Compositions and methods for homology-directed repair-based DNA modifications

Abstract

Aspects of the application relate to template nucleic acids capable of introducing edits into DNA targets. Templates of the application comprise two homology arms flanking a heterologous nucleic acid comprising nucleotides that do not base pair with a sequence in a DNA target. The nucleotides that do not base pair with the DNA target can be utilized to, for example, reduce recombination between the heterologous nucleic acid and the DNA target relative to recombination between the homology arms and the DNA target, which allows for site-specific insertion of one or more genetic edits. In some aspects, templates are described that can be used to introduce edits at a site that is separated from a DNA break by tens, hundreds, thousands, or more than thousands of nucleotides. Further aspects of the application relate to recombinant adeno-associated virus particles and methods related to template nucleic acids described herein.

Description

[0001] PCT / US25 / 41648 12 August 2025 (12.08.2025)

[0002] -1-

[0003] COMPOSITIONS AND METHODS FOR HOMOLOGY-DIRECTED REPAIR-BASED DNA MODIFICATIONS

[0004] 5 RELATED APPLICATIONS

[0005] The application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application number 63 / 682,269 filed August 12, 2024, which is incorporated by reference in its entirety.

[0006] 10 REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The content of the electronic sequence listing (U120270150WO00-SEQ-PRW.xml; Size: 196,752 bytes; and Date of Creation: August 12, 2025) is herein incorporated by reference in its entirety.

[0008] FEDERALLY SPONSORED RESEARCH

[0009] This invention was made with government support under R01 EY018139 and R01 EY028033 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0010] BACKGROUND

[0011] Genetic editing is a process that introduces substitutions, deletions, and / or insertions into a nucleic acid, thereby changing its sequence.

[0012] SUMMARY

[0013] 25 Nucleic acids of the present application improve genetic editing efficiency at a precisely determined position by utilizing homology arms that flank a heterologous nucleic acid comprising nucleotides that do not base pair with a sequence in a DNA target. The nucleotides that do not base pair with the DNA target can be utilized to, for example, reduce recombination between the heterologous nucleic acid and the DNA target relative to recombination between the homology arms and the DNA target, which allows for site-specific insertion of one or more genetic edits. This can also expand the number of DNA targets that can be edited and the selection of available gRNAs for target editing.

[0014] Aspects of the application relate to a nucleic acid comprising: a first homology arm comprising homology to a first region of a DNA target and a second homology arm comprising

[0015] 35 homology to a second region of the DNA target; and a heterologous nucleic acid flanked by the first homology arm and the second homology arm, wherein the heterologous nucleic acid

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[0017] -2- comprises a variant of a sequence in the DNA target, wherein the variant comprises one or more synonymous mutations relative to the sequence in the DNA target.

[0018] In some embodiments, the heterologous nucleic acid comprises a sequence encoding an RNA. In some embodiments, the sequence encoding the RNA comprises the variant of the

[0019] 5 sequence in the DNA target. In some embodiments, the nucleic acid is configured to integrate the sequence encoding the RNA at an insertion site that is flanked by the first region and the second region and located in a gene that comprises the sequence that corresponds to the variant. In some embodiments, the heterologous nucleic acid comprises: a splicing acceptor site, wherein a splicing donor site is located 5’ relative to an insertion site that is flanked by the first region

[0020] 10 and the second region; a sequence encoding a protease cleavage site that is located 5’ relative to the sequence encoding the RNA; a sequence encoding a self-cleaving peptide that is located 5’ relative to the sequence encoding the RNA; a transcription termination sequence that is located 3’ relative to the sequence encoding the RNA; a sequence encoding a splicing donor site, wherein a sequence comprising a splicing acceptor site is located 3’ relative to an insertion site that is flanked by the first region and the second region; a start codon that is operably linked to the sequence encoding the RNA; and / or a stop codon that is operably linked to the sequence encoding the RNA.

[0021] In some embodiments, the nucleic acid is configured to integrate a sequence in the heterologous nucleic acid at an insertion site that is separated from a protospacer adjacent motif (PAM) by at least 10 nucleotide positions in the DNA target. In some embodiments, the first region or the second region comprises a protospacer adjacent motif (PAM). In some embodiments, the variant comprises one or more non-synonymous mutations relative to the sequence in the DNA target.

[0022] In some embodiments, the nucleic acid comprises at least one regulatory sequence. In

[0023] 25 some embodiments, the least one regulatory sequence comprises: a regulatory sequence operably linked to the variant of the sequence in the DNA target; and / or a regulatory sequence operably linked to a sequence comprising the first homology arm, the heterologous nucleic acid, and the second homology arm. In some embodiments, the at least one regulatory sequence comprises a promoter.

[0024] In some embodiments, the nucleic acid comprises a sequence encoding a guide RNA (gRNA) and / or a sequence encoding an RNA-guided nuclease. In some embodiments, the gRNA comprises a sequence that hybridizes to a sequence located at least one nucleotide upstream or downstream of a protospacer adjacent motif (PAM), wherein the PAM is located in the first region or the second region. In some embodiments, the gRNA is a single-guide RNA

[0025] 35 (sgRNA). In some embodiments, the RNA-guided nuclease is a Cas nuclease selected from the

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[0027] -3- group consisting of: a Cas9 nuclease or a variant thereof; a Casl2 nuclease or a variant thereof; a Casl3 nuclease or a variant thereof; and a Casl4 nuclease or a variant thereof. In some embodiments, the sequence encoding the gRNA and / or the sequence encoding the RNA-guided nuclease are operably linked to at least one regulatory sequence.

[0028] 5 In some embodiments, the first homology arm, the second homology arm, and / or the heterologous nucleic acid comprises a heterologous protospacer adjacent motif (PAM).

[0029] In some embodiments, the nucleic acid: is a linear nucleic acid or a circular nucleic acid; comprises deoxyribonucleotides, ribonucleotides, or a combination thereof; is a single- stranded nucleic acid, a double- stranded nucleic acid, or comprises one or more single- stranded stretches

[0030] 10 of sequence and one or more double- stranded stretches of sequence; and / or comprises one or more chemical modifications.

[0031] In some embodiments, the nucleic acid comprises a sequence which is at least 80% identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-62.

[0032] Aspects of the application further relate to a guide RNA (gRNA) comprising any one of the nucleotide sequences set forth in SEQ ID NOs: 1-20. In some embodiments, the gRNA: comprises one or more chemical modifications; and / or is a single-guide RNA (sgRNA).

[0033] The application further relates, at least in some aspects, to a recombinant adeno- associated virus (rAAV) particle comprising: a nucleic acid described herein and / or a gRNA described herein; and at least one adeno-associated virus (AAV) capsid protein. The application also relates, at least in some aspects, to a cell (e.g., a mammalian cell, such as a human cell) or a cell population thereof comprising a nucleic acid described herein and / or a gRNA described herein. The application also relates, at least in some aspects, to compositions and kits comprising any of the nucleic acids, gRNAs, rAAV particles, and / or cells or cell population thereof described herein.

[0034] 25 Aspects of the application also relate to methods that utilize a nucleic acid or a gRNA described herein.

[0035] In some embodiments, a method comprises introducing a nucleic acid or a gRNA described herein into a cell (e.g., a mammalian cell, such as a human cell). In some embodiments, the cell comprises a nucleic acid encoding a guide RNA (gRNA) and / or an RNA guided-nuclease. In some embodiments, the method comprises introducing a guide RNA (gRNA) and / or an RNA-guided nuclease into the cell or a descendant thereof. In some embodiments, the gRNA hybridizes with a sequence positioned at least one nucleotide upstream or downstream of a protospacer adjacent motif (PAM) in the first region or the second region of the DNA target. In some embodiments, the gRNA and the RNA-guided nuclease is contacted

[0036] 35 with the cell as a pre-formed ribonucleoprotein (RNP) complex. In some embodiments, the

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[0038] -4- nucleic acid comprises a sequence encoding a gRNA and / or a sequence encoding an RNA- guided nuclease. In some embodiments, the introducing the nucleic acid into the cell comprises contacting the cell with a recombinant adeno-associated virus (rAAV) particle comprising the nucleic acid and at least one adeno-associated virus (AAV) capsid protein. In some

[0039] 5 embodiments, the method comprises introducing at least one genetic edit in the cell, wherein the at least one genetic edit comprises the heterologous nucleic acid or a portion thereof. In some embodiments, the cell comprises the DNA target. In some embodiments, the cell or a descendant thereof comprises an edited DNA target after being contacted with the nucleic acid, wherein the edited DNA target comprises at least one genetic edit that is absent in a counterpart cell or a

[0040] 10 sample derived from the counterpart cell not contacted with the nucleic acid. In some embodiments, the nucleic acid is contacted with a neuronal cell. In some embodiments, the nucleic acid is contacted with a non-neuronal cell. In some embodiments, the nucleic acid is contacted with a retinal cell.

[0041] In some embodiments, a method comprises administering a nucleic acid described herein to a subject (e.g., a mammalian subject, such as a human subject). In some embodiments, the administering the nucleic acid comprises administering a recombinant adeno-associated virus (rAAV) particle comprising the nucleic acid and at least one adeno-associated virus (AAV) capsid protein to the subject. In some embodiments, the nucleic acid is contacted with a neuronal cell. In some embodiments, the nucleic acid is contacted with a non-neuronal cell. In some embodiments, the nucleic acid is contacted with a retinal cell.

[0042] BRIEF DESCRIPTION OF DRAWINGS

[0043] FIGs. 1A-1E show schematics illustrating non-limiting embodiments of template nucleic acids for HDR-based gene editing. FIG. 1A shows a non-limiting example of a template nucleic

[0044] 25 acid comprising a heterologous nucleic acid that edits a DNA target. FIG. IB shows nonlimiting embodiments of an edited DNA target produced by genetic engineering using the template in FIG. 1A. FIG. 1C shows a non-limiting example of a template nucleic acid configured to insert a heterologous nucleic acid at an site in a DNA target. FIG. ID shows nonlimiting embodiments of an edited DNA target produced by genetic engineering using the template in FIG. 1C. FIG. IE shows non-limiting examples of Silently Mutate And Repair Template (SMART) designs for genome editing.

[0045] FIGs. 2A-2I show results obtained from a genetic editing approach in mouse neonatal retina. FIG. 2A shows a schematic detailing non-limiting embodiments of a CRIPSR-tagging approach in mouse neonatal retina. Cas9 protein is premixed with gRNA, which is formed by

[0046] 35 annealing crRNA and tracrRNA, to create RNP particles. Then, repair template and pCAG-GFP

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[0048] -5- plasmid are further added to the mixture. The mixture is injected into the subretinal space of retina of P0 / 1 pups followed by electroporation to deliver all the components into retina cells. Expression of GFP labels the transduced cells, and knock-in by RNP particles and repair templates that can be used to analyze protein localization. FIG. 2B shows a representative image

[0049] 5 of an electroporated retina dissected at P21. The dashed lines delineate the electroporated regions expressing GFP. FIG. 2C shows confocal images of the electroporated retina’s crosssections showing the DAPI signal, GFP signal and immunoreactivities for the HA tag inserted to the N terminus of Eamin-Bl. FIGs. 2D-2F show retinas were dissected at P2 (FIG. 2D), P8 (FIG. 2E) and P21 (FIG. 2F) after electroporation at Pl. Confocal images of the retina cross¬

[0050] 10 sections showing the DAPI signal, GFP signal and immunoreactivities for the HA tag inserted to the N terminus of Eamin-Bl. NBE: neuroblast layer, ONE: outer nuclear layer, INF: inner nuclear layer, and GCE: ganglion cell layer. FIG. 2G shows quantification of the transduction efficiency in the transduced region by cell counting at P2, P8 and P21 (the ratio of the number of GFP-positive neurons to that of D API-positive neurons). n=3 retinas from 3 mice for P2 / P8 / P21. **p<0.01, one-way ANOVA. FIG. 2H shows a quantification of the editing efficiency in the electroporated region by cell counting at P2, P8 and P21 (the ratio of the number of HA-positive neurons to that of D API-positive neurons). n=3 retinas from 3 mice for P2 / P8 / P21. **p<0.01, one-way ANOVA. FIG. 21 shows a correlation analysis between the transduction efficiency and the editing efficiency. R2=0.988. The slope is 0.341. See also FIGs. 10A-10E.

[0051] FIGs. 3A-3L show non-limiting embodiments of template design and analyses of targeted knock-in efficiency. FIG. 3A shows a schematic illustrating a traditional template used for homology-directed repair when the cut site is positioned close to the insertion site. The coding region of the gene is labeled blue, and other regions are marked gray. When the PAM site is positioned close to the start codon (black), the sequence between cut site (dashed line with

[0052] 25 a scissor) and insertion site (dashed line) in the traditional repair template is short and does not detrimentally affect homologous recombination (indicated by black dashed lines) between the two homology arms of repair template and the broken targeted DNA. In this case, the entire repair template is involved in the homology-directed repair, which leads to the insertion of the tag sequence in the targeted gene. FIG. 3B shows a schematic illustrating the principle of a traditional template used for homology-directed repair when the cut site is positioned far from the insertion site. When the cut site is located far away from the insertion site, the elongated sequence between the two sites in the repair template predominates driving homologous recombination (indicated by black dashed lines) with its adjacent homology arm. This results in the other homology arm and the insertion sequence not participating in the homology-directed

[0053] 35 repair. Consequently, the damaged DNA is restored to its original sequence. FIG. 3C shows a

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[0055] -6- schematic illustrating non-limiting embodiments of SMART used for homology-directed repair. The sequence between the cut and insertion sites is silently mutated (represented by black slashes) in the repair template, which prevents its base pairing with the broken DNA and thus forces the entire repair template to be used for the homology-directed repair. After repair, the tag

[0056] 5 is integrated downstream of the start codon followed by the silently mutated sequence. FIG. 3D shows a schematic illustrating non-limiting embodiments of SMART-RC used for homology- directed repair, such as when the cut site is positioned far from the start codon. SMART-RC can make the insertion site the same as the cut site (represented by a single dashed line). The insertion sequence of SMART-RC contains P2A sequence, tag sequence, start codon (black) and

[0057] 10 silently mutated sequence (shaded region with black slashes) between cut site and start codon. After homology-directed repair, insertion sequence is inserted at the cut site, resulting in a tag sequence positioned upstream of the complete coding sequence of the gene. P2A is to eliminate amino acids upstream of the tag. The skipping site of P2A during translation is indicated by the white dashed line. FIGs. 3E, 3G and 31 show Lamin-Bl was endogenously tagged by gRNA-17 with 3 different repair templates: traditional template (FIG. 3E), SMART (FIG. 3G), and SMART-RC (FIG. 31). The cut site of gRNA-17 is 17 bp away from the start codon. Retinas were electroporated at P0 and dissected at PIO. Confocal images of the retina cross-sections show the DAPI signal, GFP signal and immunoreactivities for the HA tag inserted to the N terminus of Lamin-Bl. FIG. 3K shows a quantification of the editing efficiency normalized to transduction efficiency by cell counting for 3 different repair templates when gRNA-17 was used (the ratio of the number of HA -positive neurons to that of GFP-positive neurons). n=4 retina sections from 2 mice for traditional template / SMART / SMART-RC. *p<0.05, ***p<0.001, ****p<0.0001, one-way ANOVA. FIGs. 3F, 3H and 3J show Lamin-Bl was endogenously tagged by gRNA-142 with 3 different repair templates: traditional template (FIG.

[0058] 25 3F), SMART (FIG. 3H), and SMART-RC (FIG. 3J). The cut site of the gRNA-142 is 142 bp away from the start codon. Retinas were electroporated at P0 and dissected at PIO. FIG. 3L shows a quantification of the editing efficiency normalized to transduction efficiency by cell counting for 3 different repair templates when gRNA-142 was used (the ratio of the number of HA-positive neurons to that of GFP-positive neurons). n=3 retina sections from 2 mice for traditional template / SMART / SMART-RC. ***p<0.001, ****p<0.0001, one-way ANOVA. See also FIGs. 11A, FIGs. 12A-12C, and FIGs. 13-13D.

[0059] FIGs. 4A-4B show schematics illustrating non-limiting embodiments of SMART templates for modifying terminal ends of genes and / or proteins. FIG. 4A shows a schematic illustrating non-limiting embodiments of SMART-RC / CT used for homology-directed repair in

[0060] 35 C-terminus tagging. SMART-RC / CT makes the insertion site the same as the cut site

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[0062] -7-

[0063] (represented by a single dashed line). The insertion sequence contains silently mutated sequence (shaded region with black slashes) between cut site and stop codon, tag sequence and stop codon (black). After homology-directed repair, insertion sequence is inserted at the cut site, resulting in a tag sequence positioned downstream of the complete coding sequence of the gene. The

[0064] 5 inserted stop codon is to terminate the translation downstream of the tag. FIG. 4B shows a schematic illustrating non-limiting embodiments of SMART-RC / NT for homology-directed repair when the cut site is positioned far from the start codon. SMART-RC / NT makes the insertion site the same as the cut site. The insertion sequence contains a self-cleaving peptide sequence (e.g., the P2A sequence), a tag sequence, a start codon, and a silently mutated

[0065] 10 sequence between the cut site and start codon. After homology-directed repair, the insertion sequence is inserted at the cut site, resulting in a tag sequence positioned upstream of the complete coding sequence of the gene. The self-cleaving peptide is to eliminate amino acids upstream of the tag. The skipping site of self-cleaving peptide during translation is indicated by the white dashed line between the P2A and tag sequences.

[0066] FIGs. 5A-5E show non-limiting embodiments of a lineage reprogramming approach for targeting post-mitotic neurons with SMART. FIG. 5A shows a schematic illustrating nonlimiting embodiments of a lineage reprogramming strategy. Plasmids encoding eCAS9, gRNA targeting Nrl and reporter protein GFP are mixed with RNP particles and repair templates for tagging and delivered into rod precursors via subretinal injection and electroporation. Rod precursors are reprogrammed into cone-like cells upon Nrl knock out allowing for the study of localization of tagged proteins in cones. FIG. 5B shows reprogramming rod precursors into cone-like cells. Retinas were electroporated at Pl and dissected at P21. Confocal images of the retina cross-sections show the DAPI signal, GFP and immunoreactivities for PNA. FIG. 5C shows S-opsin labeling by SMART-RC / CT in cones. Retinas were electroporated at Pl and

[0067] 25 dissected at P21. Confocal images of the retina cross-sections show the GFP signal and immunoreactivities for the HA tag. FIG. 5D shows HA-tagged S-opsin co-localization with endogenous S-opsin. Confocal images of the retina cross-sections show the DAPI signal and immunoreactivities for the HA tag and S-opsin. Dashed line box indicates the region of the merged image reported with a higher magnification. FIG. 5E shows cone-arrestin tagging by SMART-RC / CT in cones. Retinas were electroporated at Pl and dissected at P21. Confocal microscopic images of the retina cross-sections show the DAPI signal and immunoreactivities for the HA tag.

[0068] FIGs. 6A-6C show results obtained from editing in photoreceptors that establishes a pattern of selective pre-synaptic targeting. Three pre-synaptic proteins were tagged in

[0069] 35 photoreceptors: PSD95 (FIG. 6A), ELFN1 (FIG. 6B), ELFN2 (FIG. 6C). Retinas were

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[0071] -8- electroporated at P0 / P1 and dissected at P21+. Confocal images of the retina cross-sections showing the DAPI signal and immunoreactivities for HA tag, PSD95 , ELFN1 and PNA. Dashed line box indicates the region of the merged image reported with a higher magnification. The white arrowheads point to representative HA signals colocalizing with the endogenous

[0072] 5 proteins’ immunoreactivities. See also FIGs. 15A-15B.

[0073] FIGs. 7A-7D show results obtained from editing in bipolar cells that defines the organization of post-synaptic compartment. Four post-synaptic proteins were labeled in bipolar cells: Gao (FIG. 7A), Trpml (FIG. 7B), mGluR6 (FIG. 7C) and GPR179 (FIG. 7D). Retinas were electroporated at P0 / P1 and dissected at P21. Confocal images of the retina cross-sections

[0074] 10 show the DAPI signal and immunoreactivities for the HA tag, Gao, Trpml and PKCa. See also FIGs. 16A-16B.

[0075] FIGs. 8A-8C show sparse neuron- selective labeling allows unequivocal attribution of synaptic molecules to pre- vs. post-synaptic compartments. FIG. 8A shows schematics showing the different labeling patterns of pre-synaptic protein vs post-synaptic protein. When pre- synaptic protein is tagged in one rod, only one punctum will be detected in the synapse. In contrast, when post-synaptic protein is labeled in one bipolar cell, a tree of puncta will be visualized in the OPL (dark shaded arrows indicate cell labeling and light shaded clusters below cell bodies indicate puncta). FIG. 8B shows sparse labeling of Lrit3 as a single punctum. Retinas were electroporated at Pl and dissected at P21. Confocal microscopic images of the retina cross-sections show the DAPI signal and immunoreactivities for the HA tag and PKCa. FIG. 8C shows Nyctalopin tagging reveals clusters of puncta. Retinas were electroporated at Pl and dissected at P21. Confocal microscopic images of the retina cross-sections show the DAPI signal and immunoreactivities for the HA tag and PKCa. White dashed lines delineate the cell bodies of the edited cell. See also FIGs. 17A-17B.

[0076] 25 FIGs. 9A-9C show a combination of SMART tagging with traditional gene disruption by CRISPR. FIG. 9A shows schematics of the combined approach to achieve protein tagging while knocking-out another protein. RNP particles and repair templates are used for tagging and plasmids encoding eCas9 and gRNAs are used for knock-out. The components are mixed, injected and electroporated together. FIG. 9B shows localization of tagged Trpml when nothing is knocked out. Retinas were electroporated at Pl and dissected at P21. Confocal microscopic images of the retina cross-sections show the DAPI signal, GFP and immunoreactivities for the HA tag and mGluR6. FIG. 9C shows localization of tagged Trpml upon mGluR6 knock out. Retinas were electroporated at Pl and dissected at P21. Confocal microscopic images of the retina cross-sections show the DAPI signal, GFP and immunoreactivities for the HA tag and

[0077] 35 mGluR6.

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[0079] -9-

[0080] FIGs. 10A-10E show verification of the in vivo tagging of Lamin-Bl, related to FIGs.

[0081] 2A-2I. FIG. 10A shows a graphical representation of the mouse genomic locus of Lmnbl before and after CRISPR-editing and the repair template sequence. The insertion site (indicated by dashed lines) was positioned downstream of the start codon (black). The PAM site and target

[0082] 5 sequence were selected in the non-coding strand, which results in a cut site (indicated by a dashed line with a scissor) located 1 bp away from the start codon. The repair template includes two homology arms (within brackets) surrounding the insertion site. Between the two arms, there is the insertion sequence encoding an HA tag. Following homology-directed repair, the HA tag sequence is inserted just downstream of the start codon. Corresponding amino acid

[0083] 10 sequences are displayed under the DNA sequences. FIG. 10B shows the highest knock-in efficiency was reached with the 117-volt electroporation program. Retina was electroporated at Pl and dissected at P8. Confocal microscopic images of the retina cross-sections show the DAPI signal, GFP signal and immunoreactivities for the HA tag inserted to the N terminus of Lamin- Bl. FIG. 10C shows a quantification of the transduction efficiency and editing efficiency by cell counting when using 117-volt electroporation program (n=3 retina cross-sections from 1 mouse). FIG. 10D shows a schematic of the mouse genomic locus of Lmnbl showing two pairs of primers for PCR and DNA sequencing. The first pair of primers, Fl and Rl, amplified the DNA sequence upstream of the HA tag, which contains the whole HA tag. The second pair of primers, F2 and R2, amplified the DNA sequence downstream of the HA tag, which also contains the whole HA tag. DNA sequencing results from the two amplicons are combined and shown together under the scheme. HA tag sequence is highlighted in with a shaded box. FIG. 10E shows colocalization between HA-Lamin-Bl and endogenous Lamin-Bl. Confocal microscopic images of the retina cross-sections show the immunoreactivities for the HA tag and endogenous Lamin-Bl. The white arrows point to representative neurons where HA-lamin-Bl

[0084] 25 and endogenous Lamin-Bl share the same localizations.

[0085] FIGs. 11A-11C show the sequences of three non-limiting examples of repair templates when gRNA-17 was used for editing Lmnbl, related to FIGs. 3A-3L. FIG. 11A shows a graphical representation of the mouse genomic locus of Lmnbl before and after CRISPR-editing using the traditional template. The insertion site (indicated by dashed lines) was positioned downstream of the start codon (black). The PAM site and target sequence were selected in the non-coding strand, which results in a cut site (indicated by a dashed line with a scissor) located 17 bp away from the start codon. The traditional template includes two homology arms (within brackets) surrounding the insertion site. Between the two arms, there is the insertion sequence encoding an HA tag. To prevent re-cutting by Cas9 after homology-directed repair, a silent

[0086] 35 mutation (nucleotide positions with light shading) was introduced in the homology arm to

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[0088] -10- disrupt the PAM site. Following homology-directed repair, the HA tag sequence is inserted just downstream of the start codon. Corresponding amino acid sequences are displayed under the DNA sequences. FIG. 11B shows a graphical representation of the mouse genomic locus of Lmnbl before and after CRISPR-editing using SMART. A difference between SMART and

[0089] 5 traditional template is that the sequence between the insertion and cut sites (encircled by black rectangle) was silently mutated (nucleotide positions with light shading) in the homology arm, which prevents the sequence (encircled by a black rectangle) from base pairing with the broken DNA and thereby forces the entire repair template to be used for the homology-directed repair. After homology-directed repair, the tag sequence is integrated downstream of the start codon

[0090] 10 followed by the silently mutated sequence. FIG. 11C shows a graphical representation of the mouse genomic locus of Lmnbl before and after CRISPR-editing using SMART-RC / NT. Different from SMART and traditional templates, SMART-RC / NT makes the insertion site the same as the cut site (represented by a single dashed line), and its homology arms were designed surrounding the cut site. The insertion sequence was directly inserted at the cut site, which contains a GSG linker sequence for increasing the cleavage efficiency of P2A, P2A sequence, HA tag sequence, ATG, and the sequence with silent mutations (nucleotide positions with light shading) between the cut site and start codon (encircled by a black rectangle). The insertion also includes a nucleotide, C, upstream of the GSG linker sequence, ensuring that the inserted sequence aligns in-frame with the open reading frame of Lmnbl after integration. After

[0091] 20 homology-directed repair, the coding sequence of the Lmnbl is completed with the silently mutated sequence, and the HA tag sequence is added at the very N terminus, downstream of the P2A sequence. The repeating sequence upstream of the insertion region will be removed via the ribosomal skipping induced by P2A during translation. The skipping site of P2A is indicated by a white dashed line.

[0092] FIGs. 12A-12C show the sequences of three non-limiting examples of repair templates when the gRNA-142 was used for tagging Lmnbl, related to FIGs. 3A-3L. FIGs. 12A-12C show a graphical representation of the mouse genomic locus of Lmnbl before and after CRISPR-editing with three repair template sequences: traditional template (FIG. 12A), SMART (FIG. 12B), and SMART-RC / NT (FIG. 12C). Two single- stranded DNA templates were

[0093] 30 designed with 50-bp overlap to form a long repair template. The target sequence is silently mutated (nucleotide positions with light shading) in each template to avoid being recognized again after repair. The inserted nucleotides, TG, between the left homology arm and GSG linker sequence, are to align the insertion sequence in-frame with the open reading frame of Lmnbl after integration (FIG. 12C).

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[0095] -11-

[0096] FIGs. 13A-13D show analyses of P2A cleavage efficiency in retinal neurons, related to FIGs. 3A-3L. FIGs. 13A-13B show a graphical representation of the mouse genomic locus of Lmnbl before and after CRISPR-editing with two repair template sequences: Myc-P2A-HA (FIG. 13A) and Myc-scrambled P2A-HA (FIG. 13B). The optimally positioned gRNA targeting

[0097] 5 Lmnbl was used for CRISPR-editing. In the insertion region of repair template, P2A sequence is placed between Myc tag sequence and HA tag sequence. The inserted nucleotides, GT, between the left homology arm and the Myc tag sequence, are to align the insertion sequence inframe with the open reading frame of Lmnbl after integration. After repair, two tag sequences are inserted downstream of the start codon of Lmnbl. Myc tag is separated from HA tag

[0098] 10 appending to the N terminus of Lamin-Bl due to the ribosomal skipping induced by P2A peptide during translation. In the Myc-scrambled P2A-repair template, missense mutations (marked in dark shading) were introduced in P2A sequence, disrupting the ribosomal skipping function of the P2A peptide. Consequently, Lamin-B 1 is expressed in neurons with both Myc and HA tags. FIG. 13C shows Myc tag was efficiently separated from HA-Lamin-Bl by P2A peptide. Retinas were electroporated at P0 and dissected at PIO. Confocal microscopic images of the retina cross-sections show the DAPI signal, GFP signal (white) and immunoreactivities for the HA tag and Myc tag inserted to the N terminus of Lamin-B 1. White arrowhead points to a cell where Myc tag was not 100% separated from HA-Lamin-Bl and detected weakly. FIG. 13D shows Myc tag was not separated from HA-Lamin-B 1 by scrambled P2A peptide. Retinas were electroporated at P0 and dissected at P10. Confocal microscopic images of the retina crosssections show the DAPI signal, GFP signal and immunoreactivities for the HA tag and Myc tag inserted to the N terminus of Lamin-Bl. White arrowheads point to representative cells where Myc signal colocalized with HA signal.

[0099] FIG. 14 shows a graphical representation of the mouse genomic locus of Ctbp2 and Glul

[0100] 25 before and after CRISPR-editing with SMART-RC / CT. Similar to SMART-RC / NT, SMART- RC / CT makes the insertion site the same as the cut site (represented by a single dashed line), and its homology arms were designed surrounding the cut site. The insertion sequence was directly inserted at the cut site, which contains the sequence with silent mutations (nucleotide positions with light shading) between the cut site and stop codon (within brackets), HA tag sequence, and stop codon (black). Following homology-directed repair, the coding sequence of the targeted gene is completed with the silently mutated sequence, and the HA tag sequence is added at the very C terminus, followed by a stop codon. The redundant sequence downstream of the inserted stop codon will not be translated.

[0101] FIGs. 15A-15B show a graphical representation of the mouse genomic locus of Dlg4

[0102] 35 (FIG. 15A) and Elfnl (FIG. 15B) before and after CRISPR-editing with SMART-RC / CT.

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[0104] -12-

[0105] FIGs. 16A-16B show non-limiting repair template designs of post-synaptic genes, related to FIGs. 7A-7D. FIG. 16A shows a graphical representation of the mouse genomic locus of Gnaol before and after CRISPR-editing with SMART (nucleotide positions with light shading). Four HA tags were inserted after the 122nd amino acid, Alanine, in Gao. FIG. 16B

[0106] 5 shows a graphical representation of the mouse genomic locus of Gprl79 before and after CRISPR-editing with SMART-RC / CT.

[0107] FIGs. 17A-17B show repair template design and selective labeling patterns of synaptic proteins, related to FIGs. 8A-8C. FIG. 17A shows a graphical representation of the mouse genomic locus of Lrit3 before and after CRISPR-editing with SMART-RC / CT. FIG. 17B shows

[0108] 10 a graphical representation of the mouse genomic locus of Nyx before and after CRISPR-editing with SMART (nucleotide positions with light shading). HA tag was inserted after the 19th amino acid, Threonine, in Nyctalopin. The signal peptide sequence is indicated by a bracket, and the cleavage site of the signal peptide is indicated by an arrow.

[0109] FIG. 18 shows an in silico analysis of 145,442 sites across the coding sequences of the human genome quantifying the PAM sites within 10 bp distance of the intended modification site. Using 60% on-target efficiency as a threshold, the percentage of sites with at least one effective gRNA was calculated.

[0110] FIGs. 19A-19D show SMART template design greatly improves targeted knock-in efficiency. FIG. 19A shows targeting Lmnbl using an efficient gRNA, and an EcoRI site was inserted at various positions using either traditional templates or the SMART. FIGs. 19B-19C show analyses of the knock-in efficiency (KI%) as a function of distance between the cut and insertion sites for Lmnbl (left panel) and the CXCR4 editing (right panel). FIG. 19D shows quantification of knock-in efficiency for assays targeting Lmnbl (left panel) and CXCR4 (right panel) with decreasing silent mutations.

[0111] 25 FIGs. 20A-20B shows a comparison of editing efficiency between traditional and SMART templates. FIG. 20A shows SMART templates dramatically enhance editing efficiency compared to traditional templates. FIG. 20B shows SMART templates dramatically enhanced the normalized editing efficiency compared to traditional templates. For FIGs. 20A-20B, editing results that were observed for traditional template are represented by the leftmost bars in each set of two bars above the four target genes along the x-axis. Editing results that were observed for SMART templates are represented by the rightmost bars in each set of two bars above the four target genes along the x-axis.

[0112] FIG. 21 is a schematic illustrating non-limiting embodiments of SMART-RC / CDS used for homology-directed repair in inserting a full coding sequence (CDS) of a gene, or a portion

[0113] 35 thereof, into a DNA target.

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[0115] -13-

[0116] DETAILED DESCRIPTION

[0117] Aspects of the application relate to compositions and methods for nucleic acid editing.

[0118] 5 Traditional gene editing strategies can be limited due to the position of protospacer adjacent motif (PAM) sequences relative to the target (see, e.g., FIG. 18 and Example 2). Nucleic acids of the present application overcome these limitations by utilizing homology arms that flank a heterologous nucleic acid comprising nucleotides that do not base pair with a sequence in DNA target (see, e.g., bottom nucleic acid depicted in FIGs. 1A and 1C; FIGs. 19-20 and Example 2).

[0119] 10 The nucleotides that do not base pair with the DNA target can be utilized to, for example, reduce recombination between the heterologous nucleic acid and the DNA target relative to recombination between the homology arms and the DNA target, which allows for site-specific insertion of one or more genetic edits into a DNA target. This can also expand the number of targets that can be edited and the selection of available gRNAs for target editing, thereby improving editing efficiency at a precisely determined position.

[0120] In some embodiments, a heterologous nucleic acid comprises a sequence which is a variant (also referred to herein as a “variant sequence”) of a corresponding sequence (also referred to herein as a counterpart sequence) in a DNA target, wherein the variant sequence differs from the corresponding sequence by one or more nucleotides. In some embodiments, a variant sequence nucleotide that do not base pair with a sequence in a DNA target, such as nucleotides utilized to reduce recombination between a heterologous nucleic acid and a DNA target relative to recombination between homology arms and the DNA target.

[0121] In some embodiments, a variant sequence comprises synonymous mutations relative to corresponding nucleotide positions in a counterpart sequence. For example, in some

[0122] 25 embodiments, a variant sequence comprises a “silently mutated sequence”. In some embodiments, a silently mutated sequence is used to redesign (e.g., by replacing) the targeted sequence to comprise a sequence encoding an amino acid sequence that is identical to an amino acid sequence encoded by the sequence in the target prior to editing. In some embodiments, a variant sequence comprising synonymous mutations is used to integrate a sequence (e.g., a sequence located upstream or downstream of the variant sequence in the template) into the DNA target.

[0123] However, in some aspects of the application, a variant sequence comprises synonymous mutations and non- synonymous mutations relative to corresponding nucleotide positions in a counterpart sequence in a DNA target. In some embodiments, a variant sequence comprising

[0124] 35 non-synonymous mutations relative to a counterpart sequence in a DNA target encodes a variant

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[0126] -14- of a protein which is encoded by the sequence in the DNA target (e.g., a protein comprising a conservative mutation at one or more amino acid positions and / or a non-conservative mutation at one or more amino acid positions). Thus, some aspects of the application relate to template nucleic acids that can be used to genetically modify a DNA target and express a variant of a

[0127] 5 coding sequence in the DNA target which comprises mutations at specific positions of interest (e.g., positions in a DNA target that comprise disease-associated mutations).

[0128] These aspects and others are illustrated, for example, in the non-limiting embodiments shown in FIGs. 1A-1E and also further described below.

[0129] In some aspects, the disclosure relates to template nucleic acids which comprise a variant

[0130] 10 of a sequence located in a target region in a DNA target, wherein the target region is located between regions of a DNA target that comprise sequences that hybridize with homology arms in the template nucleic acid (see, e.g., FIG. 1A). In some embodiments, the compositions and methods can be used for inserting a sequence at an insertion site in a DNA target which is separated from a site of DNA damage (e.g., a site cut by an RNA-guided nuclease) by tens of nucleotides, hundreds of nucleotides, or more (e.g., 1,000 nucleotides or greater than 1,000 nucleotides). In some embodiments, the site of DNA damage is located near a sequence that binds to a nuclease, such as a PAM that can bind to a CRISPR / Cas molecule, a zinc-finger nuclease binding sequence, or a transcription activator-like effector nuclease (TALEN).

[0131] In some aspects, the application relates to template nucleic acids which comprise a variant of a sequence in a DNA target and can integrate the variant at an insertion site located between two regions of a DNA target that comprise sequences that hybridize with the homology arms of the template nucleic acid (see, e.g., FIG. 1C). As such, compositions and methods of the application can comprise a template nucleic acid that is configured to integrate a heterologous nucleic acid between a first region and a second region of a DNA target, thereby extending the

[0132] 25 distance between the first region and the second region in the edited DNA target (see, e.g., FIGs. 1C-1E). In some embodiments, a gene sequence or a portion thereof can be in the first region or the second region. However, in some embodiments, a first portion of a gene sequence is in, for example, a first region of a DNA target and a second portion of the gene sequence is in a second region the DNA target.

[0133] In some embodiments, a template nucleic acid comprises a regulatory sequence. In some embodiments, a template nucleic acid comprises a regulatory sequence which is operably linked to a sequence in the heterologous nucleic acid and / or is configured to become operably linked to a gene or a portion thereof in a target (see, e.g., FIG. ID). In some embodiments, sequences which are operably linked in a template and / or in an edited DNA target alter the expression of a

[0134] 35 gene in the edited DNA target. Altered expression of the gene can occur through a variety of

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[0136] -15- mechanisms using the template nucleic acids of the application including, but not limited to, altering transcription, pre-mRNA processing (e.g., alternative splicing), translation, and / or post- translational modifications. In some embodiments, a regulatory sequence in a template can regulate production of a first RNA sequence and / or a first amino acid sequence which is

[0137] 5 encoded by a gene in an edited DNA target and which is not covalently linked to a second RNA sequence and / or a second amino acid sequence encoded by a heterologous nucleic acid (see, e.g., FIG. ID).

[0138] Further aspects of the application relate to cells (e.g., mammalian cells, such as human cells, including but not limited to mitotic cells, post-mitotic cells, in vitro cells, and ex vivo cells)

[0139] 10 and recombinant adeno-associated virus (rAAV) particles comprising a template nucleic acid. The application also relates to methods of introducing template nucleic acids into a cell (e.g., mammalian cells, such as human cells, including but not limited to mitotic cells, post-mitotic cells, in vitro cells, and ex vivo cells), such as methods of genetically engineering cells, and methods of administering template nucleic acids to a subject (e.g., a mammalian subject, such as a human subject) which can be used, for example, to edit targets in vivo. Additional aspects of the application relate to kits that can be used for target editing and can be used in methods of the application as described herein.

[0140] DNA Target Editing

[0141] “Homology-directed repair” or “HDR” refers to a process that synthesizes new DNA in a first nucleic acid using the sequence comprised in a second nucleic acid (also referred to as a template nucleic acid). HDR is mediated by a variety of proteins. Briefly, nucleases produce single- stranded overhangs on the first nucleic acid. The overhangs hybridize with sequences in the second nucleic acid which comprise homology to the overhangs. Repair polymerases extend

[0142] 25 the overhangs of the first nucleic acid using the second nucleic acid as a template. Gaps in the polymerized strands of the first nucleic acid are filled by gap polymerases and ligases. Thus, the template nucleic acid can restore the original sequence if the template sequence is identical to the first nucleic acid, but the process can introduce insertions, deletions, and / or nucleotide substitutions if the template is not identical to the first nucleic acid (e.g., if the template includes insertions, deletions, and / or nucleotide substitutions relative to the first nucleic acid). However, HDR is not limited in this regard as this process can also result in substitution of one or more nucleotides, deletion of one or more nucleotides, and / or insertion of one or more nucleotides.

[0143] A “template” or “template nucleic acid” refers to a nucleic acid that serves as a substrate for HDR and, for example, promote integration of one or more nucleic acids into a DNA target.

[0144] 35 Generally, templates of the application comprise two homology arms flanking a heterologous

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[0146] -16- nucleic acid (see, e.g., bottom nucleic acid depicted in FIGs. 1A and 1C). A “homology arm” refers to a sequence configured to stably and / or specifically hybridize to a sequence in a DNA target and a “heterologous nucleic acid” refers to a nucleic acid comprising one or more sequences which are not found in the DNA target (and that will not stably and / or specifically

[0147] 5 hybridize to the target DNA).

[0148] Sequences configured to introduce edits into a DNA target can be comprised in a heterologous nucleic acid. As a non-limiting example, a heterologous nucleic acid can comprise a sequence which is a variant of a sequence in a DNA target, such as a variant of a target region. A “variant” refers to a sequence that differs from a corresponding counterpart sequence by one

[0149] 10 or more nucleotides. Generally, a variant sequence comprises one or more synonymous mutations relative to corresponding nucleotide positions in a counterpart sequence. A “synonymous mutation” refers to a nucleotide substitution in a codon that does not alter the sequence of the amino acid encoded by the codon. Thus, some aspects of the application relate to templates that can be used to replace a sequence in a DNA target with one or more silently mutated sequences. However, the application also provides for heterologous nucleic acids comprising non- synonymous mutations, such as conservative or non-conservative mutations and / or other heterologous sequences. In some embodiments, a variant sequence in a heterologous nucleic acid comprises non- synonymous mutations and synonymous mutations relative to a corresponding sequence in a DNA target.

[0150] 20 In some embodiments, a template is configured to edit a DNA target at an insertion site located between a first and second region of the target (see, e.g., FIGs. 1A-1B). In some embodiments, a template is configured to generate an insertion in the DNA target (see, e.g., FIGs. 1C-1D). In some embodiments, the DNA target is edited to produce an edited DNA target, wherein the edited DNA target comprises a heterologous gene sequence (e.g., a variant gene sequence) and / or other heterologous sequence (see, e.g., FIGs. 1A-1D). In some embodiments, editing a DNA target comprises mutating a peptide or a protein coding sequence. In some embodiments, editing a DNA target comprises introducing a sequence that results in an alteration in the N-terminus of a peptide or a protein. In some embodiments, editing a DNA target comprises introducing a sequence that results in an alteration in the C-terminus of a

[0151] 30 peptide or a protein. In some embodiments, a template nucleic acid integrates a heterologous nucleic acid between a first region and a second region of a DNA target (see, e.g., FIGs. 1C-1D). In some embodiments, the template comprises a regulatory sequence which is operably linked to a sequence in the heterologous nucleic acid and / or is configured to become operably linked to a gene or a portion thereof in a target (see, e.g., FIG. ID).

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[0153] -17-

[0154] As used herein, “operably linked” refers to a nucleic acid sequence and a regulatory sequence are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences. As a nonlimiting example, two DNA sequences are said to be operably linked if activation of a promoter

[0155] 5 in a 5’ regulatory sequence result in the transcription of a coding sequence. In some embodiments, sequences which are operably linked in a template and / or in an edited DNA target alter the expression of a gene in the edited DNA target. Altered expression of the gene can occur through a variety of mechanisms using the template nucleic acids of the application including, but not limited to, altering transcription, pre-mRNA processing (e.g., alternative splicing),

[0156] 10 translation, and / or post-translational modifications.

[0157] In some embodiments, a regulatory sequence in a template can regulate production of a first RNA sequence and / or first amino acid sequence which is encoded by a gene in an edited DNA target and which is not covalently linked to a second RNA sequence and / or second amino acid sequence encoded by a heterologous nucleic acid (see, e.g., FIG. ID). In some embodiments, a gene sequence or a portion thereof can be in the first region or the second region. However, in some embodiments, a first portion of a gene sequence (e.g., a portion of the gene encoding an N-terminal domain of a protein) is in, for example, a first region of a DNA target and a second portion of the gene sequence (e.g., a portion of the gene encoding a C- terminal domain of a protein) is in a second region of the DNA target.

[0158] In some embodiments, template nucleic acids can edit a DNA target at an insertion site separated from a protospacer adjacent motif by tens of nucleotides, hundreds of nucleotides, thousands of nucleotides, or more. As used herein, a “protospacer adjacent motif ‘ or “PAM” refers to a sequence that targets an RNA-guided nuclease to a polynucleotide sequence. An “RNA-guided nuclease” refers to a protein comprising a nuclease domain or a variant thereof

[0159] 25 that physically interacts with an RNA molecule that localizes the nuclease to a sequence in a DNA target. In some embodiments, an RNA-guided nuclease comprises a CRISPR / Cas nuclease or a variant thereof. In some embodiments, an RNA molecule bound by an RNA-guided nuclease comprises a “guide RNA” or “gRNA” which refers to a nucleic acid comprising a sequence that hybridizes to a sequence in a DNA target and directs cleavage of the DNA target when bound to an RNA-guided nuclease. In some embodiments, a gRNA is a single-guide RNA (sgRNA) comprising a fusion of a sequence in a gRNA that binds to a DNA target and a “trans activation RNA” or “tracrRNA” that binds to an RNA-guided nuclease. In some embodiments, a template comprises one or more heterologous PAMs that can be integrated into a DNA target at a first insertion site, wherein the one or more heterologous PAM are then used for introducing an

[0160] 35 edit at a second insertion. In some embodiments, the edit at the first insertion site (which is

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[0162] -18- edited to comprise the PAM) is made using a first RNA-guided nuclease and the edit at the second insertion site is made using a second RNA-guided nuclease.

[0163] DNA Targets

[0164] 5 A “target” or “DNA target” refers to a nucleic acid comprising sequences that are homologous to (e.g., comprise a sequence that is the reverse complement of) the homology arms of a template nucleic acid. Contacting a target with a template nucleic acid under conditions that introduce at least one mutation into the target is referred to herein as “genetic editing”, “genetic engineering”, or “genetic modification.” As such, when the at least one mutation is introduced

[0165] 10 into the target, an “edited DNA target” or “edited target” is produced and the at least one mutation is referred to herein as an “edit.”

[0166] DNA targets can comprise an “insertion site” which refers to a nucleotide position in the DNA target that can be genetically engineered to comprise an edit (see, e.g., top nucleic acid depicted in FIGs. 1A and 1C). In some embodiments, an insertion site is in a gene (e.g., in an exon or an intron). In some embodiments, an insertion site is in an intergenic sequence. A DNA target can also comprise a plurality of sequences that are organized into what is referred to herein as “regions” of the DNA target (see, e.g., top nucleic acid depicted in FIGs. 1A and 1C). In some embodiments, a DNA target comprises a first region and a second region. The terms “first region” and a “second region” refers to different sequences in a DNA target or an edited DNA target and, unless stated otherwise herein, should not be interpreted to refer to a specific strand of DNA in the target or a specific directionality (see, e.g., top nucleic acid depicted in FIGs. 1A and 1C). The first and second regions can be continuous with each other or separated by one or more nucleotides. For example, in some embodiments, a DNA target comprises a “target region” which refers to one or more sequences in a DNA target which are flanked by the

[0167] 25 first and second regions (see, e.g., top nucleic acid depicted in FIG. 1A).

[0168] Template nucleic acids of the application can be used to edit a variety of DNA targets. In some embodiments, a DNA target comprises a sequence of a wild-type gene. In some embodiments, a DNA target comprises a mutation, such as a mutation associated with a disease, disorder, or condition. In some embodiments, a sequence that has been subjected to a first set of genetic engineering conditions produces a DNA target comprising a first mutation that can be subjected to a second set of genetic engineering conditions involving a template nucleic acid configured to introduce a second mutation into the target. In some embodiments, a DNA target is a genomic sequence, such as a sequence comprised in the genome of a cell. In some embodiments, a DNA target is comprised in a nucleic acid this is not naturally found in a cell,

[0169] 35 such as a vector (e.g., a plasmid) that has been transformed or transfected into the cell (e.g., a

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[0171] -19- circular vector that can be maintained in a cell without being integrated into the genome or a vector which has been linearized and integrated into the genome of the cell). In some embodiments, a DNA target comprises a sequence in a mammalian genome, such as a human genome. However, the application is not limited in this regard, as any sequence that is selected

[0172] 5 as a target can be edited according to the embodiments described herein.

[0173] Further to the non-limiting embodiments of DNA targets described herein (see, e.g., FIGs. 1A and 1C), non-limiting examples of a DNA target are set forth in Formula (I), Formula (II), and Formula (III) below:

[0174] 5’ [Gene or Portion Thereof] -[First Region] -[Second Region] 3’

[0175] Formula (I)

[0176] 5’ [First Region] -[Second Region] -[Gene or Portion Thereof] 3’

[0177] Formula (II)

[0178] 5’ [First Region] -[Target Region Comprising a Gene or Portion Thereof] -[Second Region] 3’

[0179] Formula (III)

[0180] In some embodiments, a gene, a portion of a gene, and / or one or more regulatory sequences are in the first region and / or the second region in Formulas (I)-(III). In some embodiments, the one or more regulatory sequences comprise at least one regulatory sequence (e.g., a native promoter or a mutant thereof) that is operably linked to a gene or a portion thereof 10 in the DNA target. In some embodiments, the one or more regulatory sequences comprise at least one regulatory sequence that is not operably linked to a gene or a portion thereof in the DNA target (e.g., a start codon or a stop codon that is out of frame with the gene, a mutated splicing regulatory sequence, etc.).

[0181] In some embodiments, a DNA target comprising the components set forth in Formula (I) or Formula (II) comprises an insertion site that is 1-10,000 nucleotides or more away from a gene or a portion thereof in the DNA target. In some embodiments, a DNA target comprises the components set forth in Formula (I) or Formula (II) and an insertion site that is 1-100 nucleotides, 100-200 nucleotides, 200-500 nucleotides, 500-1,000 nucleotides, 1,000-2,500 nucleotides, 2,500-5,000 nucleotides, 5,000-10,000 nucleotides, or more than 10,000 nucleotides (e.g., 5,000-10,000, 10,000-20,000, or 20,000-30,000 nucleotides) away from a gene or a portion thereof in the DNA target. In some embodiments, when a single-stranded template is used to edit a DNA target comprising the components set forth in Formula (I) or Formula (II), homology

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[0183] -20- arms and a gRNA are designed to hybridize to the strand that does not comprise a gene or a portion thereof that comprises an insertion site.

[0184] In some embodiments, a DNA target comprising the components set forth in Formula (III) comprises an insertion site that is 1-1,000 nucleotides or more (e.g., 1,000-1,250, 1,250- 1,500, 1,500-1,750, 1,750-2,000, 2,000-2,500, or more than 2,500 nucleotides) away from the first region and / or the second region. In some embodiments, a DNA target comprises the components set forth in Formula (III) and an insertion site that is 5-10 nucleotides, 10-20 nucleotides, 20-30 nucleotides, 30-40 nucleotides, 40-50 nucleotides, 50-60 nucleotides, 60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides, 90-100 nucleotides, 125-150 nucleotides, 150-200 nucleotides, or 200-500 nucleotides away from the first region and / or the second region. In some embodiments, when a single-stranded template is used to edit a DNA target comprising the components set forth in Formula (III) and a gRNA hybridizes to a sequence that is downstream relative to an insertion site, homology arms are designed to hybridize to the strand that comprises a gene or a portion thereof. In some embodiments, when a single- stranded template is used to edit a DNA target comprising the components set forth in Formula (III) and a gRNA hybridizes to a sequence that is upstream relative to an insertion site, homology arms are designed to hybridize to the strand that does not comprise a gene or a portion thereof.

[0185] Further to the non-limiting embodiments of DNA templates and corresponding edited DNA targets described herein (see, e.g., FIGs. 1A-1E, FIGs. 3A-3D, FIGs. 4A-4B, and FIG. 21), a template nucleic acid can comprise the components set forth in Formula (IV) below:

[0186] [First Homology Arm] -[Heterologous Nucleic Acid] -[Second Homology Arm]

[0187] Formula (IV)

[0188] In this non-limiting example, a template is configured to integrate a heterologous nucleic acid between the first and second regions to produce an edited DNA target corresponding to the DNA targets of Formula (I), Formula (II), and Formula (III) and comprising the components set forth in Formula (V), Formula (VI), and Formula (VII), respectively, below:

[0189] 5’ [Gene or Portion Thereof] -[First Region] -[Heterologous Nucleic Acid] -[Second Region] 3’

[0190] Formula (V)

[0191] 5’ [First Region] -[Heterologous Nucleic Acid] -[Second Region] -[Gene or Portion Thereof] 3’

[0192] Formula (VI)

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[0194] -21-

[0195] 5’ [First Region] -[Heterologous Nucleic Acid] -[Second Region] 3’

[0196] Formula (VII)

[0197] In some embodiments, a template comprises at least one regulatory sequence in a heterologous nucleic acid that is configured to be operably linked to a gene or a portion thereof in an edited DNA target. As non-limiting examples, an edited DNA target can comprise the components set forth in any one of Formulas (VIII)-(XII) below:

[0198] 5’ [Gene or Portion Thereof] -[First Region] -[At Least One Heterologous Regulatory Sequence] - [Second Region] 3’

[0199] Formula (VIII)

[0200] 5’ [First Region] -[At Least One Heterologous Regulatory Sequence] -[Second Region] -[Gene or Portion Thereof]- 3’

[0201] Formula (IX)

[0202] 5’ [First Region] -[At Least One Heterologous Regulatory Sequence] -[Gene or Portion Thereof] - [Second Region] 3’

[0203] Formula (X)

[0204] 5’ [First Region] -[Gene or Portion Thereof] -[At Least One Heterologous Regulatory Sequence] - [Second Region] 3’

[0205] Formula (XI)

[0206] 5’ [First Region] -[At Least One Heterologous Regulatory Sequence] -[Gene or Portion Thereof] - [At Least One Heterologous Regulatory Sequence] -[Second Region] 3’

[0207] Formula (XII)

[0208] 5 Editing a DNA target can comprise introducing at least one site of DNA damage in a

[0209] DNA target. Non-limiting examples of DNA damage include DNA alkylation, base deamination, base depurination, incidence of abasic sites, single- stranded breaks (SSBs), and double-stranded breaks (DSBs). Various conditions can induce sites of DNA damage, including, but not limited to, subjecting DNA to chemical agents (e.g., alkylating agents, DSB-inducers,

[0210] 10 SSB-inducers, etc.), ionizing radiation, oxidative damage, and treatment with nucleases (e.g., zinc-finger nucleases, transcription-activator like effector nucleases (TALENs), and RNA- guided nucleases).

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[0212] -22-

[0213] In some embodiments, introducing a template nucleic acid in the presence of DNA damage can promote HDR to use the template as a substrate for the repair of the damaged DNA. In some embodiments, introducing a template nucleic acid promotes HDR to repair the damaged DNA as opposed to other DNA repair mechanisms, such as non-homologous end-joining

[0214] 5 (NHEJ) or microhomology-mediated end joining (MMEJ), and / or promotes HDR to use the template as a substrate as opposed to a different homologous sequence (e.g., a sister chromatid).

[0215] DNA breaks (e.g., SSBs and / or DSBs) in a DNA target can be generated either at an insertion site or at a position that is proximal to an insertion site. As used herein, “a DNA break at a position that is proximal to a target position” refers to a site of a DNA break that is within

[0216] 10 2,000 nucleotides upstream or downstream of the insertion site. In some embodiments, a DNA break at a position that is proximal to an insertion site is 500 nucleotides or less upstream or downstream (e.g., 1-10, 10-20, 20-30, 30-40, 60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-175, 175-200, 200-250, or 250-500 nucleotides). In some embodiments, a DNA break is generated in a first region and / or a second region of a DNA target.

[0217] In some embodiments, a DNA break is generated downstream of a start codon of a gene in a DNA target. In some embodiments, the DNA break can be used to promote integration of a heterologous sequence that is 5’ relative to a sequence encoding an RNA or a portion thereof in the gene (e.g., to edit an N-terminal portion of a peptide or protein encoded by the DNA target).

[0218] In some embodiments, a DNA break is generated upstream of a stop codon of a gene in a DNA target. In some embodiments, the DNA break can be used to integrate a heterologous sequence that is 3’ relative to a sequence encoding an RNA or a portion thereof in the gene (e.g., to edit a C-terminal portion of a peptide or protein encoded by the DNA target).

[0219] In some embodiments, a DNA break is generated at an insertion site, or at a position proximal to an insertion site, in a sequence that binds by a zinc-finger nuclease or a TALEN.

[0220] 25 In some embodiments, a DNA break is generated at an insertion site, or at a position proximal to an insertion site, in a sequence that binds by a gRNA.

[0221] In some embodiments, introducing a DNA break in a DNA target comprises contacting an RNA-guided nuclease and a gRNA with the DNA target. In some embodiments, a template nucleic acid can be used to edit a DNA target in combination with an RNA-guided nuclease and a gRNA.

[0222] CRISPR / Cas Systems

[0223] CRISPR is a family of DNA sequences (CRISPR clusters) in bacteria and archaea comprising nucleic acid fragments corresponding to prior infections by a virus. The nucleic acid

[0224] 35 fragments are used by the prokaryotic cell to detect and destroy DNA from subsequent attacks

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[0226] -23- by similar viruses. In nature, CRISPR clusters are transcribed and processed into crRNA. In certain types of CRISPR systems (e.g., type II CRISPR systems), correct processing of pre- CRISPR RNA (pre-crRNA) involves a tracrRNA, endogenous ribonuclease 3 (me), and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA.

[0227] 5 Subsequently, Cas9 / crRNA / tracrRNA cleaves a linear or circular dsDNA target complementary to the RNA. However, sgRNAs can be engineered to incorporate aspects of both the crRNA and tracrRNA into a single RNA species (e.g., a single-guide RNA (sgRNA)).

[0228] In some embodiments, an RNA-guided nuclease is a Cas nuclease or a variant thereof. Non-limiting examples of Cas nucleases include Cas9 nucleases and variants thereof, Cas 12

[0229] 10 nucleases and variants thereof, Cas 13 nucleases and variants thereof, and Cas 14 nucleases and variants thereof. In some embodiments, a Cas nuclease is a Cas9 nuclease or a variant thereof (e.g., SpCas9, SaCas9, StCas9, NmCas9, CjCas9, SpyCas9, etc.). In some embodiments, a Cas nuclease is a Cas 12a nuclease or a variant thereof (e.g., AsCasl2a, FnCasl2a, LbCasl2a, PaCasl2a, the MAD7™ system (MAD7™, Inscripta, Inc.), the Alt-R Casl2a (Cpfl) Ultra nuclease (Alt-R® Cas 12a Ultra; Integrated DNA Technologies, Inc.). In some embodiments, a Cas nuclease is a Cas 12b nuclease or a variant thereof. In some embodiments, a Cas nuclease is a Casl3 or Casl4.

[0230] In some embodiments, an RNA-guided nuclease comprises endonuclease activity that cleaves both strands of the DNA target, thereby introducing at least one DSB. However, in some

[0231] 20 embodiments, an RNA-guided nuclease is used to generate at least one SSB in a DNA target. In some embodiments, an RNA-guided nuclease that generates a SSB in a DNA target comprises a Cas enzyme with nickase activity. In some embodiments, at least one DNA break is induced in a region of a DNA target that can base-pair with a homology arm in a template. In some embodiments, a plurality of DNA breaks is generated in a DNA target. In some embodiments, the plurality of DNA breaks comprises two DSBs, two SSBs, or one DSB and one SSB. In some embodiments, the plurality of DNA breaks comprises a first DNA break and a second DNA break which flank an insertion site. In some embodiments, each DNA break in the plurality is generated using a respective gRNA. In some embodiments, introducing more than one DNA break comprises using two different RNA-guided nucleases, wherein each RNA-guided nuclease

[0232] 30 binds to a different gRNA and / or recognizes a different PAM.

[0233] In some embodiments, a PAM in a DNA target comprises 3-10 nucleotides in length. Non-limiting examples of PAM sequences that can be located in a DNA target include: NGG; NNAGAAW; NNGRRT; NNNNGATT; NVNDCCY; BRTTTTT; NR(A or G)TTTT; NNAAAR(G or A); N(N or A)G; NAAN; NAAAAY; NHDTCCA; NNNVRYM; NNNNRYAC; NAA; GNNNNCNNA; NNGTGA; NNNNGTA; NNGGG; NNNCAT;

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[0235] -24-

[0236] NNRHHHY; NRRNAT; NNNNCNAA; NNNNCMCA; NNNNCRAA; NNNNGMAA; NNNCC; NGGNG; NNNNCNDD; NYAAA; NRGNN; N(C or D)GGN(T or A or G or C)NN; NRTAW; N(C or K or A)AARC; NAAAG; NV(A or G or C)R(A or G)ACCN; NNGAC; NATGNT; N(T or V)NTAAW(A or T); NNGW(A or T)AY(T or C); NCAA(H(Y or A)B(Y or G); NH(T or C or A)AAAA; NNNATTT; NATAWN(A or T or S); NATARCH; B(T or G or C)GGD(A or T or G)TNN; N(G or T or M)GGAH(T or A or C)N(A or C or K)N; NRG; N(B or A)GG; NGGD(A or K)W(T or A); N(T or C or R)AGAN(A or K or QNN; NGGD(A or T or G)H(T or M); NGGDT; NGGD(A or T or G)GNN; NNGTAM(A or C)Y; NNGH(W or QAAA; NTGAR(G or A)N(A or Y or G)N(Y or R); NNGAAAN; NNGAD; NHARMC; NNAAAG; NHGYNAN(A or B); NNAGAAA; NHAAAAA; NH(T or M)AAAAA; NHGYRAA; NNAAACN; NN(H or G)D(A or K)GGDN(A or B); NNNNCTA; NNNNCVGAA; NNNNGYAA; NNNNATN(W or S)ANN; NNWHR(G or A)TA(not G)AA; YHHNGTH;

[0237] NNNNCDAANN; NNNNCTAA; N(C or D)NNTCCN; NNNNCCAA; NAGRGN(T or V)N(T or C); NNAH(T or M)ACN; CN(C or W or G)AV(A or S)GAC; NAR(G or A)H(W or C)H(A or T or C)GN(C or T or R); NAGNGC; NATCCTN; NGTGANN; HGCNGCR; NAR(A or G)W(T or A)AC; N(C or D)M(A or C)RN(A or B)AY(C or T); NNNCAC; BGGGTCD; NNRRCC; NRRNTT; KARDAT; BRRTTTW; NARNCCN; NAR(A or G)TC; NAAN(A or T or S)RCN; HHAAATD; NNNNGNA; TTV; TTTV; YYV; KKYV; TTTM; TTYV; TTTN; TTTTA; TTN; BTTV; YTV; YTN; NYTV; DTTD; ATTN; RTTNT; HATT; ATTW; RTTN; TVT; TG; TN; TR; TA; TTCN; TTAT; TTTR; TTR; YTTR; YTTN; CTT; TTC; CCD; RTR; VTTR; TBN; VTTN; NGTT; CGTT; AGG; CGG; GTT; or RGTG, wherein: “N” is any nucleotide or base; “W” is adenine (A) or thymine (T); “R” is A or guanine (G); “V” is A, cytosine (C), or G; “Y” is C or T; “M” is C or A; “K” is G or T; “D” is A, G, or T; “S” is G or C; “B” is C, G, or T; and “H” is A, C, or T.

[0238] In some embodiments, an RNA-guided nuclease will cut a DNA target at a position that is at least one nucleotide upstream or one nucleotide downstream of a PAM that the RNA- guided nuclease is bound to. In some embodiments, a PAM selected for editing a DNA target is at a position that is at least one nucleotide upstream or downstream of the insertion site. In some embodiments, a PAM is at a position in a DNA target that is separated from an insertion site by a plurality of nucleotides (e.g., 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, etc.). In some embodiments, the plurality of nucleotides comprises at least 10 nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 10-15, 15-20, 20-25, 25-35, 35-45, 45-55, 55-65, 65-75, 75-85, 85-95, 1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700 700-800,

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[0240] -25-

[0241] 800-900, 900-1,000, or more than 1,000 nucleotides, such as 1,000-2,500, 2,500-5,000, or more than 5,000 nucleotides, such as 5,000-10,000, 10,000-20,000, or 20,000-30,000 nucleotides).

[0242] In some embodiments, a gRNA comprises a sequence that hybridizes with a sequence in a DNA target. In some embodiments, a sequence in a gRNA that hybridizes with a sequence in a DNA target comprises no more than 40 nucleotides. In some embodiments, a sequence in a gRNA that hybridizes with a DNA target comprises 10-35 nucleotides, such as 15, 16, 17, 18,

[0243] 5 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0244] In some embodiments, a sequence in a gRNA that hybridizes with a DNA target comprises at least 15 nucleotides that are 100% identical to a sequence in a DNA target, such as 15-18, 18-20, 20-22, or 22-24 nucleotides that are 100% identical to a sequence in a DNA target. Sequence identity, including determination of sequence complementarity for nucleic acid

[0245] 10 sequences, may be determined by sequence comparison and alignment algorithms known in the art (e.g., in BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), COBALT, OPAL, Multlin, Clustal Omega, Clustal W2.0, or Clustal X2.0 software). To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. In some embodiments, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., % homology=# of identical positions / total # of positionsxlOO), optionally

[0246] 20 penalizing the score for the number of gaps introduced and / or length of gaps introduced.

[0247] However, in some embodiments, a sequence in a gRNA that hybridizes with a DNA target comprises one or more mismatches relative to the sequence in the DNA target that it hybridizes to.

[0248] An RNA guided-nuclease and / or the gRNA can be encoded on the sample nucleic acid or a separate nucleic acid, such as by being transformed or transfected with a vector comprising sequences encoding the RNA-guided nuclease and / or the gRNA. The nucleic acid encoding an RNA-guided nuclease, or a gRNA can comprise DNA, RNA, a DNA-RNA hybrid, or be a modified nucleic acid, such as a chemically modified nucleic acid. Alternatively, an RNA- guided nuclease and / or a gRNA can be introduced into a cell as a pre-formed complex

[0249] 30 comprising the RNA-guided nuclease and the gRNA.

[0250] In some embodiments, a gRNA comprises one or more chemical modifications. In some embodiments, the one or more chemical modifications comprises chemically modified nucleosides and / or sugar-phosphate backbone chemical modifications. In some embodiments,

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[0252] -26- the one or more chemical modifications in a gRNA comprises a phosphorothioate moiety, a 2’- O-Me-modification, a 2’F-modification, and / or a thioPACE moiety. In some embodiments, the one or more chemical modifications comprises modified nucleotides at the 3’ end and / or the 5’ of the gRNA and / or modified nucleotides that are located internally (e.g., in the sequence that binds the DNA target).

[0253] Template Nucleic Acids

[0254] A template nucleic acid can comprise DNA, RNA, or any combination of DNA and RNA nucleotides. In some embodiments, a template nucleic acid comprises 15,000 nucleotides

[0255] 5 or less in length. In some embodiments, a template comprises 5,000 nucleotides or less (e.g., 20- 30, 30-40, 25-50, 40-50, 50-75, 75-100, 50-100, 100-150, 100-200,150-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1,000, 1,000-1,500, 1,500-2,000, 2,000- 2,500, 2,500-3,000, 3,000-3,500, 3,500-4,000, 4,000-4,500, or 4,500-5,000 nucleotides) in length. A nucleic acid comprising a template can comprise a double-stranded nucleic acid, a

[0256] 10 single- stranded nucleic acid, or one or more stretches of double- stranded sequence and one or more stretches of single- stranded sequence. In some embodiments, when a template comprising single- stranded sequences are used to edit a DNA target, the single- stranded template comprises fewer nucleotides relative to a double-stranded template that can be used to introduce the same edit into a DNA target. However, in some embodiments, a template nucleic acid comprises more than 15,000 nucleotides (e.g., 15,000-20,000, 20,000-25,000, 25,000-30,000, or more than 30,000 nucleotides or more) in length.

[0257] In some embodiments, a nucleic acid is linear. In some embodiments, a nucleic acid is circular. In some embodiments, a nucleic acid comprises one or more single- stranded nicks. In some embodiments, a nucleic acid comprises a vector, such as a plasmid (e.g., a circular

[0258] 20 plasmid, a nanoplasmid, or a minicircle plasmid), a cosmid, or an artificial chromosome. In some embodiments, a nucleic acid comprising a template is a self-cleaving nucleic acid (e.g., a nucleic acid comprising one or more sites that bind a nuclease including, but not limited to, selfcleaving nucleic acid that comprises a sequence encoding the nuclease) which is circular prior to liberating the template as a linear template nucleic acid.

[0259] In some embodiments, a nucleic acid comprising a template comprises one or more chemical modifications. Non-limiting examples of chemical modifications include chemically modified nucleosides and sugar-phosphate backbone chemical modifications. In some embodiments, a nucleic acid comprises one or more nucleotides comprising a chemically modified sugar, a chemically modified nucleobase, and / or a chemically modified phosphate

[0260] 30 group. In some embodiments, a nucleic acid comprises a phosphate analog. In some

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[0262] -27- embodiments, a nucleic acid comprises one or more chemically modified internucleotide linkages. In some embodiments, chemical modifications in a nucleic acid are in homology arm and / or a heterologous nucleic acid. In some embodiments, chemical modifications in a nucleic acid can be used to improve cellular uptake, improve stability, reduce immunogenicity, improve 5 potency, improve target hybridization, and / or reduce susceptibility to cleavage by endogenous nucleases of a template.

[0263] In some embodiments, a template is operably linked to at least one regulatory sequence in a nucleic acid. The at least one regulatory sequence can comprise 1, 2, 3, 4, or more than 4 regulatory sequences. The at least one regulatory sequence can comprise regulatory sequences capable of promoting transcription (e.g., any one or more of a promoter, an enhancer, a transcription factor binding sequence, a transcriptional start sequence, etc.) and / or terminating transcription (e.g., any one or more of a transcription termination sequence, etc.). In some embodiments, one or more regulatory sequences are operably linked to a sequence comprising a first homology arm, a heterologous nucleic acid, and a second homology arm. In some embodiments, regulatory sequences can be used to control stability, expression, and / or degradation of a template in a cell. In some embodiments, a template is operably linked to a promoter. In some embodiments, a promoter is operably linked to a template for the purposes of expressing the template as an RNA in a cell. In some embodiments, a promoter operably linked to a template is one that is active in a cell, or a cell of a similar cell type, that comprises a DNA target to which the homology arms hybridize. In some embodiments, a promoter operably linked to a template is an inducible promoter, such as a small-molecule inducible promoter.

[0264] Homology Arms

[0265] In some embodiments, a homology arm comprises 2,000 nucleotides or less in length. In some embodiments, a homology arm comprises 1,000 nucleotides or less in length. In some

[0266] 10 embodiments, a homology arm comprises 500 nucleotides or less (e.g., 10-20, 20-30, 30-40, 40- 50, 50-60, 60-70, 70-80, 80-100, 25-100, 100-250, 100-125, 125-150, 150-200, 200-500, 200- 250, 250-300, 300-350, 350-400, 400-450, or 450-500 nucleotides) in length.

[0267] In some embodiments, a homology arm comprises homology to a region of a DNA target comprising a PAM.

[0268] In some embodiments, a homology arm comprises a PAM. In some embodiments, a PAM in a homology arm comprises a sequence which is 3-10 nucleotides in length. In some embodiments, a PAM in a homology arm comprises a sequence of any one of: NGG; NNAGAAW; NNGRRT; NNNNGATT; NVNDCCY; BRTTTTT; NR(A or G)TTTT; NNAAAR(G or A); N(N or A)G; NAAN; NAAAAY; NHDTCCA; NNNVRYM;

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[0270] -28-

[0271] NNNNRYAC; NAA; GNNNNCNNA; NNGTGA; NNNNGTA; NNGGG; NNNCAT; NNRHHHY; NRRNAT; NNNNCNAA; NNNNCMCA; NNNNCRAA; NNNNGMAA; NNNCC; NGGNG; NNNNCNDD; NYAAA; NRGNN; N(C or D)GGN(T or A or G or C)NN; NRTAW; N(C or K or A)AARC; NAAAG; NV(A or G or C)R(A or G)ACCN; NNGAC; NATGNT; N(T or V)NTAAW(A or T); NNGW(A or T)AY(T or C); NCAA(H(Y or A)B(Y or G); NH(T or C or A)AAAA; NNNATTT; NATAWN(A or T or S); NATARCH; B(T or G or C)GGD(A or T or G)TNN; N(G or T or M)GGAH(T or A or C)N(A or C or K)N; NRG; N(B or A)GG; NGGD(A or K)W(T or A); N(T or C or R)AGAN(A or K or QNN; NGGD(A or T or G)H(T or M); NGGDT; NGGD(A or T or G)GNN; NNGTAM(A or C)Y; NNGH(W or QAAA; NTGAR(G or A)N(A or Y or G)N(Y or R); NNGAAAN; NNGAD; NHARMC; NNAAAG; NHGYNAN(A or B); NNAGAAA; NHAAAAA; NH(T or M)AAAAA; NHGYRAA; NNAAACN; NN(H or G)D(A or K)GGDN(A or B); NNNNCTA; NNNNCVGAA; NNNNGYAA; NNNNATN(W or S)ANN; NNWHR(G or A)TA(not G)AA; YHHNGTH; NNNNCDAANN; NNNNCTAA; N(C or D)NNTCCN; NNNNCCAA; NAGRGN(T or V)N(T or C); NNAH(T or M)ACN; CN(C or W or G)AV(A or S)GAC; NAR(G or A)H(W or C)H(A or T or C)GN(C or T or R); NAGNGC; NATCCTN; NGTGANN; HGCNGCR; NAR(A or G)W(T or A)AC; N(C or D)M(A or C)RN(A or B)AY(C or T); NNNCAC; BGGGTCD; NNRRCC; NRRNTT; KARDAT; BRRTTTW; NARNCCN; NAR(A or G)TC; NAAN(A or T or S)RCN; HHAAATD; NNNNGNA; TTV; TTTV; YYV; KKYV; TTTM; TTYV; TTTN; TTTTA; TTN; BTTV; YTV; YTN; NYTV; DTTD; ATTN; RTTNT; HATT; ATTW; RTTN; TVT; TG; TN; TR; TA; TTCN; TTAT; TTTR; TTR; YTTR; YTTN; CTT; TTC; CCD; RTR; VTTR; TBN; VTTN; NGTT; CGTT; AGG; CGG; GTT; or RGTG, wherein: “N” is any nucleotide or base; “W” is adenine (A) or thymine (T); “R” is A or guanine (G); “V” is A, cytosine (C), or G; “Y” is C or T; “M” is C or A; “K” is G or T; “D” is A, G, or T; “S” is G or C; “B” is C, G, or T; and “H” is A, C, or T. In some embodiments, a PAM binds to an RNA-guided nuclease. In some embodiments, a PAM binds to a CRISPR / Cas nuclease or a variant thereof. In some embodiments, a PAM binds to a Cas9 nuclease or a variant thereof, a Casl2 nuclease or a variant thereof, a Casl3 nuclease or a variant thereof, or a Casl4 nuclease or a variant thereof.

[0272] However, in some embodiments, a homology arm is designed not to comprise a PAM. In some embodiments, a homology arm does not comprise a PAM which is located in a DNA target selected for editing and / or does not comprise a PAM that binds an RNA-guided nuclease that is selected for editing.

[0273] In some embodiments, a homology arm comprises homology to a region of a DNA target that comprises a sequence that hybridizes to a gRNA. However, in some embodiments, a homology arm comprises homology to a sequence proximal to a sequence that hybridizes to a

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[0275] -29- gRNA. A “sequence proximal to a sequence capable of hybridizing to a gRNA” refers to sequences that are approximately 1-1,000 nucleotides or more nucleotides upstream or downstream of the sequence in a DNA target that is capable of hybridizing to the gRNA. In some embodiments, a sequence proximal to a sequence capable of hybridizing to a gRNA in a DNA target are 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-100, 25-100, 100- 250, 100-125, 125-150, 150-200, 200-500, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 nucleotides upstream or downstream of the sequence that hybridizes to the gRNA.

[0276] In some embodiments, a first homology arm and a second homology arm comprise an equal number of nucleotides. In other embodiments, a first homology arm and a second homology arm comprise a nonequal number of nucleotides. In some embodiments, the lengths of the first and second homology arms may differ by approximately 1-5, 5-10, 10-20, 20-30, 30-

[0277] 5 40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, 275-300, 300-325, 325-350, 350-375, 375-400, 400-425, 425-450, 450-475, 475-500, or more nucleotides in length. In some embodiments, the homology arm that is longest comprises homology to a sequence in a DNA target comprising a PAM. In some embodiments, when a single- stranded template is used, a homology arm is designed to hybridize to the same

[0278] 10 strand as a gRNA (e.g., to prevent hybridization of the gRNA with a homology arm).

[0279] In some embodiments, a homology arm comprises one or more stretches of sequence that are identical to a region of a DNA target and / or one or more stretches of sequence that differ by at least one nucleotide from a region of the DNA target that the homology arm hybridizes to. In some embodiments, the one or more stretches of sequence that are identical to the region of the DNA target can comprise 2-20, 20-50, 50-100, 100-200, 200-500, or more than 500 nucleotides in length. In some embodiments, the one or more stretches of sequence that differ from the region of the DNA target comprises one or more insertions, one or more deletions, and / or one or more substitutions relative to the region. In some embodiments, the one or more stretches of sequence that differ from the region of the DNA target is positioned closer to the end of the

[0280] 20 homology arm that is closest to a heterologous nucleic acid in the template (e.g., 200 nucleotides or less away of the heterologous nucleic acid, such as 100-200 nucleotides, 50-100 nucleotides, 50-25 nucleotides, 15-25 nucleotides, 10-15 nucleotides, 5-10 nucleotides, 4 nucleotides, 3 nucleotides, 2 nucleotides, or 1 nucleotide away from the heterologous nucleic acid).

[0281] In some embodiments, a homology arm comprises one or more stretches of sequence that

[0282] 25 are identical to a region of a DNA target and / or one or more stretches of sequence that differ by at least one nucleotide from a region of the DNA target, wherein the at least one nucleotide differing from the region in the DNA target comprises a heterologous PAM. In some embodiments, a heterologous PAM binds to an RNA-guided nuclease that does not bind to a

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[0284] -30-

[0285] PAM in a DNA target. In some embodiments, the at least one nucleotide differing from the region in the DNA target comprises a sequence configured to mutate a PAM in a DNA target, such as to disable a PAM from binding an RNA-guided nuclease.

[0286] In some embodiments, a first homology arm comprises 100% sequence identity to a first region of a DNA target and a second homology arm comprises the at least one nucleotide which differs from a second region of the DNA target. In some embodiments, wherein a first homology arm and a second homology arm comprise a non-equal number of nucleotides, the longer homology arm comprises the at least one nucleotide differing from the region in the DNA target. However, in other embodiments, a first homology arm comprises 100% sequence identity to a first region of a DNA target and a second homology arm comprises 100% sequence identity to a second region of a DNA target. Sequence identity, including determination of sequence complementarity for nucleic acid sequences, may be determined by sequence comparison and alignment algorithms known in the art (e.g., in BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), COBALT, OPAL, Multlin, Clustal Omega, Clustal W2.0, or Clustal X2.0 software). To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. In some embodiments, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., % homology=# of identical positions / total # of positionsxlOO), optionally penalizing the score for the number of gaps introduced and / or length of gaps introduced.

[0287] In some embodiments, a homology arm comprises homology to a region of a DNA target comprising a sequence that binds a zinc-finger nuclease or a TALEN. In some embodiments, a homology arm comprises homology to a sequence proximal to a sequence that binds a zinc- finger nuclease or a TALEN. A “sequence proximal to a sequence that binds a zinc-finger nuclease or a TALEN” refers to sequences that are approximately 1-1,000 nucleotides or more nucleotides upstream or downstream of the sequence in a DNA target that binds to the zinc- finger nuclease or the TALEN. In some embodiments, a sequence proximal to a sequence in a DNA target that binds to a zinc-finger nuclease or a TALEN is 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-100, 25-100, 100-250, 100-125, 125-150, 150-200, 200-500, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 nucleotides upstream or downstream of the sequence that binds to the zinc-finger nuclease or the TALEN.

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[0289] -31-

[0290] Heterologous Nucleic Acids

[0291] In some embodiments, a heterologous nucleic acid comprises 10,000 nucleotides or less in length. In some embodiments, a heterologous nucleic acid comprises 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 25-50, 50-60, 55-65, 50-75, 60-70, 65-75, 70-80, 75-85, 80-

[0292] 5 90, 85-95, 95-100, 75-100, 100-400, 400-800, 800-1000, 900-1000, 950-1000, 1000-1300, 1100-1400, 1200-1500, 1300-1600, 1400-1700, 1500-1800, 1600-1900, 1700-2000, 1800-2100, 1900-2200, 2000-2500, 2100-2600, 2200-2700, 2300-2800, 2400-2900, or 2500-3000 nucleotides in length. In some embodiments, a heterologous nucleic acid comprises 1,000- 10,000 nucleotides in length. In some embodiments, a heterologous nucleic acid comprises

[0293] 10 1,000 nucleotides or less in length. In some embodiments, a heterologous nucleic acid comprises 500 nucleotides or less in length. In some embodiments, a heterologous nucleic acid comprises 100 nucleotides or less in length. However, in some embodiments, a template nucleic acid comprises more than 10,000 nucleotides (e.g., 10,000-15,000, 15,000-20,000, 20,000-25,000, 25,000-30,000, or more than 30,000 nucleotides or more) in length.

[0294] Variant Sequences in Heterologous Nucleic Acids

[0295] Variant sequences of the application can comprise one or more synonymous mutations relative to a corresponding counterpart sequence in a DNA target. In some embodiments, a variant sequence encodes an RNA which comprises one or more synonymous mutations relative to a sequence in a DNA target which encodes an RNA. In some embodiments, an RNA encoded by a variant sequence in a heterologous nucleic acid is an mRNA encoding a peptide or a protein. In some embodiments, a variant sequence comprises one or more non-expressed sequences (e.g., regulatory sequences, intergenic sequences, etc.) and one or more expressed sequences (e.g., sequences encoding RNAs, such as those that encode a peptide or protein).

[0296] 25 In some embodiments, a variant sequence comprises a nucleotide sequence that is at least 1% identical to a corresponding gene or a portion thereof. In some embodiments, a variant sequence comprises a nucleotide sequence that is greater than 1% identical to a corresponding gene or a portion thereof, such as 2%, 3%, 4%, 5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, or 95-99% identical to the corresponding gene or the portion thereof. In some embodiments, a variant sequence comprises a nucleotide sequence that is 100% identical to a corresponding gene or a portion thereof and one or more stretches of sequence that differ by at least one nucleotide relative to a sequence in a DNA target (e.g., wherein the one or more stretches of sequence comprise a nucleotide sequence that is at least 1% identical to the sequence in the DNA target). In some embodiments, a variant sequence

[0297] 35 comprises one or more heterologous codons (e.g., codons comprising substitutions, such as

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[0299] -32- conservative and non-conservative substitutions, and / or inserted codons) relative to a corresponding gene or a portion thereof.

[0300] Sequence identity, including determination of sequence complementarity for nucleic acid sequences, may be determined by sequence comparison and alignment algorithms known in the

[0301] 5 art (e.g., in BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), COBALT, OPAL, Multlin, Clustal Omega, Clustal W2.0, or Clustal X2.0 software). To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides at corresponding nucleotide positions are then compared. When a position in the

[0302] 10 first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. In some embodiments, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., % homology=# of identical positions / total # of positionsxlOO), optionally penalizing the score for the number of gaps introduced and / or length of gaps introduced.

[0303] In some embodiments, a variant sequence comprises a first and a second nucleotide sequence (e.g., wherein the first nucleotide sequence in the variant sequence corresponds to a first portion of a gene and the second nucleotide sequence corresponds to a second portion of the gene) and the first and the second nucleotide sequences have different levels of percent identity to a corresponding gene or a portion thereof. In some embodiments, the second nucleotide

[0304] 20 sequence has a greater percent identity to a corresponding gene or a portion thereof relative to the percent identity that the first nucleotide sequence has relative to the corresponding gene or a portion thereof (e.g., wherein the second nucleotide sequence has a greater percent identity to a corresponding second portion of a gene than the first nucleotide sequence has to a corresponding first portion of the gene). In some embodiments, the second nucleotide sequence has at least 1% (e.g., at least 1-20%, 1%, 2%, 3%, 4%, 5%, 5-10%, or 10-20%) more identity to a corresponding gene or a portion thereof than the first nucleotide sequence has to the corresponding gene or a portion thereof (e.g., wherein the second nucleotide sequence has at least 1-20% more identity to a corresponding second portion of the gene as compared to the percent identity that the first nucleotide sequence has to a corresponding first portion of the gene). In some embodiments, the

[0305] 30 first and second nucleotide sequences are equal in length. In other embodiments, the first nucleotide sequence is longer than the second nucleotide sequence (e.g., wherein the first nucleotide sequence corresponds to a sequence that is closer to a PAM, a gRNA cut site, and / or a insertion site in the DNA target relative to the sequence that the second nucleotide sequence corresponds to in the DNA target).

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[0308] In some embodiments, a variant sequence comprises synonymous mutations and / or non- synonymous mutations relative to a corresponding gene or portion thereof. Generally, nucleotide positions in a variant sequence which comprise synonymous mutations will encode an amino acid sequence that is identical to the amino acid sequence encoded by the sequence in the DNA

[0309] 5 target which corresponds to the variant sequence. In some embodiments, synonymous mutations in a variant sequence are comprised in heterologous codons that have similar or closely matching usage frequency in a cell type that comprises a DNA target. In some embodiments, a variant sequence comprises or consists of a codon optimized sequence (e.g., a sequence that codon-optimized for expression in a mammalian cell, which includes, but is not limited to, a

[0310] 10 human cell). In some embodiments, a variant sequence comprises a silently mutated sequence, wherein the silently mutated sequence comprises one or more synonymous mutations relative to a corresponding gene or portion thereof. In some embodiments, each codon in a variant sequence comprises a synonymous mutation or a non-synonymous mutation relative to a corresponding gene or portion thereof. Non-synonymous mutations include nucleotide insertions, nucleotide deletions, and nucleotide substitutions that change the amino acid sequence encoded by a polynucleotide. A non-synonymous mutation in a variant sequence can comprise a nucleotide insertion, a nucleotide deletion, or a nucleotide substitution that results encodes an amino acid sequence comprising a conservative mutation or a non-conservative mutation. In some embodiments, a conservative mutation comprises an amino acid substitution. In some embodiments, a conservative amino acid substitution comprises a first amino acid being substituted for a second amino acid having similar side chain size and side chain chemistry (e.g., a substitution of glycine for alanine or a substitution of aspartate for glutamate). In some embodiments, a non-conservative mutation comprises an amino acid deletion. In some embodiments, a non-conservative mutation comprises an amino acid insertion. In some

[0311] 25 embodiments, a non-conservative mutation comprises an amino acid substitution. In some embodiments, a non-conservative substitution comprises a first amino acid being substituted for a second amino acid having significantly different side chain size and / or side chain chemistry (e.g., a substitution of tryptophan for lysine or a substitution of alanine for arginine). In some embodiments, a variant sequence encodes an amino acid sequence that is 1-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-99% identical to a corresponding amino acid sequence. In some embodiments, a variant sequence encodes an amino acid sequence that is 100% identical to a corresponding amino acid sequence, wherein the amino acid sequence encoded by the variant sequence comprises one or more synonymous mutations relative to the corresponding sequence in the DNA. In some

[0312] 35 embodiments, a variant sequence comprises a silently mutated sequence, wherein the silently

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[0314] -34- mutated sequence encodes an amino acid sequence that is 100% identical to a corresponding amino acid sequence encoded by the corresponding gene or a portion thereof.

[0315] In some embodiments, producing an edited DNA target comprising the components of Formula (V) or Formula (VI) comprises integrating one or more variant sequences into an insertion site, wherein the one or more variant sequences correspond to a gene or a portion thereof in the DNA target. In some embodiments, a variant sequence in an edited DNA target comprising the components set forth in Formula (V) or Formula (VI) encodes an amino acid sequence that is at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to an amino acid sequence encoded by the corresponding gene or the portion thereof.

[0316] In some embodiments, producing an edited DNA target comprising the components of Formula (VII) comprises replacing one or more sequences in a gene or a portion thereof found in 5 the target region of Formula (III) with a variant sequence. In some embodiments, replacing a gene or a portion thereof comprises editing at least 1% (e.g., 1-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%) of the gene or the portion thereof. In some embodiments, a variant sequence in an edited DNA target comprising the components of Formula (VII) encodes an amino acid sequence that is at least 1%, at least 5%, at 10 least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to an amino acid sequence encoded by the corresponding gene or the portion thereof.

[0317] In some embodiments, a variant sequence in a heterologous nucleic acid described herein is used to mutate a DNA target when an insertion site is separated from a PAM by at least one nucleotide. In some embodiments, a variant sequence in a heterologous nucleic acid described herein is used to mutate a DNA target when an insertion site is separated from a PAM by a plurality of nucleotides. In some embodiments, the plurality of nucleotides comprises 10 or more nucleotides. In some embodiments, the plurality of nucleotides comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 10-15, 10-25, 15-20, 20-25, 25-35, 25-50, 35-45, 45-55, 55-65, 65-75, 75-85, 85-95, 1-100, 50-100, 100-150, 100-200, 150-200, 200-300, 300-400, 400- 500, 500-600, 600-700700-800, 800-900, 900-1000, or more than 1000 nucleotides (e.g., 1,000- 10,000 nucleotides, or more than 10,000 nucleotides).

[0318] Expression Products Encoded by Heterologous Nucleic Acids

[0319] 15 In some embodiments, a heterologous nucleic acid comprises a sequence encoding an

[0320] RNA. In some embodiments, a heterologous nucleic acid encodes a plurality of RNAs (e.g., 2, 3, 4, or more RNAs).

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[0322] -35-

[0323] In some embodiments, a heterologous nucleic acid comprises a sequence encoding a therapeutic RNA and / or a therapeutic peptide or protein. The term “therapeutic” RNA, peptide, or protein refers to an RNA, peptide, or protein molecule that leads to a physiological change in a cell that can improve a biological process in a cell and / or the organism comprising the cell. In

[0324] 5 some embodiments, a therapeutic RNA, peptide, or protein is associated with or expected to at least partially, if not fully, prevent and / or remedy at least one symptom associated with a disease, disorder, or condition. In some embodiments, the disease, disorder, or condition comprises a genetic disease, cancer, inflammatory disease or an inflammatory condition, autoimmune disease, spleen disease, lung disease, hematological disease, neurological disease,

[0325] 10 painful condition, psychiatric disorder, metabolic disorder, immune disorder, infection of a pathogen, a kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, retinal disease, neuromuscular disease, musculoskeletal disease, lysosomal storage disease, or other disease, or any combination thereof.

[0326] Non-limiting examples of RNAs that can be encoded by a heterologous nucleic acid include a small-hairpin RNA (shRNA), a short-interfering RNA (siRNA), a prokaryotic- interfering RNA (pro-siRNA), a micro-RNA (miRNA), a long non-coding RNA (IncRNA), a Piwi-interacting RNA (piRNA), an exon-skipping RNA, an enzymatic RNA, a guide RNA (gRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), a ribosomal RNA (rRNA), a transfer RNA (tRNA), an RNA aptamer, and a messenger RNA (mRNA). In some embodiments, an RNA encoded by a sequence in a heterologous nucleic acid is an inhibitory RNA, such as a shRNA, an siRNA, a miRNA, a IncRNA, an exon- skipping RNA, an RNA aptamer, or an enzymatic RNA. In some embodiments, an inhibitory RNA can be used to regulate expression of a gene in a cell, such as a cell comprising a DNA target. In some embodiments, an inhibitory RNA can be used to regulate an RNA and / or protein, wherein

[0327] 25 increased levels of the RNA or protein in a cell is associated with a disease, disorder, or condition.

[0328] In some embodiments, an RNA encoded by a sequence in a heterologous nucleic acid is an mRNA encoding a peptide or a protein. Non-limiting examples of peptides and proteins that can be encoded by a sequence in a heterologous nucleic acid include a cell-, organelle-, or tissue-targeting peptide or protein, an antibody, an antigen-binding fragment, an antigen, an enzyme or an enzymatic domain, a proteinaceous enzyme substrate, a glycoprotein, a lipoprotein, a secreted protein, an extracellular matrix protein or a fragment thereof, a viral coat protein, a transmembrane receptor or a fragment thereof, a toxin or a fragment thereof, a hormone, receptors, a peptibody, a growth factor, a clotting factor, a cytokine, a chemokine, an

[0329] 35 activating or inhibitory peptide, a thrombolytic, a bone morphogenetic protein, an Fc-fusion

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[0331] -36- protein, an anticoagulant, a signaling protein, a cell surface protein, a nucleic acid-binding protein, and a reporter. In some embodiments, a reporter is mNeonGreen, GFP, EGFP, Superfold GFP, Azami Green, mWasabi, TagGFP, TurboGFP, acGFP, zsGreen, T-sapphire, EBFP, EBFP2, mTagBFP, ECFP, Cerulean, mTurquoise, CyPet, AmCyanl, TagCFP, Mtfpl,

[0332] 5 EYFP, mCitrine, TagYFP, phiYFP, zsYellowl, mBanana, Kusabira Orange, mOrange, dTomato, DsRed, mTangerine, mRuby, mApple, mStrawberry, AsRed2, Mrfpl, mCherry, HcRedl, or smURFP. In some embodiments, an antibody is a monoclonal antibody, a polyclonal antibody, a nanobody, or a single-chain antibody (e.g., an scFv). In some embodiments, a protein comprising an antigen-binding fragment is a chimeric antigen receptor, such as one

[0333] 10 comprising an antibody. In some embodiments, an enzyme is a protease, a signaling protein, a polymerase, a transcriptional regulator, a nuclease, an RNA-guided nuclease, a metabolic enzyme, a kinase, a phosphatase, a lipid-transferase, a glycosylase, a DNA ligase, a ubiquitin ligase, a methyltransferase, an acetyltransferase, a SUMO transferase, a foldase, a reductase, a lyase, a dehydrogenase, a phosphorylase, a decarobxylase, a dephosphorylase, a kinase, a synthase, or a hydrolase. In some embodiments, a nucleic acid-binding protein is a transcription regulator (e.g., a transcription factor), a splicing regulator, a translation regulator (e.g., a ribosome-binding protein, a signaling protein that post-translationally modifies RNA subunits, etc.), a nuclease (e.g., an RNA-guided nuclease, a zinc finger nuclease, or a transcription activator- like effector nuclease (TALEN). In some embodiments, a signaling protein is an

[0334] 20 enzyme (e.g., a kinase), a ligand (e.g., a ligand of a receptor), a secreted protein, a morphogen, a cell differentiation regulator. In some embodiments, a cell surface protein is a membrane protein, such as a receptor, a channel, a lineage- specific antigen, an extracellular matrix protein, or protein comprising a sequence that binds to an antigen-binding fragment, an antibody, or a chimeric antigen receptor. In some embodiments, a hormone is insulin, glucagon, growth hormone, thyroid-stimulating hormone, adrenocorticotropic hormone, follicle-stimulating hormone, luteinizing hormone, prolactin, oxytocin, vasopressin, parathyroid hormone, cortisol, erythropoietin, luteinizing hormone p, and aldosterone. In some embodiments, an antigen is a protein or a fragment thereof on a pathogen (e.g., an immunogenic protein or fragment thereof) or a tumor antigen.

[0335] 30

[0336] Engineering Targets for Subsequent Editing with Heterologous Nucleic Acids

[0337] In some embodiments, a heterologous nucleic acid can be used to introduce a heterologous PAM into a DNA target. In some embodiments, a heterologous nucleic acid can be used to introduce a plurality of heterologous PAMs into a DNA target. In some embodiments, a heterologous PAM in a heterologous nucleic acid comprises a sequence which is 3-10

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[0339] -37- nucleotides in length. In some embodiments, a heterologous PAM comprises a sequence of any one of: NGG; NNAGAAW; NNGRRT; NNNNGATT; NVNDCCY; BRTTTTT; NR(A or G)TTTT; NNAAAR(G or A); N(N or A)G; NAAN; NAAAAY; NHDTCCA; NNNVRYM; NNNNRYAC; NAA; GNNNNCNNA; NNGTGA; NNNNGTA; NNGGG; NNNCAT; NNRHHHY; NRRNAT; NNNNCNAA; NNNNCMCA; NNNNCRAA; NNNNGMAA; NNNCC; NGGNG; NNNNCNDD; NYAAA; NRGNN; N(C or D)GGN(T or A or G or C)NN; NRTAW; N(C or K or A)AARC; NAAAG; NV(A or G or C)R(A or G)ACCN; NNGAC; NATGNT; N(T or V)NTAAW(A or T); NNGW(A or T)AY(T or C); NCAA(H(Y or A)B(Y or G); NH(T or C or A)AAAA; NNNATTT; NATAWN(A or T or S); NATARCH; B(T or G or C)GGD(A or T or G)TNN; N(G or T or M)GGAH(T or A or C)N(A or C or K)N; NRG; N(B or A)GG; NGGD(A or K)W(T or A); N(T or C or R)AGAN(A or K or QNN; NGGD(A or T or G)H(T or M); NGGDT; NGGD(A or T or G)GNN; NNGTAM(A or C)Y; NNGH(W or QAAA; NTGAR(G or A)N(A or Y or G)N(Y or R); NNGAAAN; NNGAD; NHARMC; NNAAAG; NHGYNAN(A or B); NNAGAAA; NHAAAAA; NH(T or M)AAAAA; NHGYRAA;

[0340] NNAAACN; NN(H or G)D(A or K)GGDN(A or B); NNNNCTA; NNNNCVGAA; NNNNGYAA; NNNNATN(W or S)ANN; NNWHR(G or A)TA(not G)AA; YHHNGTH; NNNNCDAANN; NNNNCTAA; N(C or D)NNTCCN; NNNNCCAA; NAGRGN(T or V)N(T or C); NNAH(T or M)ACN; CN(C or W or G)AV(A or S)GAC; NAR(G or A)H(W or C)H(A or T or C)GN(C or T or R); NAGNGC; NATCCTN; NGTGANN; HGCNGCR; NAR(A or G)W(T or A)AC; N(C or D)M(A or C)RN(A or B)AY(C or T); NNNCAC; BGGGTCD; NNRRCC; NRRNTT; KARDAT; BRRTTTW; NARNCCN; NAR(A or G)TC; NAAN(A or T or S)RCN; HHAAATD; NNNNGNA; TTV; TTTV; YYV; KKYV; TTTM; TTYV; TTTN; TTTTA; TTN; BTTV; YTV; YTN; NYTV; DTTD; ATTN; RTTNT; HATT; ATTW; RTTN; TVT; TG; TN; TR; TA; TTCN; TTAT; TTTR; TTR; YTTR; YTTN; CTT; TTC; CCD; RTR; VTTR; TBN; VTTN; NGTT; CGTT; AGG; CGG; GTT; or RGTG, wherein: “N” is any nucleotide or base; “W” is adenine (A) or thymine (T); “R” is A or guanine (G); “V” is A, cytosine (C), or G; “Y” is C or T; “M” is C or A; “K” is G or T; “D” is A, G, or T; “S” is G or C; “B” is C, G, or T; and “H” is A, C, or T.

[0341] Introduction of a heterologous PAM into a DNA target using a heterologous nucleic acid in a template can be used for subsequent editing of an edited DNA target (e.g., to make the edited DNA target accessible to an RNA-guided nuclease). In some embodiments, a heterologous PAM is capable of binding to a CRISPR / Cas nuclease or a variant thereof. In some embodiments, a heterologous PAM is capable of binding to a Cas9 nuclease or a variant thereof, a Casl2 nuclease or a variant thereof, a Casl3 nuclease or a variant thereof, or a Casl4 nuclease or a variant thereof.

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[0343] -38-

[0344] In some embodiments, a heterologous nucleic acid can be used to introduce a sequence into a DNA target that is capable of hybridizing to a gRNA. In some embodiments, the sequence capable of hybridizing to a gRNA comprises 40 nucleotides or less. In some embodiments, the sequence capable of hybridizing to a gRNA comprises 10-35 nucleotides, such as 15, 16, 17, 18,

[0345] 5 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the sequence capable of hybridizing to a gRNA comprises at least 15 nucleotides (e.g., 15-18, 18-20, 20-22, or 22-24 nucleotides) that are the reverse complement of a gRNA. However, in some embodiments, the sequence capable of hybridizing to a gRNA comprises one or more mismatches relative to the gRNA. In some embodiments, the gRNA that the heterologous nucleic acid is capable of hybridizing to is not used in the genetic editing that introduces the heterologous nucleic acid and is instead used in a subsequent genetic editing method or step.

[0346] In some embodiments, a heterologous nucleic acid can be used to introduce a sequence into a DNA target that binds a zinc-finger nuclease or a TALEN.

[0347] Regulatory Sequences and Heterologous Nucleic Acids

[0348] In some embodiments, a heterologous nucleic acid comprises one or more regulatory

[0349] 15 sequences. In some embodiments, a heterologous nucleic acid comprises a plurality of regulatory sequences (e.g., 2, 3, 4, 5, 6, or more than 6 regulatory sequences). Non-limiting examples of regulatory sequences that can be comprised in a heterologous nucleic acid include a promoter, an enhancer, a silencer, a transcription factor binding sequence, a 5’ untranslated region (UTR), a 3’ UTR, a translation initiation regulatory sequence, a transcriptional start

[0350] 20 sequence, a transcription termination sequence, a splicing acceptor site, a splicing donor site, a small nuclear ribonucleoprotein binding site, a mRNA degradation or decay signal, a polyadenylation signal, a Kozack sequence, a Shine-Dalgamo sequence, a start codon, a RNA- binding protein binding site, a ribosome binding site, a ribozyme, an intron, a translation termination sequence, a stop codon, a self-cleaving peptide, an intein (e.g., a split intein), and a protease cleavage site.

[0351] In some embodiments, a heterologous nucleic acid comprises at least one regulatory sequence which is operably linked to a sequence encoding an RNA (e.g., a variant sequence encoding an RNA having one or more synonymous mutations relative to a sequence in a DNA target). In some embodiments, the at least one regulatory sequence is capable of regulating

[0352] 30 transcription (e.g., any one or more of a promoter, an enhancer, a transcription factor binding sequence, a transcriptional start sequence, transcription termination sequence, etc.), regulating translation (e.g., any one or more of a 5’ UTR, a translation initiation regulatory sequence, a Kozack sequence, a Shine-Dalgamo sequence, a start codon, a ribosome binding site, a 3’ UTR,

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[0354] -39- a translation termination sequence, a stop codon, etc.), or regulating splicing (e.g., binding sites for small nuclear ribonucleoproteins, splicing acceptor sites, splicing donor sites, etc.) of an RNA encoded by a sequence in a heterologous nucleic acid. In some embodiments, a promoter comprised in a heterologous nucleic acid is one that is active in a cell, or a cell of a similar cell

[0355] 5 type, comprising a DNA target. In some embodiments, a heterologous nucleic acid comprises a constitutive promoter (e.g., a Herpes Simplex virus (HSV) promoter, a thymidine kinase (TK) promoter, a Simian Virus 40 (SV40) promoter, a Mouse Mammary Tumor Virus (MMTV) promoter, a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), a cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), a dihydrofolate reductase promoter, a P-actin promoter, a phosphoglycerol kinase (PGK) promoter, a CAG promoter, a human elongation factor- 1 alpha (EFla) promoter, an RNA pol II promoter, an RNA pol III promoter, a U6 promoter, a Hl promoter, an RNA pol II promoter, a chicken P-actin (CBA) promoter, etc.), an inducible promoter (e.g., cytochrome P450 gene promoters, heat shock protein gene promoters, metallo thionein gene promoters, hormone- inducible gene promoters, a zinc-inducible sheep metallothionine (MT) promoter, a dexamethasone (Dex) -inducible mouse mammary tumor virus (MMTV) promoter, a T7 polymerase promoter system, a ecdysone insect promoter, a tetracycline-inducible promoter, a rapamycin-inducible promoter, etc.), or a tissue- specific promoter (e.g., a neuron- specific promoter, a skeletal muscle- specific promoter, a smooth muscle-specific promoter, an immune cell-specific promoter, a cardiac muscle-specific promoter, etc.).

[0356] In some embodiments, a heterologous nucleic acid comprises one or more sequences capable of regulating splicing. In some embodiments, a sequence capable of regulating splicing comprises a splicing acceptor site, a splicing donor site, and / or a small nuclear ribonucleoprotein binding site.

[0357] In some embodiments, a heterologous nucleic acid comprises one or more sequences capable of regulating post-translational modifications of an amino acid sequence. Sequences that can regulate the translation and / or post-translational modifications of an amino acid sequence can be positioned in a heterologous nucleic acid 5’ and / or 3’ relative to a sequence encoding an RNA. Non-limiting examples of a sequences capable of regulating translation and / or post- translational modifications of an amino acid sequence include a protease cleavage, a selfcleaving peptide (e.g., a 2A peptide, such as a P2A peptide, a T2A peptide, an E2A peptide, or an F2A peptide), an intein (e.g., a split intein), and a degron.

[0358] 25 Modulating Gene Expression in an Edited DNA Target

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[0360] -40-

[0361] Modulating expression of a gene or a portion thereof in an edited DNA target can comprise, but is not limited to, modulating transcription, modulating translation, and / or modulating post-translational modifications (see, e.g., FIG. IB and FIG. ID). Non-limiting examples of regulatory sequences capable of modulating expression of an edited DNA target

[0362] 5 includes sequences capable of regulating transcription (e.g., any one or more of a promoter, an enhancer, a transcription factor binding sequence, a transcriptional start sequence, transcription termination sequence, etc.), regulating translation (e.g., any one or more of a 5’ UTR, a translation initiation regulatory sequence, a Kozack sequence, a Shine-Dalgarno sequence, a start codon, a ribosome binding site, a 3’ UTR, a translation termination sequence, a stop codon,

[0363] 10 etc.), regulating splicing (e.g., binding sites for small nuclear ribonucleoproteins, splicing acceptor sites, splicing donor sites, etc.), or regulating post-translational modifications (e.g., a protease cleavage, a self-cleaving peptide, an intein (e.g., a split intein), a degron, a ubiquitination signal, etc.).

[0364] In some embodiments, a heterologous nucleic acid comprises a sequence encoding an RNA that is configured to be operably linked to at least one regulatory sequence after it is integrated. In some embodiments, the at least one regulatory sequence comprises a regulatory sequence that is in the DNA target prior to editing (e.g., the native promoter of a gene in the DNA target, a splicing regulatory sequence in the DNA target, etc.). In some embodiments, the at least one regulatory sequence comprises a regulatory sequence in the heterologous nucleic

[0365] 20 acid. In some embodiments, a heterologous nucleic acid comprises a variant sequence and at least one regulatory sequence configured to be operably linked to a gene or a portion thereof (e.g., a portion comprising a regulatory sequence, such as a splicing regulatory sequence) in an edited DNA target. In some embodiments, a heterologous nucleic acid in an edited DNA target promotes expression of a variant sequence encoding an RNA.

[0366] In some embodiments, a heterologous nucleic acid in an edited DNA target promotes expression of a variant sequence encoding an RNA and operably links a corresponding gene or a portion thereof (e.g., a portion comprising an exon and / or an intron) to a regulatory sequence that modulates expression (e.g., increases expression, decreases expression, or results in alternative splicing) of the corresponding gene or the portion thereof.

[0367] 30

[0368] Modulating Transcription with Heterologous Nucleic Acids

[0369] In some embodiments, integration of a heterologous nucleic acid modulates transcription of a gene in an edited DNA target. In some embodiments, modulating transcription of a gene in an edited DNA target comprises mutating (e.g., generating substitutions, deletions, and / or

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[0371] -41- insertions at) a promoter, an enhancer, a transcription factor binding sequence, a transcriptional start sequence, and / or a transcriptional termination sequence.

[0372] In some embodiments, a heterologous nucleic acid comprising a transcription termination sequence is integrated at an insertion site (e.g., to prematurely terminate

[0373] 5 transcription of an RNA upregulated in a disease, disorder, or condition). In some embodiments, the transcription termination sequence is 3’ relative to a variant sequence encoding an RNA. In some embodiments, the transcription termination sequence suppresses transcription of a gene or a portion thereof that is located in the edited DNA target.

[0374] In some embodiments, a heterologous nucleic acid comprising one or more sequences

[0375] 10 capable of promoting transcription is integrated at an insertion site (e.g., to upregulate transcription of a transcript that is therapeutic for a disease, disorder, or condition). In some embodiments, the one or more sequences capable of promoting transcription comprise a promoter, an enhancer, a transcription factor binding sequence, and / or a transcriptional start sequence.

[0376] Modulating Splicing with Heterologous Nucleic Acids

[0377] In some embodiments, integration of a heterologous nucleic acid modulates splicing of a gene in an edited DNA target. In some embodiments, modulating splicing of a gene in an edited DNA target comprises mutating (e.g., generating substitutions, deletions, and / or insertions at)

[0378] 20 splicing regulatory sequences, such as splicing acceptor sites, splicing donor sites, and / or small nuclear ribonucleoprotein binding sites. In some embodiments, modulating splicing of a gene in an edited DNA target comprises introducing a mutation into a splicing regulatory sequence that makes the splicing regulatory sequence non-functional. In some embodiments, modulating splicing of a gene in an edited DNA target comprises integrating a splicing regulatory sequence. In some embodiments, integration of a heterologous nucleic acid can result in exon inclusion, intron inclusion, and / or exon-skipping during expression of a gene or a portion thereof in the edited DNA target (e.g., to reduce levels of a mRNA that is encoded in the DNA target and associated with a disease, disorder, or condition and / or increase levels of an alternatively spliced RNA encoded by a variant sequence and that is therapeutic for a disease, disorder, or condition).

[0379] 30 In some embodiments, a heterologous nucleic acid comprising a splicing acceptor site is integrated at an insertion site. In some embodiments, a splicing donor site is located 5’ relative to the insertion site (e.g., an insertion site located in a gene). In some embodiments, a heterologous nucleic acid is integrated that comprises a splicing acceptor site that is 5’ relative to a variant sequence encoding an RNA is integrated at the insertion site, thereby resulting in

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[0381] -42- expression of a transcript wherein a gene or a portion thereof is skipped during splicing and the variant sequence is retained.

[0382] In some embodiments, a heterologous nucleic acid comprising a splicing donor site is integrated at an insertion site. In some embodiments, a splicing acceptor site is located 3’

[0383] 5 relative to the insertion site (e.g., an insertion site located in a gene). In some embodiments, a heterologous nucleic acid is integrated that comprises a splicing donor site that is 3’ relative to a variant sequence encoding an RNA is integrated at the insertion site, thereby resulting in expression of a transcript wherein a gene or a portion thereof is skipped during splicing and the variant sequence is retained.

[0384] 10 In some embodiments, a heterologous nucleic acid comprising a splicing donor site and a splicing acceptor is integrated, wherein the splicing donor site is integrated at a first insertion site (e.g., an insertion site located in a first portion of a gene) and the splicing acceptor site is integrated at a second insertion site (e.g., an insertion site located in a second portion of the gene). In some embodiments, a splicing acceptor site is located 3’ relative to the first insertion site and a splicing donor site is located 5’ relative to the second insertion site. In some embodiments, a variant sequence is located 5’ relative to the splicing donor site in the heterologous nucleic acid and / or a variant sequence is located 3’ relative to the splicing acceptor site in the heterologous nucleic acid, thereby retaining the variant sequence(s) in the spliced RNA product.

[0385] In some embodiments, a heterologous nucleic acid comprising a splicing acceptor site and a splicing donor is integrated, wherein the splicing acceptor site is integrated at a first insertion site (e.g., an insertion site located in a first portion of a gene) and the splicing donor site is integrated at a second insertion site (e.g., an insertion site located in a second portion of the gene). In some embodiments, a splicing donor site is located 5’ relative to the first insertion

[0386] 25 site and a splicing acceptor site is located 3’ relative to the second insertion site. In some embodiments, the splicing acceptor site, and the splicing donor site in the heterologous nucleic acid flank a variant sequence, thereby retaining the variant sequence in the spliced RNA product.

[0387] Modulating Translation and Post-Translation Modifications with Heterologous Nucleic Acids

[0388] In some embodiments, integration of a heterologous nucleic acid modulates translation of an RNA encoded by a gene in an edited DNA target. In some embodiments, modulating translation of an RNA encoded by a gene in an edited DNA target comprises mutating (e.g., generating substitutions, deletions, and / or insertions in) a 5’ UTR, a translation initiation regulatory sequence, a Kozack sequence, a Shine-Dalgarno sequence, a start codon, a ribosome

[0389] 35 binding site, a coding sequence, a stop codon, and / or a 3’ UTR. In some embodiments,

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[0391] -43- modulating translation of an RNA encoded by a gene in an edited DNA target comprises integrating a 5’ UTR, a translation initiation regulatory sequence, a Kozack sequence, a Shine- Dalgamo sequence, a start codon, a ribosome binding site, a sequence encoding a protease cleavage site, a sequence encoding a self-cleaving peptide, a stop codon, and / or a 3’ UTR. In

[0392] 5 some embodiments, integration of a heterologous nucleic acid moves a start codon into frame with a gene in an edited DNA target (e.g., to upregulate translation of a peptide or protein that is therapeutic for a disease, disorder, or condition). In some embodiments, integration of a heterologous nucleic acid moves a start codon out of frame with a gene in an edited DNA target (e.g., to decrease translation of a peptide or protein that is therapeutic for a disease, disorder, or

[0393] 10 condition). In some embodiments, integration of a heterologous nucleic acid moves a premature stop codon into frame with a gene in an edited DNA target (e.g., to decrease translation of a peptide or protein that is therapeutic for a disease, disorder, or condition). In some embodiments, integration of a heterologous nucleic acid moves a premature stop codon out of frame with a gene in an edited DNA target (e.g., to increase translation of a peptide or protein that is therapeutic for a disease, disorder, or condition). In some embodiments, integration of a heterologous nucleic acid comprising a sequence encoding a protease cleavage site and / or a sequence encoding a self-cleaving peptide alters one or more mutations (e.g., corrects one or more mutations) in an amino acid sequence encoded by a DNA target that are associated with a disease, disorder, or condition.

[0394] In some embodiments, one or more sequences that regulate the post-translational modification of an amino acid sequence is integrated at an insertion site positioned 5’ and / or at an insertion site positioned 3’ relative to a gene or a portion thereof in a DNA target. In some embodiments, the one or more sequences that regulate the post-translational modification of an amino acid sequence become operably linked to a gene or a portion thereof in the DNA target

[0395] 25 upon generating the edited DNA target. In some embodiments, integration of the one or more sequences that regulate the post-translation modification of an amino acid sequence produces an amino acid sequence encoded by a gene or a portion thereof in the edited DNA target that is physically separate from an amino acid sequence encoded by a variant sequence RNA encoded by a heterologous nucleic acid. In some embodiments, the one or more sequences that regulate the post-translational modification of an amino acid sequence are located 5’ relative to the variant sequence in the heterologous nucleic acid. In some embodiments, the one or more sequences that regulate the post-translational modification of an amino acid sequence comprises a sequence encoding a protease cleavage site and / or a self-cleaving peptide. In some embodiments, a sequence encoding a self-cleaving peptide is integrated to induce ribosomal

[0396] 35 skipping. In some embodiments, a sequence encoding a self-cleaving peptide is integrated to

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[0398] -44- terminate translation of a peptide or protein associated with a disease, disorder, or condition. In some embodiments, a self-cleaving peptide is a 2A peptide, such as a P2A peptide, a T2A peptide, an E2A peptide, or an F2A peptide. In some embodiments, a start codon and / or a stop codon is operably linked to a variant sequence in the heterologous nucleic acid. In some

[0399] 5 embodiments, the variant sequence RNA and the sequence encoding the protease cleavage site and / or the sequence encoding the self-cleaving peptide are separated by a linker sequence (e.g., a flexible linker, such as a glycine- and / or serine-rich linker).

[0400] Recombinant Adeno-associated Viruses

[0401] 10 In some aspects, the application relates to AAVs and recombinant AAVs comprising a nucleic acid (e.g., a template) described herein. As used herein, the term “adeno-associated virus” or the abbreviation “AAV” refers to the virus itself or derivatives thereof. The term covers all AAV subtypes including both naturally occurring and recombinant forms, unless otherwise indicated. The term “recombinant AAV (rAAV)” refers to recombinant adeno-associated virus which refers to AAV comprising a nucleic acid not of AAV origin. In some embodiments, the nucleic acid not of AAV origin comprises a template and / or a sequence encoding a gRNA and / or a sequence encoding an RNA-guided nuclease. In some embodiments, a nucleic acid sequence found within an rAAV is an “rAAV genome” which refers to a nucleic acid comprising a heterologous nucleic acid flanked by 5' and 3' AAV inverted terminal repeats (ITRs). In some embodiments, a nucleic acid flanked between AAV ITRs comprises at least one transgene. As used herein, “transgene” refers to a DNA sequence which encodes at least one RNA to be expressed in a cell. The term “AAV particle” or “rAAV particle” refers to a particle formed by one or more AAV capsid proteins. In some embodiments, AAV particles and rAAV particles comprise an encapsidated nucleic acid (e.g., an rAAV particle comprising an rAAV genome).

[0402] 25 In some embodiments, an rAAV particle comprises a serotype of mammalian AAV1, mammalian AAV2, mammalian AAV3, mammalian AAV4, mammalian AAV5, mammalian AAV6, mammalian AAV7, mammalian AAV8, mammalian AAV9, or mammalian AAV10. In some embodiments, an rAAV particle is a pseudotyped rAAV particle. Non-limiting examples of rAAV pseudotypes include mammalian AAV2 / 1, mammalian AAV2 / 5, mammalian AAV2 / 6, mammalian AAV2 / 8, mammalian AAV2 / 9, mammalian AAV3 / 1, mammalian AAV 3 / 5, mammalian AAV3 / 8, and mammalian AAV 3 / 9, wherein the slash denotes an rAAV genome comprising ITRs of one serotype packaged in the capsid from a different serotype (e.g., an rAAV genome comprising AAV2 ITRs packaged in a capsid of AAV5 would be AAV2 / 5). In some embodiments, a pseudotyped rAAV particle can be engineered to comprise a hybrid or a mutant

[0403] 35 mammalian AAV capsid protein derivate, such as AAVrh.10, AAVrh.74, AAVhu.14, AAV3a / 3b,

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[0405] -45-

[0406] AAVrh32.33, AAV-HSC15, AAV- HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV218, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6(Y445F / Y731F), AAV2.5T, AAV- HAE1 / 2, AAV clone 32 / 83, AAVShHIO, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, AAV2(pentaYF), AAV2-BCDG(T491V+K556R), AAV5-M2, AAV5(Y719F),

[0407] 5 AAV6(T492V+S663V), AAV6(T492V+Y705F+Y731F), AAV6(S551V+S663V), AAV8- C&G(T494V), AAV8-M3, AAV8(Y733F), AAV8(T494V+Y733F), AAV8(Y275F+Y447F+Y733F), AAV9-PHP.B, or AAVr3.45.

[0408] In some embodiments, rAAV particles are packaged using a packaging nucleic acid and / or a helper nucleic acid. As used herein, a “helper nucleic acid” refers to a nucleic acid (e.g.,

[0409] 10 a helper vector or a nucleic acid provided in a helper virus) comprising one or more genes (e.g., El, E2A, E4, and / or VA) which functions in trans for productive AAV replication and encapsidation. In some embodiments, a helper nucleic acid is an adenovirus helper nucleic acid (e.g., a mammalian adenovirus helper nucleic acid). In some embodiments, a helper nucleic acid is provided by a helper virus, such as an adenovirus (e.g., human Ad2, human Ad5, etc.). As used herein, a “packaging nucleic acid” refers to a nucleic acid (e.g., a packaging vector) which provides nucleotide sequences (e.g., AAV rep and AAV capsid protein gene sequences) upon which an AAV is dependent for replication (e.g., accessory functions).

[0410] Preferably, a helper nucleic acid supports efficient rAAV particle production without generating any detectable wild-type AAV particles (e.g., AAV particles containing functional rep and capsid protein genes). Nucleic acids for rAAV particle production have been previously described and are commercially available (see, e.g., pDM, pDG, pDPlrs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene,

[0411] 25 Cambridge, MA; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno- Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072-11081.; Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini , Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, R.O. (2008), International efforts for recombinant adeno-associated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188). These references are incorporated by

[0412] 35 reference herein for their disclosures related to compositions and methods for rAAV production.

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[0414] -46-

[0415] In some embodiments, the components cultured in a cell to package a rAAV genome in a capsid may be provided to the cell in trans. In some embodiments, rAAV particles may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650 which is incorporated by reference herein for its disclosures related to compositions and

[0416] 5 methods for rAAV production). In some embodiments, rAAV particles are produced by transfecting a cell with an AAV vector (comprising a nucleic acid, such as a template nucleic acid, flanked by ITR elements) to be packaged into rAAV particles, and at least one AAV helper or packaging nucleic acid. In some embodiments, two nucleic acids are used which include a helper nucleic acid and a packaging nucleic acid. Alternatively, in some embodiments, any one

[0417] 10 or more of the required components (e.g., template nucleic acid flanked by AAV ITRs, rep sequences, cap sequences, and / or helper nucleic acids) may be provided by a cell which has been engineered to stably contain one or more of the required components (e.g., via genomic integration). In some embodiments, the cell will contain the required component(s) under the control of either an inducible promoter or a constitutive promoter.

[0418] Methods used to construct rAAV particles have been previously described (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on this application. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745). In addition, AAV serotypes, AAV derivatives, and AAV pseudotypes, as well as methods for producing them, have been previously described (see, e.g., Mol. Ther. 2012 Apr;20(4): 699- 708. doi: 10.1038 / mt.2011.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan Al, Schaffer DV, Samulski RJ; Duan et al, J. Virol., 75:7662-7671, 2001; Halbert et al, J. Virol., 74:1524-1532, 2000; Zolotukhin et al, Methods, 28: 158-167, 2002; and Auricchio et al., Hum. Molec. Genet.,

[0419] 25 10:3075-3081, 2001; see, e.g., US Patent Publication No. : US 2005 / 0100890 Al; International Publication No.: WO 01 / 83692 A2; US Patent Publication No. : US 2003 / 0103939 Al; and Miller (1996). Proc. Natl. Acad. Sci., 93: 11407-11413). These references are incorporated by reference herein for their disclosures related to compositions and methods for recombinant nucleic acid production and rAAV production.

[0420] Compositions

[0421] Other aspects of the present application relate to compositions. In some embodiments, a composition comprises a nucleic acid described herein, such as a template nucleic acid or a gRNA. In some embodiments, a composition comprises an RNA-guided nuclease. In some

[0422] 35 embodiments, a composition comprises a template nucleic acid in addition to an RNA-guided

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[0424] -47- nuclease and / or a gRNA (e.g., wherein the composition comprises a ribonucleoprotein (RNP) complex that comprises the RNA-guided nuclease and the gRNA). In some embodiments, a composition comprises a cell or, a cell population comprising a template nucleic acid, a gRNA, and / or an RNA-guided nuclease. In some embodiments, the cell or the cell population comprises

[0425] 5 eukaryotic cells. In some embodiments, the cell or the cell population comprises mammalian cells, such as human cells. In some embodiments, the cell or the cell population comprises mitotic cells. In some embodiments, the cell or the cell population comprises post- mitotic cells. In some embodiments, the cell or the cell population comprises neuronal cells. In some embodiments, the cell or the cell population comprises non-neuronal cells. In some

[0426] 10 embodiments, the cell or the cell population comprises retinal cells. In some embodiments, the cell or the cell population comprises in vitro cells. In some embodiments, the cell or the cell population comprises ex vivo cells. In some embodiments, the cell or the cell population comprises prokaryotic cells. In some embodiments, a composition comprises an rAAV particle described herein (e.g., an rAAV particle comprising a template nucleic acid and / or a sequence encoding a gRNA and / or a sequence encoding an RNA-guided nuclease). Additionally, the application relates to compositions comprising a genetically engineered cell or a genetically engineered cell population produced using embodiments described herein. In some embodiments, a composition (e.g., pharmaceutical compositions) can be used to administer a nucleic acid described herein to a cell, such as a cell in a subject (e.g., a mammalian subject, such as a human subject). The application also provides combinations of compositions (e.g., wherein the combination comprises a plurality of compositions, such as two, three, or more than three compositions) that can be used to deliver a template nucleic acid to a cell, such as an in vitro cell, an ex vivo cell, or a cell in a subject (e.g., wherein the cell is a mammalian cell, such as a human cell).. In some embodiments, a combination of compositions include one

[0427] 25 composition that comprises a template nucleic acid and another composition that comprises a gRNA and / or an RNA-guided nuclease (e.g., an RNP complex comprising the gRNA and the RNA-guided nuclease). In some embodiments, a combination of compositions include a composition comprising an rAAV particle described herein. Accordingly, at least in some embodiments, a composition comprises any or all of the components necessary to genetically engineer a cell, such as an in vitro cell, an ex vivo cell, or a cell in a subject (e.g., wherein the cell is a mammalian cell, such as a human cell).

[0428] In some embodiments, a composition comprises an rAAV particle described herein at an amount of at least 1 x 102vector genomes (vg). In some embodiments, the amount of an rAAV particle in a composition comprises at least 1 x 103vg, 1 x 104vg, 1 x 105vg, 1 x 106vg, 1 x 107

[0429] 35 vg, 1 x 108vg, 1 x 109, 1 x IO10vg, 1 x 1011vg, 1 x 1012vg, 1 x 1013vg, 1 x 1014vg, 1 x 1015vg,

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[0431] -48-

[0432] 1 x 1016vg, 1 x 1017vg, 1 x IO18vg, or more. In some embodiments, the amount of an rAAV particle in a composition comprises 1 x 105vg to 1 x 1018. In some embodiments, the amount of an rAAV particle in a composition comprises about 1 x 107vg, 1 x 108vg, 1 x 109vg, 1 x IO10vg, 1 x 1011vg, 1 x 1012vg, 1 x 1013vg, 1 x 1014vg, 1 x 1015vg, 1 x 1016vg, 1 x 1017vg, or 1 x

[0433] 5 1018vg.

[0434] Compositions of the application can be suitable for treatment regimens and thereby administered to a subject via a variety of methods described herein. Such compositions can be formulated for use in a variety of therapies, such as, for example, in the amelioration, prevention, and / or treatment of a disease, disorder, or condition described herein. In some

[0435] 10 embodiments, a composition comprises a liposome, a lipid, a lipid complex, a lipid nanoparticle, a microsphere, a microparticle, a nanosphere, and / or a nanoparticle, or may be otherwise formulated for administration to a cell, biological sample, tissue, organ, or body of a subject in need thereof. In some embodiments, a composition comprises one or more agents that are useful for administration of a composition to a subject, such as anti-pruritic s, astringents, local anesthetics, anti-inflammatory agents (e.g., antihistamine, diphenhydramine), or any combination thereof. Accordingly, compositions described herein may be administered to a subject, such as mammalian subjects (e.g., humans). In some embodiments, a composition is provided in a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In some embodiments, a composition can be buffered, if necessary, with sufficient saline, polyalcohols, or glucose.

[0436] In some embodiments, a composition comprises a pharmaceutical excipient. In some embodiments, pharmaceutically acceptable excipients (excipients) that do not exert or are not intended to exert a therapeutic effect. In some embodiments, excipients act to a) aid in

[0437] 25 processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and / or d) enhance any other attribute of the overall safety, effectiveness, or delivery of a therapeutic agent (e.g., a template nucleic acid or an rAAV particle or a cell comprising the same) during storage or use.

[0438] Methods of Administration

[0439] In other aspects, the present application relates to methods of administering a nucleic acid described herein to a subject. In some embodiments, a subject is a mammalian subject. In some embodiments, a mammalian subject is a human subject. In other embodiments, a

[0440] 35 mammalian subject is non-human, such as a mouse, a rat, or a non-human primate. In some

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[0442] -49- embodiments, a method comprises administering a template nucleic acid to a subject. In some embodiments, a method comprises administering a gRNA and / or a gRNA nuclease to a subject. In some embodiments, a nucleic acid comprising a template, a sequence encoding a gRNA, and / or a sequence encoding an RNA-guided nuclease is administered to a subject in an rAAV

[0443] 5 particle described herein. In some embodiments, a template, a sequence encoding a gRNA, and / or a sequence encoding an RNA-guided nuclease is comprised in a composition (e.g., a pharmaceutical composition) and / or a kit described herein.

[0444] In some embodiments, a method comprises administering a composition (e.g., a nucleic acid) described herein to a subject. In some embodiments, a subject (e.g., a subject in need

[0445] 10 thereof) is a subject that has, is suspected of having, or at risk of developing a disease, disorder, or condition. In some embodiments, a subject that has, is suspected of having, or at risk of developing a disease, disorder, or condition comprises one or more mutations in a DNA target that a template nucleic acid is designed to edit. In some embodiments, the disease, disorder, or condition comprises a genetic disease, cancer, inflammatory disease or an inflammatory condition, autoimmune disease, spleen disease, lung disease, hematological disease, neurological disease, painful condition, psychiatric disorder, metabolic disorder, immune disorder, infection of a pathogen, a kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, retinal disease, neuromuscular disease, musculoskeletal disease, lysosomal storage disease, or other disease, or any combination thereof.

[0446] In some embodiments, a composition (e.g., a nucleic acid) is administered to a subject to treat a disease, disorder, or condition. The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein. In some embodiments, a nucleic acid is administered to a subject after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, a

[0447] 25 nucleic acid is administered in the absence of signs or symptoms of the disease. As a nonlimiting example, a nucleic acid can be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of exposure to a pathogen). In some embodiments, a nucleic acid is administered after symptoms have resolved, for example, to delay or prevent recurrence. In some embodiments, treating a subject comprises administering a template nucleic acid that is designed to edit a DNA target comprising a mutated sequence (e.g., a gene comprising one or more mutations associated with a disease, disorder, or condition) by introducing therapeutic edits (e.g., nucleotide changes that correct nucleotide positions comprising a mutation associated with a disease, disorder, or condition).

[0448] In some embodiments, administration of a template nucleic acid described herein

[0449] 35 achieves one, two, three, four, or more of the following effects, including, for example: (i)

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[0451] -50- reduction or amelioration the severity of disease, disorder, or condition or symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease, disorder, or condition; (iii) protection against the progression of a disease or disorder or symptom associated therewith; (iv) regression of a disease, disorder, or condition or symptom associated therewith;

[0452] 5 (v) protection against the development or onset of a symptom associated with a disease, disorder, or condition; (vi) protection against the recurrence of a symptom associated with a disease; (vii) reduction in the hospitalization of a subject; (viii) reduction in the hospitalization length; (ix) an increase in the survival of a subject with a disease; (x) a reduction in the number of symptoms associated with a disease, disorder, or condition; (xi) an enhancement,

[0453] 10 improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy.

[0454] In some embodiments, an rAAV particle comprising a nucleic acid described herein is administered to a subject in need thereof. In some embodiments, a method comprises administering an rAAV particle described herein at a dose of at least 1 x 105vector genomes (vg) per kilogram (kg) (vg / kg) of a subject’s body weight. In some embodiments, an rAAV particle is administered in a composition (e.g., a pharmaceutical composition) comprising a dose described herein. In some embodiments, a method comprises administering an rAAV particle described herein at a dose of about 1 x 106vg / kg, 2 x 106vg / kg, 3 x 106vg / kg, 4 x 106vg / kg, 5 x 106vg / kg, 6 x 106vg / kg, 7 x 106vg / kg, 8 x 106vg / kg, 9 x 106vg / kg, 1 x 107vg / kg, 2 x 107vg / kg, 3 x 107vg / kg, 4 x 107vg / kg, 5 x 107vg / kg, 6 x 107vg / kg, 7 x 107vg / kg, 8 x 107vg / kg, 9 x 107vg / kg, 1 x 108vg / kg, 2 x 108vg / kg, 3 x 108vg / kg, 4 x 108vg / kg, 5 x 108vg / kg, 6 x 108vg / kg, 7 x 108vg / kg, 8 x 108vg / kg, 9 x 108, 1 x 109vg / kg, 2 x 109vg / kg, 3 x 109vg / kg, 4 x 109vg / kg, 5 x 109vg / kg, 6 x 109vg / kg, 7 x 109vg / kg, 8 x 109vg / kg, 9 x 109vg / kg, vg / kg, 1 x IO10vg / kg, 2 x IO10vg / kg, 3 x IO10vg / kg, 4 x IO10vg / kg, 5 x IO10vg / kg, 6 x IO10vg / kg, 7 x IO10vg / kg, 8 x

[0455] 25 IO10vg / kg, 9 x IO10vg / kg, or more. In some embodiments, a method comprises administering an rAAV particle described herein at a dose of about 1 x 106vg / kg to 1 x 1015vg / kg. In some embodiments, a method comprises administering an rAAV particle described herein at a dose of about 1 x 107vg / kg to 1 x 1014vg / kg. In some embodiments, a method comprises administering an rAAV particle described herein at a dose of about 1 x 108vg / kg to 1 x 1013vg / kg. In some embodiments, a method comprises administering an rAAV particle described herein at a dose of about 1 x 109vg / kg to 1 x 1012vg / kg.

[0456] In some embodiments, administration of a nucleic acid described herein or an rAAV particle and / or composition thereof is performed subcutaneously, intraretinally, intraocularly, subretinally, intravitreally, parenterally, subcutaneously, intravenously,

[0457] 35 intracerebroventricularly, intramuscularly, intracranially, intrathecally, intraperitoneally, or by

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[0459] -51- direct injection to one or more cells, tissues, or organs. In some embodiments, direct injection is performed concurrently with a surgical procedure or interventional procedure. In some embodiments, compositions are administered to a subject through one administration route. In some embodiments, multiple administration routes are exploited (e.g., serially, or

[0460] 5 simultaneously).

[0461] Kits

[0462] In some aspects, the application relates to kits. In some embodiments, a kit comprises a nucleic acid described herein, such as a template nucleic acid or a gRNA. In some embodiments,

[0463] 10 a kit comprises an RNA-guided nuclease. In some embodiments, a kit comprises a cell or a cell population comprising a template nucleic acid, a gRNA, and / or an RNA-guided nuclease. In some embodiments, the cell or the cell population comprises mammalian cells, such as human cells. In some embodiments, a kit comprises an rAAV particle described herein (e.g., an rAAV particle comprising a template nucleic acid and / or a sequence encoding a gRNA and / or a sequence encoding an RNA-guided nuclease). Additionally, the application relates to kits comprising a genetically engineered cell or a genetically engineered cell population produced using embodiments described herein. In some embodiments, a kit can be used to administer a nucleic acid described herein to a cell, such as a cell in a subject (e.g., via administration to a mammalian subject, such as a human subject). Accordingly, at least in some embodiments, a kit comprises any or all of the components necessary to genetically engineer a cell, such as a cell in a subject.

[0464] In some embodiments, a kit comprises one or more containers housing components for performing the methods described herein, and optionally instructions for use. The components may be prepared sterilely, packaged in a syringe, and shipped refrigerated. Alternatively, they

[0465] 25 may be housed in a vial or other container for storage. A second container may have other components prepared sterilely. Alternatively, the kits may include the active agents premixed and shipped in a vial, tube, or other container. The kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc.

[0466] In some embodiments, a kit further comprises a set of instructions for carrying out the methods described herein. As used herein, “instructions” can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of this application. Instructions also can include any oral or electronic instructions provided in any

[0467] 35 manner such that a user will clearly recognize that the instructions are to be associated with the

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[0469] -52- kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and / or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use or sale for animal administration. As

[0470] 5 used herein, “promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral, and electronic communication of any form, associated with this application. Additionally, the kits may include other components depending on the specific application, as described herein.

[0471] In some embodiments, a kit comprises a blister pouch, a shrink-wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box, or a bag. The kits may be sterilized after the accessories are added, thereby allowing the individual accessories

[0472] 15 in the container to be otherwise unwrapped. The kits, or any of its components, can be sterilized using any appropriate sterilization techniques, such as filtration, radiation sterilization, heat sterilization, or other sterilization methods known in the art.

[0473] EXAMPLES

[0474] 20 Example 1: Template redesign for homology-based DNA repair enables flexible and efficient in vivo editing

[0475] A ribonucleoprotein (RNP) strategy was used, combining recombinant Cas9 protein with gRNA assembled from crRNA and tracrRNA and repair template. Resulting RNP particles were further mixed with a genetic construct expressing GFP under the control of ubiquitous neuronal CAG promoter to facilitate the identification of transduced regions. The whole system was then delivered into retinas at postnatal day 0 or 1 (P0 or Pl) mice via subretinal injection, followed by electroporation (FIG. 2A). Traditional template design was used for editing the Lmnbl gene that encodes nuclear lamina protein Lamin-Bl which is ubiquitously expressed by all retina cell types. The manipulation added an N-terminal HA-tag (FIG. 2A) to Eamin-Bl with an efficient

[0476] 30 gRNA relying on an optimally positioned PAM sequence. Retinas were dissected at various developmental stages after the manipulation and sectioned (FIG. 2B). The transduced regions were identified by GFP fluorescence (FIG. 2C, left panel), and editing was ascertained by immunohistochemical detection of the HA immunofluorescence (FIG. 2C, right panel). Under high editing efficiency conditions, more than 40% delivery and almost 30% editing efficiencies

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[0478] -53- were achieved (FIGs. 10B-10C). The use of high voltage compromised the viability of pups and was reduced to 105-volt level. This still yielded 30% delivery efficiency and was well tolerated by mice.

[0479] The timeline of construct expression and editing was evaluated. It was found that Lmnbl

[0480] 5 can be correctly edited (FIG. 10D) to express HA-Lamin-Bl at its expected nuclear localization (FIG. 10E) within -1 day after surgery (FIG. 2D). When retinas were harvested a week later (P8), a further increase in the efficiency was observed (FIGs. 2E, 2G, 2H). No significant increase in editing efficiency was observed beyond this stage, and adult retinas displayed similar number of edited cells (FIGs. 2F, 2G, 2H). It was found that there was a nearly perfect

[0481] 10 correlation between electroporation efficiency and knock-in efficiency (FIG. 21).

[0482] Variations were modeled in traditional template design for Lmnbl for which the original gRNA and template combination are highly efficient as the cut site is positioned within 1 base pair from the HA tag insertion site (FIG. 3A). Two alternative repair templates were designed for it, aiming to insert HA tag at the exact same location. In one, a gRNA targeting Lmnbl was selected, with its cut site 17 bp downstream from the insertion site (gRNA- 17) (FIG. 3A). In another gRNA, the cut sits moved further down the sequence, 142 bp away from the insertion site (gRNA-142) (FIG. 3B). CRISPR-tagging components were delivered into the retina using the optimized RNP-electroporation protocol at P0, and retinas were harvested at P10 followed by the analysis of Lamin-Bl tagging with an HA by immunohistochemistry. It was found that gRNA- 17 was not as efficient for editing Lmnbl (~5% efficiency) as the original gRNA (-30% efficiency) when normalized to transduction efficiency (GFP+ cells) (FIGs. 3K). No editing was found occurring with the gRNA-142 with the traditional repair template (FIGs. 3L).

[0483] A strategy for designing repair template was devised, called Silently Mutate And Repair Template (SMART). In SMART, the sequence was silently mutated between the cut site and

[0484] 25 insertion site, which prevents the base pairing between the homology arm of repair template and the targeted DNA without changing the sequence of amino acids (FIG. 3C). This was used to test if the probability that double-stranded breaks are repaired by the whole repair template is increased. SMART template contains a large segment of non-homologous sequence between the insertion and cut sites in the targeted DNA, repair of which may require breaking extended hydrogen bonding. Another version of the SMART was also designed which aligns the insertion and the cut sites and then reconstructs the gene with silently mutated template, SMART- ReConstruct (SMART-RC) (FIG. 3D). Different from SMART and traditional templates, the homology arms of SMART-RC were designed surrounding the cut site, and the designed sequence was precisely inserted at the cut site while reconstructing the gene, which thereby

[0485] 35 eliminated the dependence on PAM site and gRNA positioning. Initially, this strategy was

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[0487] -54- implemented for the N-terminal labeling (SMART-RC / NT) by including the P2A sequence in the insertion part of repair template, followed by the tag sequence and the silently mutated reconstructive template for the gene repair (FIG. 3D). This enabled elimination of the redundant amino acids encoded by the sequence upstream from the cut site. After ribosomal skipping, the

[0488] 5 protein sequence is the same as before except for a proline and tag added at the very N-terminus (FIG. 3D).

[0489] Both SMART strategies were tested using the same gRNAs that were used for the traditional approach positioned 17 bp (FIGs. 11B-11C) and 142 bp (FIGs. 12B-12C) away from the insertion site. The results with gRNA-17 showed that SMART enhanced the knock-in

[0490] 10 efficiency, achieving a threefold improvement compared to the traditional template (FIGs. 3G, 3K). SMART-RC was also superior to traditional design offering improvement in editing efficiency (FIGs. 31, 3K). When gRNA-142 was used with a distant cut site significant editing efficiency was achieved with SMART-RC performing the best (FIGs. 3H, 3 J, 3L). DNA sequencing confirmed the correct editing of Lmnbl locus targeted by both gRNAs with SMART and SMART-RC / NT.

[0491] To further test the SMART-RC / NT approach, the P2A-mediated cleavage efficiency was directly evaluated. For this purpose, double-tag repair templates were designed for Lmnbl inserting a P2A sequence between Myc and HA tags (FIGs. 13A-13B). The results showed virtually no Myc tag presence following editing confirming efficient separation of the polypeptides by the P2A sequence and degradation of the Myc-tag containing leader sequence (FIG. 13C). This manipulation was specific as scrambling the P2A sequence in the repair template resulted in Myc and HA reactivity being simultaneously detected on nuclear Lamin-B 1 (FIG. 13D).

[0492] Double labeling with rod photoreceptor marker - Gatl confirmed Lmnbl editing in rods.

[0493] 25 Below the rods, the inner nuclear layer (INL) mostly contains various types of bipolar neurons. In line with this, labeling of Lamin-B 1 was observed in these neurons using ON-bipolar marker PKCa. The inner nuclear layer also contains Muller cells and amacrine cells, and their labeling was similarly positively identified with the Glutamine synthetase (GS) marker and AP-2P marker, respectively. GFP-filled edited amacrine cells also had characteristic morphology supporting their identification. Finally, non-neuronal retina pigment epithelial cells are notably located at the tips of the photoreceptors in a dedicated layer and as other cell types their identity was confirmed by double labeling with RPE65 marker. Labeling could not be identified in any ganglion cells, horizontal cells and cone photoreceptors which are post-mitotic at birth.

[0494] Next, different cell types were targeted more directly by labeling marker proteins in them

[0495] 35 with an HA tag using a SMART-RC approach. The SMART template design was further

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[0497] -55- modified to tailor it for introducing sequences at the very end of the molecules (SMART- RC / CT). Specifically, a stop codon was introduced downstream from the HA tag sequence in the insertion position while silently mutating the sequence between the cut site and stop codon (FIG. 14). The function of the inserted stop codon is to terminate the translation of the redundant

[0498] 5 sequence downstream from the cut site. Using this strategy, an HA tag was first introduced at the C-terminus of CTBP2 (FIG. 14). DNA sequencing confirmed that CTBP2 was edited precisely as designed. Analysis of retina cross-sections revealed a characteristic pattern of HA staining as a horseshoe shaped puncta corresponding to rod ribbons and diffuse labeling of bipolar neuron cell bodies in the INL. In GFP-positive rod photoreceptors, horseshoe-shaped

[0499] 10 HA-labeled elements co-localized with marker of synaptic ribbons Bassoon, suggesting that HA-tagged CTBP2 was correctly transported and assembled at the presynaptic release sites. Using this strategy, Glutamine synthase and AP-2P were further successfully labeled, positively identifying Muller glia and amacrine cells, respectively.

[0500] Lineage reprogramming approach for targeting post-mitotic neurons with SMART

[0501] Rod photoreceptor precursors are accessible to CRISPR and can be reprogrammed into cones by silencing transcription factor Nrl that commits them to terminal differentiation into mature rods. This observation was exploited by performing CRISPR-based knockout of Nrl in neonatal retinas via electroporating a cDNA construct that expresses Cas9, GFP reporter and

[0502] 20 gRNA targeting Nrl (FIG. 5A). Reprogramming of a significant number of cells into cones was achieved, as assessed by cone-like morphology of the cells and the appearance of cone- specific marker PNA in cells expressing GFP (FIG. 5B).

[0503] SMART editing was next performed in cones. For this, reprogramming cDNA construct was combined with RNP particles containing gRNA and repair templates for knock-in (FIG. 5 A). Following electroporation at neonatal stage, retinas were dissected and analyzed at P21. First, cone-specific visual pigment that initiates phototransduction, S-opsin, was edited by appending a C-terminal tag to it using the SMART-RC / CT method. Efficient labeling was observed in cone photoreceptors (FIG. 5C). Detailed analysis revealed that HA-tagged S-opsin was co-localized with endogenous S-opsin stained by specific antibodies in the outer segment of

[0504] 30 cones, as expected (FIG. 5D). Next, cone-arrestin, which participates in phototransduction cascade shut off, was targeted by introducing C-terminal HA tag to it with SMART-RC / CT. HA staining appeared in cones where it spread throughout the cell, filling the cytoplasm as expected from the distribution of endogenous cone-arrestin (FIG. 5E). These results demonstrate that editing in post mitotic cells can be achieved using a SMART approach (for example, by

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[0506] -56- combining it with traditional CRISPR-based manipulations targeting factors that determine cell fate).

[0507] Interrogating organization of photoreceptor synapses with SMART editing.

[0508] 5 The entire axonal terminals of rods were first labeled by introducing an HA tag into the C-terminus of PSD95 which demarks this compartment (FIG. 15A). Retinas were electroporated at P0 and dissected at P33. Immuno staining revealed HA signal to clearly outline what morphologically appeared to be rod spherules in a distinctive sparse manner (FIG. 6A). HA immunoreactivity was found in electroporated GFP-positive cells and co-localized with the

[0509] 10 PSD95 signal when detected with specific antibodies against the protein PSD95 (FIG. 6A).

[0510] Next, localization of cell adhesion molecules ELFN1 and ELFN2 expressed by rod and cone photoreceptors, respectively, and involved in trans- synaptic alignment of photoreceptor synapses, were analyzed. For labeling ELFN1, retinas were electroporated at Pl, dissected at P21, sectioned and double- stained with anti-HA and anti-ELFNl antibodies. The C-terminus of ELFN1 was targeted for the incorporation of an HA tag (FIG. 15B) and correct editing was confirmed by DNA sequencing. HA signal was detected as discrete puncta sparsely dotting axonal terminals of rods revealed by electroporation marker GFP. Notably, HA signal was confined to ELFN1 -positive puncta confirming correct targeting of modified protein (FIG. 6B). For labeling the cone specific synaptic molecule ELFN2, a SMART-RC / CT strategy was used, combining the insertion of a C-terminal tag into ELFN2 with reprogramming cellular lineage into cones. Upon immuno staining prominent clustering of HA-labeled synaptic puncta was found within the PNA-positive cone pedicles expressing GFP indicating successful labeling and correct synaptic targeting of ELFN2 (FIG. 6C).

[0511] Next, postsynaptic compartment was defined by editing synaptic genes in ON-bipolar

[0512] 25 neurons. A Gao, a key transducer of neurotransmitter signals abundantly present in the entire dendrites of ON-bipolar cells, was used. Because both termini of Ga proteins are essential for their function, four HA tags were targeted to be inserted at the internal safe harbor position of the protein (FIG. 16A). HA signal was found across the entire cell with prominent labeling of the dendrites where it colocalized with native Gao (FIG. 7A). Trpml, an effector channel that mediates the depolarizing response of ON-bipolar cells to light, was further labeled by introducing a C-terminal HA tag to its sequence. Retinas were electroporated at Pl and harvested at P21. Its HA signal was detected prominently in the synaptic puncta at the tips of the dendrites with a reserve pool detected at the intracellular sites, consistent with reported localization pattern and colocalization with native Trpml (FIG. 7B). Finally, two synaptic

[0513] 35 GPCRs were tagged, mGluR6 and GPR179 involved in the generation of the postsynaptic

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[0515] -57- response. In both cases, SMART-RC / CT templates were used to incorporate C-terminal HA (FIG. 16B). Again, prominent synaptic targeting was detected of both mGluR6 and GPR179 to discrete synaptic puncta at the tips of the ON-bipolar dendrites, consistent with native pattern of these receptors’ localization (FIGs. 7C-7D).

[0516] 5 After confirming the localization of well-established molecules, outstanding issues with synaptic molecules were probed. Previous reports indicate it is difficult to define whether synaptic molecules are localized on the pre-synaptic or post-synaptic site. When detected by immunohistochemical approaches, synaptic molecules appear as discrete puncta at all synapses and the resolution of traditional confocal microscopy is not sufficient to assign their location to

[0517] 10 pre- vs. post-synaptic compartment. It was reasoned that a SMART approach should allow significant insights into resolving these issues by virtue of the sparse labeling it achieves. This is exemplified by photoreceptor synapses where dendritic arbor of a single bipolar neuron contacts multiple (-25-35) photoreceptors (FIG. 8A). This creates a situation where the cellular origin of the synaptic molecule could be discerned by confocal microscopy from a pattern of its distribution in the synaptic layer. For example, when a gene encoding synaptic protein is edited in a rod, this is expected to generate sparse synaptic puncta evenly spaced throughout multiple dendritic trees of bipolar cells. In contrast, when a gene encoding synaptic protein is edited in a bipolar cell, tagged protein is expected to appear in a closely spaced cluster within a dendritic tree of a bipolar cell (FIG. 8A).

[0518] This reasoning was used to determine the site of localization for two synaptic molecules for which the site of origin has not been unequivocally established. LRIT3, a cell adhesion molecule which leads to complete congenital stationary night blindness (cCSNB) when mutated, was labeled at the C terminus with SMART-RC / CT (FIG. 17A). Immuno staining revealed that HA signal was detected as sparsely distributed with only 1-2 puncta per each dendritic arbor of

[0519] 25 ON-bipolar cells marked by PKCa staining (FIG. 8B). Double labeling with anti-HA and anti- LRIT3 antibodies confirmed labeling specificity. These observations suggest that LRIT3 was confined to pre-synaptic compartment and was expressed by photoreceptors. Lrit3 was then labeled in cone photoreceptors using a reprograming strategy. Again, very sparse HA signal was observed, appearing as clusters within PNA-positive cone pedicles marked by GFP. This indicates that LRIT3 is prominently present in cone synapses too and confirms its presynaptic origin.

[0520] Next, Nyctalopin, a cell adhesion molecule which leads to Congenital Stationary Night Blindness (cCSNB) when mutated, was labeled with an HA tag inserted after its signal peptide at the N-terminus by SMART (FIG. 17B). HA signal was detected as clusters of closely spaced

[0521] 35 puncta in OPL. Double staining with PKCa antibody revealed that all puncta were confined

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[0523] -58- within the dendritic tree of a single ON-bipolar cell (FIG. 8C). These data suggested that Nyctalopin is postsynaptic and exclusively expressed by bipolar cells.

[0524] Multiplexing SMART tagging with CRISPR-based knockout

[0525] 5 Multiplexing the SMART approach with traditional CRISPR-mediated knockout technique was examined. For this, the well-established role of key postsynaptic neurotransmitter receptor in ON-bipolar neurons, mGluR6, in controlling the localization of its downstream effector, Trpml channel, was examined. A two-component system was used. One part of it was the exact SMART-RC / CT based RNP system that was used to tag Trpml at the C-terminus. The

[0526] 10 other comprised a plasmid encoding eCas9 nuclease separated by a T2A sequence from the GFP expression marker driven by CAG promoter and 2 different gRNAs of traditional design targeting mGluR6 without repair template driven by U6 promoter (FIG. 9A). Examination of electroporated retinas confirmed expected labeling of TRPM1 with an HA tag based on its characteristic distribution in dendritic puncta and cell bodies. Endogenous mGluR6 was further stained with specific antibodies and confirmed its punctate synaptic localization throughout the OPL. Many GFP-positive bipolar neurons were identified. The dendritic areas of the GFP- positive bipolar neurons lacked mGluR6 puncta indicating that its CRISPR-based knockout was successful.

[0527] When examining the co-localization of Trpml -HA and mGluR6 - two different patterns were observed. In GFP negative cells where mGluR6 was detected, HA signal was found in the cell body and detected in the OPL as puncta overlapping with mGluR6 signal (FIG. 9B). However, in GFP positive cells where mGluR6 was not detected, HA signal was not detected as puncta in the OPL, indicating mGluR6 is important for the synaptic localization of Trpml (FIG. 9C). These results indicated that SMART labeling approach can be readily combined with

[0528] 25 traditional CRISPR-based gene knockout techniques to study relationships between proteins in native neuronal tissues.

[0529] This allows for virtually limitless selection of gRNA thereby greatly improving editing efficiency at a precisely determined position. The use of RNP complexes were further leveraged, obviating the need to genetically express Cas9 and optimized conditions for their in vivo delivery in postnatal neurons. Genetic modifications were performed in the mammalian retina, a sensory part of the central nervous system that mediates light reception. The ability to label various types of proteins was demonstrated in nearly all neuronal and non-neuronal cell types in the retina with high efficiency. In addition, a lineage reprogramming strategy was adopted in which transcription factors that determine cell fate were concurrently targeted thereby

[0530] 35 converting the precursors of the cells in which editing had occurred into the cell type of choice.

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[0532] -59-

[0533] M at erials and Methods

[0534] Mice

[0535] C57BL / 6 mice were purchased from Jackson Laboratories and used for experiments as

[0536] 5 well as for line maintenance. Procedures involving mice strictly followed NIH guidelines and were approved by the Institutional Animal Care and Use Committees at UF Scripps Biomedical Research.

[0537] Antibodies

[0538] 10 Antibody information used in the analyses described in this Example is listed in Table 1.

[0539] Table 1. Antibody Information

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[0541] -60-

[0542] DNA Constructs pCAG-GFP plasmid: Addgene plasmid # 11150. pCAG-eCas9-GFP-U6-gRNA:

[0543] Addgene plasmid # 79145. The plasmid was engineered by removing the AAV2 ITR and fl ori

[0544] 5 elements. Then, a pair of annealed oligos (~21 bp) were cloned into the sgRNA scaffold of the plasmid to express sgRNA knocking out Nrl. To knock out Grm6, two DNA fragments containing two gRNA sequences were synthesized from Integrated DNA Technologies (IDT) and cloned into BbsI sites of the Cas9-expression plasmid by In-Fusion cloning. The sequences of oligos and DNA fragments are shown in “Oligonucleotides and DNA fragments for molecular

[0545] 10 cloning”. All the constructs were sequenced to confirm their correct sequences.

[0546] Oligonucleotides and. DNA Fragments for Molecular Cloning gRNAs were designed using both Benchling and Cas9 crRNA design tool from IDT. The crRNA and tracrRNA (IDT, 1072533) were synthesized from IDT and annealed to form gRNAs.

[0547] 15 Sequences of the gRNAs used in the analyses in this Example are shown in Table 2.

[0548] Table 2. Non-limiting Examples of gRNA Sequences

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[0550] -61-

[0551] Traditional templates and SMART-RC templates were designed based on the non-target DNA strand to avoid base pairing between gRNAs and repair templates when they are mixed together. SMART templates were designed based on the coding strand when the cut site was downstream of the insertion site (FIG. 11B, FIG. 12B and FIG. 17B). When the cut site was

[0552] 5 upstream of the insertion site, they were designed from the template strand (FIG. 16A). The rationale behind this design was to initiate DNA replication immediately after the repair template base pairs with the broken DNA strand. Otherwise, DNA replication would not be started until the 3’ flap fragment between the cut and insertion site is cleaved. Silent mutations were made based on the mouse genetic codon frequency table (genscript.com / tools / codon-

[0553] 10 frequency-table), wherein one codon was replaced by another codon with a closely matching frequency. All the repair template sequences were designed using SnapGene software.

[0554] Single-stranded DNAs were synthesized from IDT working as repair templates with a maximum length of 200 nucleotides. The template nucleic acids used in the analyses described in this Example are shown in Table 3.

[0555] 15 Table 3. Non-limiting Examples of Single-stranded DNA repair template sequences

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[0557] -62-

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[0559] -63-

[0560] *KEY for Sequences in Table 2: upper case = HA tag sequence; bold = Myc tag sequence; Italic

[0561] = P2A sequence; underline = stop codon; border = overlap between two ssODNs

[0562] The primer sequences used in the analyses described in this Example are shown in 5 Tables 4 and 5.

[0563] Table 4. PCR primer sequences (shown 5'-3')

[0564] Table 5. DNA sequencing primers (shown 5'-3')

[0565] Nucleic acids used for editing of Grm6 as described in this Example is shown in Table 6.

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[0567] -64-

[0568] Table 6. Non-limiting Example of a DNA Fragment comprising two gRNA sequences for knocking out Grm6

[0569] KEY for Table 6: sequences shown 5’-3’; upper case = gRNA sequences; bold = gRNA scaffold; underline = U6 promoter; border = overlap between two ssODNs

[0570] 5

[0571] Preparation of SMART -editing Mixture

[0572] Synthesized crRNA and tracrRNA were dissolved in Nuclease-Free Duplex Buffer (IDT, 11-01-03-01) to a concentration of 400 pM. Then crRNA and tracrRNA were hybridized in a 1:1 molar solution by incubating at 98°C for 3 min, followed by cooling to 22°C at a rate of -0.1 °C per second, resulting in a final gRNA concentration of 200 pM.

[0573] To prepare a 10 pl mixture for labeling proteins in mitotic cells:

[0574] 1. 3 pl of gRNA was combined with 2 pl of Cas9 (IDT, 1081058; with a stock concentration of 10 pg / pl) and incubated at room temperature (RT) for 30 minutes.

[0575] 2. To this mixture, 3 pl of ssDNA repair template (with a stock concentration of 200 pM, dissolved in Nuclease-Free Duplex Buffer), 1 pl of pCAG-GFP plasmid (with a stock concentration of 14 pg / pl, dissolved in double-distilled water), and 1 pl of Fast Green (with a stock concentration of 10 mg / mE, dissolved in lOxPBS) were added. The resultant mixture was then used for subretinal injection.

[0576] To prepare a 10 pl mixture for tagging cone-specific molecules, or for a combination of knock- in and knockout:

[0577] 1. 2.5 pl of gRNA was mixed with 1.5 pl of Cas9 and then incubated at RT for 30 minutes.

[0578] 25 2. To this mixture, 2.5 pl of ssDNA repair template, 2.5 pl of pCAG-eCas9-GFP-U6- gRNA plasmid (with a stock concentration of 11 pg / pl), and 1 pl of Fast Green were added. The mixture was used for subretinal injection.

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[0580] -65-

[0581] Subretinal Injections and Electroporation

[0582] P0 / P1 mice were anesthetized by chilling on ice for around 1 minute. Longer exposure to ice reduces mouse viability. A small incision was made in the eyelid and sclera near the cornea of the right eye using the tip of a sharp 30-gauge needle (BD, 305106). Subsequently, 0.5 pl of

[0583] 5 SMART-editing mixture was injected subretinally using a Hamilton syringe with a 32-gauge blunt-ended needle (catalog #87931). Following injection, tweezer- shaped electrodes (7 mm Platinum Tweezertrodes, BTX450488) coated with electrode gel (Spectra 360, Parker Laboratories, INC.) were positioned to clamp gently the pup’s head. 10 square pulses (105V, 60- ms duration, 1-s interval) were applied by using a pulse generator (Electro Square Porator, ECM

[0584] 10 830, BTX / Harvard Apparatus). Among many factors affecting genome editing efficiency in the retina, electroporation voltage plays a dominant role. Enhancing voltage can increase the editing efficiency but decrease mice viability (FIGs. 10B-10C). Electroporated mice were warmed by a heat blanket until they awoke, and then returned to their parents.

[0585] To target retinal neurons, the plus electrode should be at the injection side of the mouse head. Conversely, for genome editing in RPE cells, it should be positioned on the non-injection side.

[0586] Immunohistochemistry

[0587] The electroporated eyeballs were dissected and punctured in the sclera near the cornea, which were then fixed in 4% paraformaldehyde for 30 min and cryoprotected with 30% sucrose in PBS overnight at 4°C. The sclera and lens were kept with the retina during dehydration to preserve retina morphology. Dehydrated retinas were then carefully peeled from the eyeballs and examined for GFP fluorescence under a fluorescence microscope (Leica DM5500 B). GFP- positive retinas were incubated in 1:1 mixture of 30% sucrose and optimal cutting temperature (OCT) medium for 30 minutes, followed by immersion in pure OCT for 30 minutes at room

[0588] 25 temperature (RT) before being embedded in OCT. Twenty-micron-thick frozen sections were cut, mounted onto slides, and blocked with 10% donkey serum in PBS containing 0.1% Triton X-100 (PBST) overnight at 4°C. They then were incubated with the primary antibody in 2% donkey serum in PBST for at least 1 hour. After five washes with PBST, sections were incubated with fluorophore-conjugated secondary antibodies in 2% donkey serum in PBST for 30 min. After four washes with PBST and two with PBS, sections were mounted in DAPI Fluoromount (SouthemBiotech, 0100-20). Images were taken with Zeiss LSM 880 confocal microscope and Olympus FV3000 confocal microscope. For ELFN1-HA and Lrit3-HA staining, the slices were pretreated with Antigen Retrieval Reagents of basic condition (R&D Systems, CTS016) before the blocking step. For HA-nyctalopin staining, Antigen Retrieval Reagents of

[0589] 35 universal condition were used for pretreating retina sections. For PNA staining, slices were

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[0591] -66- incubated with Alexa Fluor™ 647 Conjugated Lectin PNA (Thermo Fisher, L32460) at a dilution of 1:100.

[0592] DNA sequencing

[0593] 5 Genomic DNA was extracted from GFP-positive retinas using DNeasy Blood & Tissue Kit (Qiagen, 69504). The edited regions of targeted genes were amplified by PCR with the primers listed in “PCR primer sequences” and indicated in FIG. 10D. To increase the yield, the PCR products were used as templates and amplified again using the same primers. These products were then purified using the Gel and PCR Clean-up kit (MACHEREY-NAGEL,

[0594] 10 740609.50) and subsequently sent to Genewiz or Eurofins for DNA sequencing using the primers listed under “DNA sequencing primers”.

[0595] Statistical Methods

[0596] GraphPad Prism 10 was used for statistical analyses. Unless otherwise noted, a minimum of three independent samples were compared. In bar graphs, statistical significance was denoted by * for P<0.05, ** for P<0.01, *** for P<0.001, and **** for P<0.0001. Where relevant, 'not significant' (ns) was marked. All data is presented as mean ± SD, with each data point indicating n.

[0597] Example 2: Improved Genome Editing Using SMART Template Design

[0598] With traditional templates, knock-in using CRISPR / Cas9 is guided by gRNAs to specific locations in the genome, which is inflexible given the need to make intended alterations. For example, epitope tags are usually introduced at the N- or C- termini of the protein to minimize the risks of disrupting its folding. This constrains the selection of gRNAs to be positioned in

[0599] 25 close proximity to the start or stop codons. This requires finding a nearby PAM site that determines the Cas9 cleavage site for the design of gRNA. The distance between the cleavage site and insertion site is critical for the recombination efficiency, and as it exceeds 10 base pairs, the knock-in efficiency decreases significantly.

[0600] Analyses were performed where putative modification sites were randomly picked across the coding regions of the human genome and were surveyed to assess the availability of nearby PAM sites. FIG. 18 shows an in silico analysis of 145,442 sites across the coding sequences of the human genome quantifying the PAM sites within 10 bp distance of the intended modification site. Using 60% on-target efficiency as a threshold, the percentage of sites with at least one effective gRNA was calculated. The results indicated that 8.2% of these sites lacked

[0601] 35 any PAM sites within 10 bp of the target location. On average, 3 PAM sites were available per

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[0603] -67- site, with two being the most common (FIG. 18). Considering that 75.7% of gRNAs on-target efficiency is lower than 60%, these results indicated that -50% of target sites lack efficient gRNAs (FIG. 18).

[0604] Therefore, using traditional templates can involve also using inefficient gRNAs, which

[0605] 5 compromises the editing efficiency.

[0606] FIGs. 19A-19D show SMART template design greatly improves targeted knock-in efficiency. FIG. 19A shows targeting Lmnbl using an efficient gRNA, and an EcoRI site was inserted at various positions using either traditional templates or the SMART. The edited region was amplified by PCR, digested with the EcoRI restriction enzyme, and analyzed by gel

[0607] 10 electrophoresis. Band migration was observed in the SMART group as the insert position moved away from the cut site. FIGs. 19B-19C show analyses of the knock-in efficiency (KI%) as a function of distance between the cut and insertion sites for Lmnbl (left panel) and the CXCR4 editing (right panel). When using traditional templates, knock-in efficiency decreased exponentially as the cut-to-insert distance increased in both cases. When using the SMART method, after the initial drop, the efficiency remained similar, n = 3 repeats. Data are presented as mean values ± SD. FIG. 19D shows a schematic illustrating the assay of decreasing silent mutations. The target gene, Lmnbl, was edited by inserting an EcoRI site 41 bp away from the cut site. FIG. 19E shows quantification of knock-in efficiency for assays targeting Lmnbl (left panel) and CXCR4 (right panel) with decreasing silent mutations, n = 4 independent biological

[0608] 20 replicates. *p<0.05, t-test (unpaired, one-tailed). Data are presented as mean values ± SD. Source data are provided as a Source Data file where exact p-values are also reported.

[0609] These results indicated that, with traditional templates, knock-in (KI) efficiency decreased exponentially as the cut-to-insert distance increased. However, with SMART, the decrease in the KI efficiency was substantially attenuated. The KI efficiency at distances between 40 and 101 bp from the cut site remained around half of that observed at the optimal position, effectively expanding the range where a gRNA can be used for modification. It further indicated that gRNAs located far from the modification site can be successfully used for genome editing when paired with SMART. These findings were further supported by next-generation sequencing (NGS) of more alleles with a PAM mutation but without EcoRI insertion when

[0610] 30 repaired by traditional templates. The results revealed repair to the original sequence confirming that the gap sequence in the traditional repair templates functioned as a homology arm during HDR. The NGS data also showed that SMART increased the proportion of desired edits without influencing on / off-target efficiencies. Additionally, these results indicated that, when the extent of silent mutagenesis in SMART was varied, fewer silent mutations lead to decreased KI

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[0612] -68- efficiency and that mutating the region near the cut site can improve editing as compared to mutating the region near the insert site.

[0613] Using a side-by-side evaluation for target genes in the retina, CRISPR / Cas9 in vivo editing performance and advantages of the SMART approach relative to traditional template

[0614] 5 design was analyzed. FIGs. 20A-20B shows a comparison of editing efficiency between traditional and SMART templates. FIG. 20A shows SMART templates dramatically enhance editing efficiency compared to traditional templates. Editing efficiency was calculated as the ratio of the number of HA puncta (for LRIT3 and ELFN1) or HA+ cells (formGluR6 and Nyctalopin) to that of LRIT3 / ELFN1 puncta (for LRIT3 and ELFN1) or PKCa+ cells (for

[0615] 10 mGluR6 and Nyctalopin). n =3 retina samples from 3 mice for each gene editing. *p<0.05, t-test (unpaired, one-tailed). Data are presented as mean values ± SD. FIG. 20B shows SMART templates dramatically enhanced the normalized editing efficiency compared to traditional templates. For FIGs. 20A-20B, editing results that were observed for traditional template are represented by the leftmost bars in each set of two bars above the four target genes along the x- axis. Editing results that were observed for SMART templates are represented by the rightmost bars in each set of two bars above the four target genes along the x-axis. The editing efficiency (H) was normalized to transduction efficiency (GFP+ cells). The normalized editing efficiency was calculated as the ratio of the number of HA puncta (for ERIT3 and EEFN1) or HA-positive cells (formGluR6 and Nyctalopin) to that of GFP+ photoreceptors (for ERIT3 and EEFN1) or

[0616] 20 GFP+ bipolar cells (for mGluR6 and Nyctalopin). n = 3 retina samples from 3 mice for each gene editing. *p<0.05, t-test (unpaired, one-tailed). Data are presented as mean values ± SD. Source data are provided as a Source Data file where exact p-values are also reported.

[0617] These results indicated that, with traditional templates, gRNA selection was limited to a 20 bp range containing ~3 gRNAs on average, whereas the SMART approach expanded this range at least ten-fold. Furthermore, SMART-RC aligned insertion and cut sites to enable gene reconstruction, thereby avoiding the positional limitations imposed by PAM sites, which significantly expanded gRNA selection. Indeed, many gRNAs within or beyond the 20-bp constrained range either resulted in inefficient labeling or completely failed to label synaptic proteins when using traditional templates. In contrast, when paired with the same gRNAs,

[0618] 30 SMART templates successfully labeled synaptic proteins, dramatically increased editing efficiency by at least an order of magnitude. This direct comparison indicated that the SMART editing approach is transformational and allowed editing of intractable genes.

[0619] Example 3: SMART-RC / CDS

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[0621] -69-

[0622] This Example relates to an application of SMART templates that can used in, for example, gene therapy, which was developed by integrating SMART-RC / NT and SMART- RC / CT. FIG. 21 shows non-limiting embodiments of a schematic illustrating non-limiting embodiments of SMART-RC / CDS used for homology-directed repair in inserting a coding

[0623] 5 sequence (CDS) of a gene, or a portion thereof, into a DNA target. SMART-RC / CDS is designed to insert the entire CDS of a gene into the target locus, combining advantages of both SMART-RC / NT and SMART-RC / CT. The template comprises an insertion cassette comprising: a self-cleaving peptide sequence (e.g., a P2A sequence); a CDS (e.g., wherein the CDS comprises a variant sequence, such as a silently mutated sequence, as described herein) or a portion thereof; and a stop codon. In some embodiments, a template comprises one or more additional regulatory sequences (e.g., one or more regulatory sequences in a heterologous nucleic acid and / or one or more regulatory sequences in a homology arm, as described herein). In some embodiments, a 3' UTR sequence can be incorporated downstream of the stop codon.

[0624] Many genetic diseases, disorders, or conditions are associated with diverse mutations

[0625] 15 across the patient population. Therapies targeting a single mutation can benefit only a small subset of patients for certain diseases, disorders, or conditions. SMART-RC / CDS enables a “one-for-all” gene therapy approach, which is capable of treating diseases, disorders, or conditions caused by different mutations by selecting a PAM site and target sequence that are located in a mutation-free region.

[0626] 20

[0627] INCORPORATION BY REFERENCE

[0628] The present application refers to various issued patent, published patent applications, scientific journal articles, and other publications, all of which are incorporated herein by reference. The details of one or more embodiments of the invention are set forth herein. Other

[0629] 25 features, objects, and advantages of the invention will be apparent from the Detailed Description, the Figures, the Examples, and the Claims.

[0630] EQUIVALENTS AND SCOPE

[0631] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described

[0632] 35 herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or

[0633] #14289420v1 PCT / US25 / 41648 12 August 2025 (12.08.2025)

[0634] -70- configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented

[0635] 5 by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present application are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if

[0636] 10 such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present application.

[0637] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0638] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0639] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0640] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically

[0641] 25 identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0642] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted

[0643] 35 items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or,

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[0645] -71- when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting

[0646] 5 essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0647] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily

[0648] 10 including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A,

[0649] 20 and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0650] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0651] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States

[0652] 30 Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also

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[0654] -72- contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

[0655] #14289420v1

Claims

CLAIMSWhat is claimed is:

1. A nucleic acid comprising:(i) a first homology arm comprising homology to a first region of a DNA target and a second homology arm comprising homology to a second region of the DNA target; and(ii) a heterologous nucleic acid flanked by the first homology arm and the second homology arm, wherein the heterologous nucleic acid comprises a variant of a sequence in the DNA target, wherein the variant comprises one or more synonymous mutations relative to the sequence in the DNA target.

2. The nucleic acid of claim 1, wherein the heterologous nucleic acid comprises a sequence encoding an RNA.

3. The nucleic acid of claim 2, wherein the sequence encoding the RNA comprises the variant of the sequence in the DNA target.

4. The nucleic acid of claim 2 or 3, wherein the nucleic acid is configured to integrate the sequence encoding the RNA at an insertion site that is flanked by the first region and the second region and located in a gene that comprises the sequence that corresponds to the variant.

5. The nucleic acid of any one of claims 2-4, wherein the heterologous nucleic acid comprises:(a) a splicing acceptor site, wherein a splicing donor site is located 5’ relative to an insertion site that is flanked by the first region and the second region;(b) a sequence encoding a protease cleavage site that is located 5’ relative to the sequence encoding the RNA;(c) a sequence encoding a self-cleaving peptide that is located 5’ relative to the sequence encoding the RNA;(d) a transcription termination sequence that is located 3’ relative to the sequence encoding the RNA;(e) a splicing donor site, wherein a sequence comprising a splicing acceptor site is located 3’ relative to an insertion site that is flanked by the first region and the second region;#14289420v1(f) a start codon that is operably linked to the sequence encoding the RNA; and / or(g) a stop codon that is operably linked to the sequence encoding the RNA.

6. The nucleic acid of any one of claims 1-5, wherein the nucleic acid is configured to integrate a sequence in the heterologous nucleic acid at an insertion site that is separated from a protospacer adjacent motif (PAM) by at least 10 nucleotide positions in the DNA target.

7. The nucleic acid of any one of claims 1-6, wherein the first region or the second region comprises a protospacer adjacent motif (PAM).

8. The nucleic acid of any one of claims 1-7, wherein the variant comprises one or more non-synonymous mutations relative to the sequence in the DNA target.

9. The nucleic acid of any one of claims 1-8, wherein the nucleic acid comprises at least one regulatory sequence.

10. The nucleic acid of claim 9, wherein the least one regulatory sequence comprises:(a) a regulatory sequence operably linked to the variant of the sequence in the DNA target; and / or(b) a regulatory sequence operably linked to a sequence comprising the first homology arm, the heterologous nucleic acid, and the second homology arm.

11. The nucleic acid of claim 9 or 10, wherein the at least one regulatory sequence comprises a promoter.

12. The nucleic acid of any one of claims 1-11, wherein the nucleic acid comprises a sequence encoding a guide RNA (gRNA) and / or a sequence encoding an RNA-guided nuclease.

13. The nucleic acid of claim 12, wherein the gRNA comprises a sequence that hybridizes to a sequence located at least one nucleotide upstream or downstream of a protospacer adjacent motif (PAM), wherein the PAM is located in the first region or the second region.

14. The nucleic acid of claim 12 or 13, wherein the gRNA is a single-guide RNA (sgRNA).#14289420v115. The nucleic acid of any one of claims 12-14, wherein the RNA-guided nuclease is a Cas nuclease selected from the group consisting of: a Cas9 nuclease or a variant thereof; a Cas 12 nuclease or a variant thereof; a Cas 13 nuclease or a variant thereof; and a Cas 14 nuclease or a variant thereof.

16. The nucleic acid of any one of claims 12-15, wherein the sequence encoding the gRNA and / or the sequence encoding the RNA-guided nuclease are operably linked to at least one regulatory sequence.

17. The nucleic acid of any one of claims 1-16, wherein the first homology arm, the second homology arm, and / or the heterologous nucleic acid comprises a heterologous protospacer adjacent motif (PAM).

18. The nucleic acid of any one of claims 1-17, wherein the nucleic acid:(a) is a linear nucleic acid or a circular nucleic acid;(b) comprises deoxyribonucleotides, ribonucleotides, or a combination thereof;(c) is a single-stranded nucleic acid, a double- stranded nucleic acid, or comprises one or more single- stranded stretches of sequence and one or more double- stranded stretches of sequence; and / or(d) comprises one or more chemical modifications.

19. The nucleic acid of any one of claims 1-18, wherein the nucleic acid comprises a sequence which is at least 80% identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-62.

20. A guide RNA (gRNA) comprising any one of the nucleotide sequences set forth in SEQ ID NOs: 1-20, optionally wherein the gRNA:(i) comprises one or more chemical modifications; and / or(ii) is a single-guide RNA (sgRNA).

21. A recombinant adeno-associated virus (rAAV) particle comprising:(i) the nucleic acid of any one of claims 1-19 and / or the gRNA of claim 20; and(ii) at least one adeno-associated virus (AAV) capsid protein.#14289420v122. A cell or a cell population thereof comprising the nucleic acid of any one of claims 1-19 and / or the gRNA of claim 20.

23. The cell or the cell population thereof of claim 22, wherein the cell is a mammalian cell.

24. A composition comprising the nucleic acid of any one of claims 1-19, the gRNA of claim 20, the rAAV particle of claim 21, and / or the cell or the cell population thereof of claim 22 or 23.

25. A kit comprising the nucleic acid of any one of claims 1-19, the gRNA of claim 20, the rAAV particle of claim 21, the cell or the cell population thereof of claim 22 or 23, and / or the composition of claim 24.

26. A method comprising introducing the nucleic acid of any one of claims 1-19 or the gRNA of claim 20 into a cell.

27. The method of claim 26, wherein:(i) the cell comprises a nucleic acid encoding a guide RNA (gRNA) and / or an RNA guided-nuclease; or(ii) the method comprises introducing a guide RNA (gRNA) and / or an RNA-guided nuclease into the cell or a descendant thereof.

28. The method of claim 27, wherein the gRNA hybridizes with a sequence positioned at least one nucleotide upstream or downstream of a protospacer adjacent motif (PAM) in the first region or the second region of the DNA target.

29. The method of claim 27 or 28, wherein the gRNA and the RNA-guided nuclease of is contacted with the cell as a pre-formed ribonucleoprotein (RNP) complex.

30. The method of any one of claims 26-29, wherein the nucleic acid comprises a sequence encoding a guide RNA (gRNA) and / or a sequence encoding an RNA-guided nuclease.

31. The method of any one of claims 26-30, wherein the introducing the nucleic acid into the cell comprises contacting the cell with a recombinant adeno-associated virus (rAAV) particle comprising the nucleic acid and at least one adeno-associated virus (AAV) capsid protein.#14289420v132. The method of any one of claims 26-31, wherein the method comprises generating at least one genetic edit in the cell, wherein the at least one genetic edit comprises the heterologous nucleic acid or a portion thereof.

33. The method of any one of claims 26-32, wherein the cell is a mammalian cell.

34. The method of any one of claims 26-33, wherein the cell comprises the DNA target.

35. The method of claim 34, wherein the cell or a descendant thereof comprises an edited DNA target after being contacted with the nucleic acid, wherein the edited DNA target comprises at least one genetic edit that is absent in a counterpart cell or a sample derived from the counterpart cell not contacted with the nucleic acid.

36. A method comprising administering the nucleic acid of any one of claims 1-19 to a subject.

37. The method of claim 36, wherein the administering the nucleic acid comprises administering a recombinant adeno-associated virus (rAAV) particle comprising the nucleic acid and at least one adeno-associated virus (AAV) capsid protein to the subject.

38. The method of claim 36 or 37, wherein the subject is a mammal.

39. The method of claim 38, wherein the mammal is a human.

40. The method of any one of claims 26-39, wherein the nucleic acid is contacted with a neuronal cell.

41. The method of any one of claims 26-39, wherein the nucleic acid is contacted with a non-neuronal cell.

42. The method of any one of claims 26-40, wherein the nucleic acid is contacted with a retinal cell.#14289420v1

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