sized EGFP templates were used as shown in the legend and were compared against a control with only integrase (no Cas9-RT). At CFTR regions 3 and 4, the EGFP cargo of 800bp displayed slightly more efficient integration than the 2981 bp cargo: around 5.5%-6% efficiency as opposed to 4.5%-5%. The ACTB 800bp control cargo exhibited the expected 22%+ efficiency, with the larger 2981 bp cargo integrating at a slightly lower efficiency.

[0189] Table 3 below shows examples of Cas9 guide and full atgRNA sequences.Table 3.Example 4Optimization With Shorter Beacons of the Integration of EGFP Insert 800 bp Using PASTE Gene Editing

[0190] The optimization with shorter beacons of the integration of EGFP Insert 800 bp using PASTE gene editing was assessed (FIGS. 1 and 5).

[0191] HEK293FT cells were plated at 5-15K the day prior to transfection in a 96-well plate. HEK293FT were transfected with Lipofectamine 3000 (Thermo Fisher Scientific) according to manufacturer’s specifications. For PASTE insertions at CFTR, 18 ng of each atgRNA guideencoding plasmid (36ng total for one forward guide and one reverse guide encoding plasmid), 64.5 ng cargo plasmid, and 97 ng SpCas9-RT-P2A-Bxb1 complex-encoding plasmid were delivered to each well. For PASTE insertion at ACTB, 25.5ng of pegRNA guide-encoding plasmid and 12.8ng of nicking guide encoding plasmid were delivered to each well along with the 64.5ng cargo plasmid and 97 ng SpCas9-RT-P2A-Bxb1 complex-encoding plasmid. Cells were harvested for genomic DNA 3 days later and subjected to digital droplet PCR analysis to determine the insertion efficiency of the full payload template.

[0192] Best pair from region 3 and region 4 in intron 1 were analyzed by ddPCR for full insertion of the payload template. One sized EGFP templates was used as shown in thelegend and is compared against a control with only integrase (no Cas9-RT). In this comparison, shorter beacons (38 bp) are compared against the normal size (46 Bp). In the case of the region 4 paired guides, we see improved insertion rates with the shorter beacon (5% efficiency opposed to 3.8% efficiency). For the region 3 paired guides, the truncated 38bp beacon exabits lower insertion rates than those of the 46bp original.

[0193] Table 4 below shows examples of Cas9 guide and full atgRNA sequences.Table 4.Example 5Optimization With Paired Guide Overlaps of the Integration of EGFP Insert 800 bpUsing PASTE Gene Editing

[0194] The optimization with paired guide overlaps of the integration of EGFP Insert 800 bp using PASTE gene editing was assessed (FIGS. 1 and 6).

[0195] HEK293FT cells were plated at 5-15K the day prior to transfection in a 96-well plate. HEK293FT were transfected with Lipofectamine 3000 (Thermo Fisher Scientific) according to manufacturer’s specifications. For PASTE insertions at CFTR, 18 ng of each atgRNA guideencoding plasmid (36ng total for one forward guide and one reverse guide encoding plasmid), 64.5 ng cargo plasmid, and 97 ng SpCas9-RT-P2A-Bxb1 complex-encoding plasmid were delivered to each well. For PASTE insertion at ACTB, 25.5ng of pegRNA guide-encoding plasmid and 12.8ng of nicking guide encoding plasmid were delivered to each well along with the 64.5ng cargo plasmid and 97 ng SpCas9-RT-P2A-Bxb1 complex-encoding plasmid. Cells were harvested for genomic DNA 3 days later and subjected to digital droplet PCR analysis to determine the insertion efficiency of the full payload template.

[0196] Best pair from region 3 and region 4 in intron 1 were analyzed by ddPCR for full insertion of the payload template. One sized EGFP templates was used as shown in the legend and is compared against a control with only integrase (no Cas9-RT). In this comparison, a 46 bp or 38 bp beacon site is used as indicated. However, the amount of overlap between the paired guides within the beacon is varied from 46 bp (full overlap) to 28 bp of overlap. We find that less overlap is sometimes better, like with region 4 where a 46bp AttB with 32 bp of overlap is ideal. For region 3 none of the overlap truncations appeared to improve the integration efficiency.

[0197] Table 5 below shows examples of Cas9 guide and full atgRNA sequences.Table 5.Example 6Insertions at Intron 1 Region 1 of CFTR Gene Using STITCHR Gene Editing

[0198] Insertions at intron 1 region 1 of CFTR gene using STITCHR gene editing were assessed (FIGS. 7 and 8).

[0199] HEK293FT cells were plated at 5-15K the day prior to transfection in a 96-well plate.HEK293FT were transfected with Lipofectamine 3000 (Thermo Fisher Scientific) according to manufacturer’s specifications. For STITCHR insertions, 30 ng of each guide encoding plasmid, 30 ng of the EGFP template, and 30 ng R2Toc-SpCas9H840A -encoding plasmidwere delivered to each well unless otherwise specified. Cells were harvested for genomic DNA3 days later and subjected to ddPCR analysis for insertion of the EGFP payload at the CFTR region. NOLC1 EGFP insertion was used as a positive control.

[0200] 5 forward facing guides were co-transfected with 3 reverse guides at CFTR intron 1 region 1 using STITCHR (R2Toc-SpCas9H840A); an EGFP payload with 50bp homology arms sharing sequence to the bases immediately upstream and downstream from the insertion site in CFTR intron 1 region 1 was co-transfected. At this region, the highest efficiency of payload integration was with forward guide 1 and reverse guide 1 , displaying a payload integration of .8%. The NOLC1 EGFP payload inserted at the expected 4.5% efficiency. The significant drop in payload integration efficiency in the absence of the STITCH R-encoding plasmid vector underscores that R2-mediated insertion is the predominant mechanism of payload integration rather than HDR.

[0201] Table 6 below shows examples of guide, CFTR region 1 STITCHR payload, controls, and NOLC1 STITCHR payload sequences.Table 6.Example 7Insertions at Intron 1 Region 2 of CFTR Gene Using STITCHR Gene Editing

[0202] Insertions at intron 1 region 2 of CFTR gene using STITCHR gene editing were assessed (FIGS. 7 and 9).

[0203] HEK293FT cells were plated at 5-15K the day prior to transfection in a 96-well plate. HEK293FT were transfected with Lipofectamine 3000 (Thermo Fisher Scientific) according to manufacturer’s specifications. For STITCHR insertions, 30 ng of each guide encoding plasmid, 30 ng of the EGFP template, and 30 ng R2Toc-SpCas9H840A -encoding plasmid were delivered to each well unless otherwise specified. Cells were harvested for genomic DNA 3 days later and subjected to ddPCR analysis for insertion of the EGFP payload at the CFTR region. NOLC1 EGFP insertion was used as a positive control.

[0204] 6 forward facing guides were co-transfected with 5 reverse guides at CFTR intron 1 region 2 using STITCHR (R2Toc-SpCas9H840A); an EGFP payload with 50bp homology arms sharing sequence to the bases immediately upstream and downstream from the insertion site in CFTR intron 1 region 2 was co-transfected. At this region, the highest efficiency of payload integration was with forward guide 6 and reverse guide 1 , displaying a payload integration of .7%. The NOLC1 EGFP payload inserted at the expected 4.5% efficiency. The significant drop in payload integration efficiency in the absence of the STITCH R-encoding plasmid vector underscores that R2-mediated insertion is the predominant mechanism of payload integration rather than HDR.

[0205] Table 7 below shows examples of guide, CFTR region 2 STITCHR payload, controls, and NOLC1 STITCHR payload sequences.Table 7.Example 8Insertions at Intron 1 Region 3 of CFTR Gene Using STITCHR Gene Editing

[0206] Insertions at intron 1 region 3 of CFTR gene using STITCHR gene editing were assessed (FIGS. 7 and 10).

[0207] HEK293FT cells were plated at 5-15K the day prior to transfection in a 96-well plate. HEK293FT were transfected with Lipofectamine 3000 (Thermo Fisher Scientific) according to manufacturer’s specifications. For STITCHR insertions, 30 ng of each guide encoding plasmid, 30 ng of the EGFP template, and 30 ng R2Toc-SpCas9H840A -encoding plasmid were delivered to each well unless otherwise specified. Cells were harvested for genomic DNA 3 days later and subjected to ddPCR analysis for insertion of the EGFP payload at the CFTR region. NOLC1 EGFP insertion was used as a positive control.

[0208] 7 forward facing guides were co-transfected with 5 reverse guides at CFTR intron 1 region 3 using STITCHR (R2Toc-SpCas9H840A); an EGFP payload with 50bp homology arms sharing sequence to the bases immediately upstream and downstream from the insertion site in CFTR intron 1 region 3 was co-transfected. At this region, the highest efficiency of payload integration was with forward guide 2 and reverse guide 2, displaying a payload integration of .3%, the least efficient editing of the 5 regions surveyed. The NOLC1 EGFP payload inserted at the expected 4.5% efficiency. The significant drop in payload integration efficiency in the absence of the STITCH R-encoding plasmid vector underscores that R2- mediated insertion is the predominant mechanism of payload integration rather than HDR.

[0209] Table 8 below shows examples of guide, CFTR region 3 STITCHR payload, controls, and NOLC1 STITCHR payload sequences.Table 8.Example 9Insertions at Intron 1 Region 4 of CFTR Gene Using STITCHR Gene Editing

[0210] Insertions at intron 1 region 4 of CFTR gene using STITCHR gene editing were assessed (FIGS. 7 and 11).

[0211] HEK293FT cells were plated at 5-15K the day prior to transfection in a 96-well plate. HEK293FT were transfected with Lipofectamine 3000 (Thermo Fisher Scientific) according to manufacturer’s specifications. For STITCHR insertions, 30 ng of each guide encoding plasmid, 30 ng of the EGFP template, and 30 ng R2Toc-SpCas9H840A -encoding plasmid were delivered to each well unless otherwise specified. Cells were harvested for genomic DNA 3 days later and subjected to ddPCR analysis for insertion of the EGFP payload at the CFTR region. NOLC1 EGFP insertion was used as a positive control.

[0212] 6 forward facing guides were co-transfected with 7 reverse guides at CFTR intron 1 region 4 using STITCHR (R2Toc-SpCas9H840A); an EGFP payload with 50bp homology arms sharing sequence to the bases immediately upstream and downstream from the insertion site in CFTR intron 1 region 4 was co-transfected. At this region, the highest efficiency of payload integration was with forward guide 5 and reverse guide 5, displaying a payload integration of .45%. The NOLC1 EGFP payload inserted at the expected 4.5% efficiency. The significant drop in payload integration efficiency in the absence of the STITCH R-encoding plasmid vector underscores that R2-mediated insertion is the predominant mechanism of payload integration rather than HDR.

[0213] Table 9 below shows examples of guide, CFTR region 4 STITCHR payload, controls, and NOLC1 STITCHR payload sequences.Table 9.Example 10Insertions at Intron 1 Region 5 of CFTR Gene Using STITCHR Gene Editing

[0214] Insertions at intron 1 region 5 of CFTR gene using STITCHR gene editing were assessed (FIGS. 7 and 12).

[0215] HEK293FT cells were plated at 5-15K the day prior to transfection in a 96-well plate. HEK293FT were transfected with Lipofectamine 3000 (Thermo Fisher Scientific) according to manufacturer’s specifications. For STITCHR insertions, 30 ng of each guide encoding plasmid, 30 ng of the EGFP template, and 30 ng R2Toc-SpCas9H840A -encoding plasmid were delivered to each well unless otherwise specified. Cells were harvested for genomic DNA 3 days later and subjected to ddPCR analysis for insertion of the EGFP payload at the CFTR region. NOLC1 EGFP insertion was used as a positive control.

[0216] Nine forward facing guides were co-transfected with 7 reverse guides at CFTR intron 1 region 5 using STITCHR (R2Toc-SpCas9H840A); an EGFP payload with 50bp homology arms sharing sequence to the bases immediately upstream and downstream from the insertion site in CFTR intron 1 region 5 was co-transfected. At this region, the highest efficiency of payload integration was with forward guide 1 and reverse guide 6, displaying a payload integration of 1 .34%; this editing was the highest of the 5 regions surveyed, and disappeared in the absence of the R2-encoding plasmid vector. The NOLC1 EGFP payload inserted at the expected 4.5% efficiency. The significant drop in payload integration efficiency in the absence of the STITCHR-encoding plasmid vector underscores that R2-mediated insertion is the predominant mechanism of payload integration rather than HDR.

[0217] Table 10 below shows examples of guide, CFTR region 5 STITCHR payload, controls, and NOLC1 STITCHR payload sequences.Table 10.Example 11Guide Combination at CFTR Introns

[0218] PASTE gene editing is a two-step system. First, prime-editing can be used to insert one or more attB sites into a genome, and the attB can be used for the integration of payloads by Bxb1 serine integrases. A ‘twin prime editing’ (twinPE) version of prime editing was utilized at the CFTR locus. A pair of pegRNAs, each targeting the sense and antisense strand of a DNA for insertion, was assessed. For PASTE gene editing, the overall integration efficiency was found to be most limited by the efficiency of attB insertion by twinPE. Therefore, to obtain the highest possible integration of a payload into CFTR, pairwise combinations of twinPE pegRNAs at different CFTR loci were tested to identify the combination that produces the highest attB insertion efficiency.

[0219] Some regions of the genome are generally more amenable to high editing efficiencies than others. Additionally, editing by CRISPR systems (including prime editing) is restricted by genomic features, such as the availability of a protospacer adjacent motif (PAM). This results in genomic loci that have higher density of potential pegRNAs editors than others. Therefore, To discover optimal pegRNA combinations, the following steps were contemplated: 1) identify CFTR loci where it is possible to test a high number of potential pegRNAs; 2) identify CFTR loci that are amenable to efficient editing; 3) within the predetermined optimal CFTR intron locus, identify the best combination of pegRNAs for efficient attB insertion; and, 4) perform further optimizations with the pre-determined optimal pegRNA combinations to maximize insertion efficiency.Comprehensive guide combination screen at CFTR introns

[0220] Four regions within the CFTR intron 1 (“intron 1 regionl , intron region 2 etc.”) with a high density of potential pegRNA editors and 1x region within intron 2 (“intron 2 region 1”) were nominated. For each region, the maximum number of potential pegRNAs encoding attB sites for the sense and antisense DNA strand at each genomic locus was designed and cloned. The pegRNA sequences are listed Table 11 below.

[0221] Table 11 below shows examples of pegRNA.Table 11.

[0222] Since twinPE requires combinations of pegRNAs, pairwise crosses of each pegRNA targeting the sense DNA strand with each pegRNA targeting the antisense strand, at each nominated CFTR locus, were performed. The pairwise combinations are listed in Table 12 below.

[0223] Table 12 below shows examples of pegRNA crosses.Table 12.

[0224] Each pegRNA cross along with the PASTE machinery were expressed inside HEK293FT cells by transfecting them with lipofectamine 3000. After transfection, the cells were incubated for 3 days, during which time the PASTE machinery and pegRNAs were expressed and were able to edit the genome. After the three days, next generation sequencing (NGS) was performed to quantify attB insertion efficiency by every pegRNA cross at each CFTR locus (Figure 13 and Figure 14). The insertion of attB sites was found to be highest at CFTR intron 1 region 1 and region 2, with a maximal insertion efficiency of 20-25% by pegRNA crosses 6 and 7 at intron 1 region 2.Large payload insertion by PASTE at CFTR intron 1 region 2

[0225] After identifying CFTR intron 1 region 2 as being amenable to high attB insertion efficiency (Figure 14), the full PASTE gene editing protocol was tested, i.e., 1) insertion of attB sites followed by 2) insertion of large cargos by Bxb1 serine integrase. The integration of GFP at CFTR intron 2 was tested using the same pegRNA crosses as in Figure 13. This was performed by delivering the PASTE machinery, pegRNA crosses, and payload into HEK293FT cells by transfection and incubating the cells for 3 days. GFP payload insertion efficiency was quantified by next generation sequencing.

[0226] PegRNA cross 6 was found to exhibit the highest integration efficiency of GFP at CFTR intron 2 (Figure 15, left) at approximately 3%, which was consistent with the high attB insertion efficiency observed for cross 6 in Figure 13. A PASTE negative control (which is incapable of attB site insertion) showed 0% insertion.

[0227] When the payload is inserted by PASTE, the attB site is converted to an attR site. In addition to quantifying the GFP payload insertion, the presence of attB sites at the end of the 96-hour incubator was also quantified, as any attB site still present suggests successful twinPE insertion of the attB sites but failure of the Bxb1 integrase to insert the GFP payload. A high degree of unused attB sites was found {e.g., almost 20% for pegRNA cross 6), suggesting that the integration of the payload by the Bxb1 integrase could be optimized (Figure 15, right).Optimization of GFP payload insertion at CFTR intron 1 region 2

[0228] The integration of the GFP payload by the Bxb1 integrase was optimized. The amount of payload expressed in cells was thought to be a limiting factor. Therefore, HEK293FT cells were transfected with the PASTE gene editing machinery (or negative control), pegRNA cross 6 (which was found to be the best cross in Figures 13-15), and increasing the amounts of the GFP payload (Figure 16 and Figure 17).

[0229] Increasing the GFP payload amount improved the efficiency of the payload insertion, plateauing at about 10% (Figure 17, left), by about 7%. Corresponding decreases in the amount of unused attB sites correlating with increasing amounts of the payload were also found (Figure 17, right).Example 12Insertion of CFTR Exons 2-27 Into Intron 1

[0230] The insertion of a cloned CFTR payload (exons 2-27, a super-exon) containing a 5’ splice acceptor site into the intron 1 locus instead of a GFP payload was performed. The sequence of the CFTR super-exon is provided in Table 13 below. CFTR exons 2-27 was cloned with the splice acceptor, and insertion into CFTR intron 1 region in HEK293FT was then tested, similarly to the GFP-based experiments described in the previous example.

[0231] The HEK293FT cells were transfected with 18ng of pDY2007 (pegRNA6_f_PBS+2), 18ng of pDY2008 (pegRNA6_r_PBS+2), 180ng of attP-splice_acceptor_CFTR_superexon_2- 27 (from pDY2173), and 100ng of pDY0216 (PASTE P2a linker plasmid) 100ng, incubated for 72hrs, and tested. Results of the tests are shown in Figure 20. When the payload was inserted by PASTE, the efficiency of the payload insertion was about 22%. However, when the payload was inserted by the Bxb1 integrase, the efficiency of the payload insertion was lower and close to 0%. These results show that, when using PASTE, the super-exon can be inserted into the first intron of CFTR and spliced into the native transcript, replacing any mutations found in the native CFTR gene. Exogenous CFTR (Exons 2-27) can be insertedinto the first exon of the endogenous CFTR gene as a potential supplementation genetic strategy. The insertion of the CFTR exons 2-27 cDNA with a splice acceptor could result in therapeutic replacement of a potential patient’s endogenous mutated CFTR. Including a splice acceptor could result in the incorporation of a payload into the patient’s transcriptome in place to the endogenous, faulty CFTR gene.

[0232] Table 13 - Below shows an example of a CFTR super-exon.Table 13.Example 13Optimization of pegRNAs

[0233] The optimal pegRNAs combinations previously identified were further optimized. pegRNAs can be optimized by varying the length of their primer binding sequences (PBS). New combinations of pegRNAs were transfected in HEK293FT cells and GFP integration was measured by NGS. A combination of pegRNAs with increased integration was identified (Figure 18, see arrows). The new pegRNA combination resulted in an approximately 100% increase in GFP integration compared to the previously identified best pegRNA combination (Figure 16, ‘old best’ vs improved ‘new best’). Example 14Optimization of PASTE Machinery Linker

[0234] The engineering of the PASTE machinery linker was found to improve integration efficiency.

[0235] The linker DNA contained in the PASTE machinery plasmid that links the M-MLV RT to the Bxb1 sequences was engineered. The new linker was engineered to encode a selfcleaving P2A peptide. HEK293FT cells were transfected with the pegRNA combination PBS- 2_aRY1808_E8; PBS-2_aRY1808_A8 (Figure 18, see arrows), and GFP integration efficiency at CFTR intron 1 region 2 by the previous PASTE machinery and the PASTE machinery containing the P2a linker were compared. The P2a sequence was found to increase integration efficiency by approximately 100%, resulting in a total integration efficiency of about 55% (see Figure 19).Example 15PASTE-Replace Correction of Cystic Fibrosis

[0236] The PASTE-replace correction of cystic fibrosis to delete and replace a region of the gene will be assessed.

[0237] PASTE-replace works by combining PRIME-del (see Choi et al., Nature Biotech., 2021 , which is incorporated by reference in its entirety) with PASTE, provides an alternative to insertion only editing, and can help mitigate non-coding mutations in the CFTR sequence. PRIME-del utilizes a pair of atgRNAs, each containing a PBS targeting the regions flanking the DNA region to be deleted. The atgRNAs contain an RNA template reverse transcribed by the M-MLV contained in the prime editing machinery. For PASTE-replace, the pair of atgRNAs contain an RNA template encoding the attB attachment site and DNA sequence complementary to corresponding region of DNA, which is to be deleted. This results in a DNA lesion, which is resolved by endogenous DNA repair mechanisms, resulting in deletion of the DNA sequenced flanked by the atgRNAs and replacement by the attB attachment site.

[0238] For CFTR, atgRNAs targeting the 5’ and 3’ end of the CFTR gene will be designed. This will result in deletion of the CFTR gene and insertion of attB attachment sites. A PASTE payload containing all CFTR exons and an attP attachment site will also be cloned. Using PASTE-replace, the endogenous CFTR gene will be replaced by the PASTE payload. These components will be transfected into HEK293FT cells, and the integration of the CFTR payload will be read out by next generation sequencing (NGS).Example 16STITCHR Correction of Cystic Fibrosis

[0239] The STITCHR correction of cystic fibrosis will be assessed.

[0240] CFTR intron 1 region 2 was previously identified as being amenable to genome editing by PASTE and similar amenability to STITCHR editing is anticipated.

[0241] The STITCHR machinery consists of an R2 retrotransposon (e.g., Talpa occidentalis, Toe, ortholog). The payload for STITCHR insertions consists of the sequence to be inserted (e.g., CFTR exons 2-27) flanked by sequences homologous to the target site and a small sequence homologous to its natural 28S insertion site. STITCHR can naturally reprogram to new sites using the payload homology arms, however, this is further stimulated by fusing the R2 to a nickase Cas9H840A and co-transfected with single guide RNAs (sgRNAs) targeting the sense and antisense strand of the target DNA site.

[0242] A STITCHR CFTR payload will be first cloned. The payload will consist of the desired insert (e.g., CFTR exons 2-27) flanked by DNA regions homologous to CFTR intron 1 region 2. sgRNAs targeting each DNA strand at CFTR intron 1 region 2 will be designed and cloned, and an all-by-all cross of sense vs anti-sense strand sgRNAs into HEK293FT cells will be transfected. sgRNA pairs that result in optimal payload insertion will be identified, where the insertion efficiency will be read out by next generation sequencing (NGS).Example 17GFP Insertion in 16Hbe Cells

[0243] GFP insertion in 16Hbe cells was assessed.

[0244] 16HbeA508 cells (human bronchial epithelial cells) are more biologically relevant cells than HEK cells and model the most common CFTR patient mutation. The GFP insertion was performed into the CFTR intron 1 using optimized pegRNAs (pegRNA6_f_PBS+2 and pegRNA6_r_PBS+2). The cells were transfected with 18ng pDY2007 (pegRNA6_f_PBS+2), 18ng of pDY2008 (pegRNA6_r_PBS+2), 180ng of pDY0181 (GFP payload), and 100ng of pDY0216 (PASTE P2a linker plasmid), incubated, and tested. Results of the tests are shown in Figure 21 , and the sequence of the GFP pDY0181 payload is shown in Table 14 below. When the Bxb1 integrase was fused to the prime editor via an XTEN linker (see “fused” in Figure 21), the efficiency of the payload insertion was about 0.5%. When the Bxb1 integrase was fused to the prime editor via a self-cleaving p2a linker (see “p2a linker” in Figure 21), the efficiency of the payload insertion was higher, at about 1.7%. These results show that insertion into physiologically relevant cell types (i.e. lung epithelium) is possible.

[0245] Table 14 shows the sequence of the GFP pDY0181 payload.Table 14.LIST OF REFERENCES

[0246] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entireties and for all purposes to the same extent as if each individual reference {e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0247] Goncz, et al., Hum Mol Genet., 7:1913-1919 (1998);

[0248] Goncz, et al., Gene Then, 8:961-965 (2001);

[0249] Bruscia, et al., Gene Then, 9:683-685 (2002);

[0250] Goncz, et al., Gene Then, 8:961-965 (2001);

[0251] Beumer, et al., Genetics, 172:2391-2403 (2006);

[0252] Lee Ciaran, et al., BioResearch Open Access, 1:99-108 (2012);

[0253] Schwank, et al., Cell Stem Cell., 13:653-658 (2013);

[0254] Scholefield et al. Gene Ther 28, 396-401 (2021);

[0255] Gerard, G. R., DNA 5:271-279 (1986);

[0256] Kotewicz, M. L., et al., Gene 35:249-258 (1985);

[0257] Karlin et al. (1990) PNAS 87: 2264-2268;

[0258] Karlin et al. (1993) PNAS 90: 5873-5877;

[0259] Altschul et al., (1990) J Mol Biol 215: 403;

[0260] Altschul et al., (1997) Nuc Acids Res 25: 3389-3402; and

[0261] Myers et al., (1988) CABIOS 4:11-17.

Claims

What is claimed:1 . An editing system capable of site-specifically integrating an exogenous nucleic acid into a cystic fibrosis transmembrane conductance regulator (CFTR) gene, the system comprising:(a) an editing polypeptide comprising a DNA binding nuclease domain linked via a linker to a reverse transcriptase domain, wherein the DNA binding nuclease domain comprises a nickase activity that nicks a strand of the CFTR gene to form a nicked strand of the CFTR gene;(b) a guide RNA (gRNA) comprising:(i) a primer binding sequence(ii) a sequence complementary to an integration sequence; and(iii) a targeting sequence that can recognize a target site of the CFTR gene and can hybridize to a strand of the CFTR gene that is complementary to the nicked strand of the CFTR gene, wherein the gRNA comprises a nucleic acid sequence that is 85%-100% identical to one of the nucleic acid sequences of Table 11 , and wherein the exogenous nucleic acid is linked to a sequence that is an integration cognate of the integration sequence; and(c) an integration enzyme.

2. The genome editing system of claim 1 , wherein the DNA binding nuclease is selected from the group consisting of Cas9-D10A, Cas9-H840A, and Cas12a / b / c / d / e / f / g / h / i / j.

3. The genome editing system of claim 1 wherein the primer binding sequence can hybridize to a 3' end of the nicked strand of the CFTR gene.

4. The genome editing system of claim 1 , wherein the integration sequence for the integration enzyme is selected from an attB sequence, an attP sequence, an attL sequence, an attR sequence, a Iox71 sequence, and a FRT sequence.

5. The genome editing system of claim 1 , wherein the integration sequence for the integration enzyme is a Bxb1 sequence.6.. The genome editing system of claim 1 , wherein the linker is cleavable or non- cleavable.

7. The genome editing system of claim 1 , wherein the linker can be replaced by two associating binding domains of the DNA binding nuclease linked to a reverse transcriptase.

8. The genome editing system of claim 1 , wherein the integration enzyme is selected from the group consisting of Cre, Dre, Vika, Bxb1 , (pC31 , RDF, FLP, (pBT1 , R1 , R2, R3, R4, R5, TP901-1 , A118, q)FC1, <pC1 , MR11 , TG1 , q)370.1, W , BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, Conceptll, Museum, Severus, Airmid, Benedict, Hinder, ICIeared, Sheen, Mundrea, BxZ2, cpRV, retrotransposases encoded by R2, L1 , Tol2 Tc1 , Tc3, Mariner (Himar 1), Mariner (mos 1), and Minos, and any mutants thereof.

9. The genome editing system of claim 1 , wherein the integration enzyme is Bxb1 or a mutant thereof.

10. The genome editing system of claim 1 , wherein the reverse transcriptase domain comprises a mutation relative to a wild-type sequence.

11. The genome editing system of claim 1 , wherein the reverse transcriptase domain is selected from the group consisting of Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain, transcription xenopolymerase (RTX), avian myeloblastosis virus reverse transcriptase (AMV-RT), and Eubacterium rectale maturase RT.

12. The genome editing system of claim 11 , wherein the M-MLV reverse transcriptase domain comprises one or more mutations selected from the group consisting of D200N, T306K, W313F, T330P, and L603W.

13. A method of site-specifically integrating an exogenous nucleic acid sequence into a cystic fibrosis transmembrane conductance regulator (CFTR) gene, the method comprising:(a) incorporating at least one integration sequence at a specific target site in the CFTR gene by introducing ex vivo into a mammalian cell:(i) an editing polypeptide, wherein the editing polypeptide comprises a DNA binding nuclease domain linked via a linker to a reverse transcriptase domain, wherein the DNA binding nuclease domain comprises a nickase activity; and(ii) a guide RNA (gRNA) comprising a targeting sequence, a primer binding sequence, a sequence complementary to the at least one integration sequence, and a nucleic acid sequence 85%-100% identical to any one of the nucleic acid sequences of Table 11 , wherein the gRNA interacts with the editing polypeptide to direct the editing polypeptide to the target site of the CFTR gene, wherein the DNA binding nuclease domain nicks a strand of the CFTR gene to form a nicked site, and wherein the reverse transcriptase domain reverse transcribes the complement of the at least one integration sequence and thereby incorporates the at least one integration sequence into the nicked site, thereby incorporating the at least one integration sequence at the specific target site of the CFTR gene; and(b) integrating the exogenous nucleic acid sequence into the CFTR gene target nucleic acid by introducing into the cell:(i) the exogenous nucleic acid sequence linked to a sequence that is an integration cognate of the at least one integration sequence; and(ii) an integration enzyme that integrates the exogenous nucleic acid sequence into the CFTR gene at the at least one integration sequence, thereby site-specifically integrating the exogenous nucleic acid sequence into the CFTR gene.

14. The method of claim 13, wherein the editing polypeptide, the gRNA, the integration enzyme, and the exogenous nucleic acid sequence are introduced into the cell concurrently or not concurrently.

15. The method of claim 13, wherein the gRNA, an expressible polynucleotide construct encoding the editing polypeptide, and an expressible polynucleotide construct encoding the integration enzyme are introduced to the cell using a virus, a RNP, an mRNA, a lipid, or a polymeric nanoparticle.

16. The method of claim 13, wherein the gRNA hybridizes to a strand of the CFTR gene.

17. The method of claim 13, wherein the exogenous nucleic acid is introduced into the cell as an adeno-associated virus (AAV) or an adenovirus (AdV).

18. The method of claim 13, wherein the exogenous nucleic acid is introduced into the cell as a minicircle, a plasmid, mRNA, or a linear DNA.

19. The method of claim 18, wherein the minicircle does not comprise a sequence of a bacterial origin.

20. The method of claim 13, wherein the integration sequence is an attB sequence, an attP sequence, an attL sequence, an attR sequence, a Iox71 sequence, a Vox sequence, or a FRT sequence.

21. The method of claim 13, further comprising introducing a nicking guide RNA (ngRNA).

22. The method of claim 13, wherein: the exogenous nucleic acid is a reporter gene; the exogenous nucleic acid is a degradation tag for programmable knockdown of proteins in the presence of small molecules; the exogenous nucleic acid is a T-cell receptor (TCR), a chimeric antigen receptor (CAR), an interleukin, a cytokine, or an immune checkpoint gene for integration into a T-cell or natural killer (NK) cell; the exogenous nucleic acid is a beta hemoglobin (HBB) gene and the cell is a hematopoietic stem cell (HSC); or the exogenous nucleic acid is a metabolic gene.

23. The method of claim 22, wherein the reporter gene is a fluorescent protein.

24. The method of claim 13, wherein the cell is a dividing cell or a non-dividing cell.

25. The method of claim 13, wherein the exogenous nucleic acid is CFTR exon 1-27.

26. A genome editing system for a cystic fibrosis transmembrane conductance regulator (CFTR) gene, the system comprising:(a) an R2 element enzyme; and(b) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5’ homology region, a 3’ homology region, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the CFTR gene, and wherein the R2 element enzyme comprises a reverse transcriptase domain, and a nickase domain.

27. The genome editing system of claim 26, wherein the R2 element enzyme further comprises a targeting domain.

28. The genome editing system of claim 27, wherein the targeting domain is a natural targeting domain or an engineered targeting domain.

29. The genome editing system of claim 27, wherein the nucleic acid insertion into the genome is a DNA or RNA insertion template.

30. The genome editing system of claim 27, wherein the R2 element enzyme is a modified R2 element enzyme.

31. The genome editing system of claim 30, wherein the coding sequence of the R2 element enzyme is modified.

32. The genome editing system of claim 30, wherein the modified R2 element enzyme is modified by an N-terminal or C-terminal truncation of the R2 element enzyme sequence.

33. The genome editing system of claim 30, wherein the modified R2 element enzyme comprises a linker.

34. The genome editing system of claim 33, wherein the linker is an XTEN linker.

35. The genome editing system of claim 30, wherein the modified R2 element enzyme is a fusion protein.

36. The genome editing system of claim 30, wherein the modified R2 element is fused to a Cas9 protein that is fully active, catalytically dead (H840A / D10A for SpCas9), or functioning as a nickase (H840A or D10A for SpCas9).37 The genome editing system of claim 30, wherein the modified R2 element is fused to a Cas12 protein that is fully active, catalytically dead, or functioning as a nickase.

38. The genome editing system of claim 30, wherein the modified R2 element is fused to a TALEN protein, zinc finger protein, argonaute, or meganuclease protein.

39. The genome editing system of claim 26, wherein the 5’ homology region, the 3’ homology region, or both the 5’ and 3’ homology region target an exogenously introduced landing sequence.

40. The genome editing system of claim 26, further comprising a guide RNA.

41. A method of inserting a nucleic acid into a cystic fibrosis transmembrane conductance regulator (CFTR) gene in a cell, wherein the method comprises supplying a Cas9 or Cas12 fusion protein to the cell, wherein the Cas9 or Cas12 fusion protein is supplied with a payload RNA template, wherein the RNA template is reverse transcribed by the Cas9 or Cas12 fusion protein prior to being inserted into the gene of the cell; and wherein the nucleic acid is inserted into the gene.

42. The method of claim 41 , wherein the Cas9 fusion protein comprises a Cas9 portion and an R2 element portion, optionally wherein the Cas9 fusion protein comprises a targeting domain, a reverse transcriptase domain, and a nickase domain; or optionally wherein the Cas12 fusion protein comprises a Cas12 portion and an R2 element portion.

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