Modified st1cas9 guide nucleic acids

Abstract

The disclosure provides, e.g., compositions, systems, and methods for targeting, editing, modifying, or manipulating a host cell's genome at one or more locations in a DNA sequence in a cell, tissue, or subject. Improved gRNA scaffolds compatible with StlCas9 are described. For instance, certain patterns of chemical modifications on gRNA scaffolds are provided.

Description

[0001] MODIFIED ST1CAS9 GUIDE NUCLEIC ACIDS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 565,379, filed March 14, 2024; the title of which is “MODIFIED ST1CAS9 GUIDE NUCLEIC ACIDS” and the content of which is incorporated herein by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML format compliant with WIPO Standard ST.26 and is hereby incorporated by reference in its entirety. Said XML copy, created on March 14, 2025, is named 2017469_0038.XML and is 15,726,980 bytes in size.

[0006] BACKGROUND

[0007] Integration of a nucleic acid of interest into a genome occurs at low frequency and with little site specificity, in the absence of a specialized protein to promote the insertion event. Some existing approaches, like CRISPR / Cas9, are more suited for small edits that rely on host repair pathways and are less effective at integrating longer sequences. Other existing approaches, like Cre / loxP, require a first step of inserting a loxP site into the genome and then a second step of inserting a sequence of interest into the loxP site. There is a need in the art for improved compositions (e.g., proteins and nucleic acids) and methods for inserting, altering, or deleting sequences of interest in a genome.

[0008] Alpha- 1 antitrypsin deficiency (AATD) is characterized by low circulating levels of alpha- 1 antitrypsin (AAT). AAT is produced primarily in liver cells and secreted into the blood, but it is also made by other cell types including lung epithelial cells and certain white blood cells. AAT inhibits several serine proteases secreted by inflammatory cells (most notably neutrophil elastase [NE], proteinase 3, and cathepsin G) and thus protects organs, such as the lung, from protease-induced damage, especially during periods of inflammation.

[0009] The two most common clinical variants of AAT are E264V (PiS) and E342K (PiZ). The clinical E342K (PiZ) mutation (also referred to as the Z mutation) is caused by a single base-pair substitution in the SERPINA gene (referred to as the Z allele) and results in a glutamic acid to lysine mutation at position 342 of AAT. Inheritance of the Z allele is autosomal codominant and more than half of AATD patients harbor at least one copy of the Z allele. The E342K mutation leads to structurally unstable and / or inactive AAT-Z protein that causes toxicity in the liver and is inactive in the lungs. The E342K mutation is located at the hinge between the beta sheet and the Reactive Center Loop (RCL) of the AAT protein and causes a loop-sheet dimer that can extend to form long chains of loop-sheet polymers. These polymers form aggregates that accumulate inside the rough endoplasmic reticulum of hepatocytes during translation and are therefore not secreted into the bloodstream. Consequently, circulating AAT levels in individuals homozygous for the Z allele (PiZZ) are markedly reduced; only approximately 15% of mutant AAT-Z protein folds correctly and is secreted by the cell. An additional consequence of the Z mutation is that the secreted AAT-Z protein has reduced activity compared to wild-type protein, with 40% to 80% of normal antiprotease activity (American thoracic society / European respiratory society, Am J Respir Crit Care Med. 2003; 168(7):818-900; and Ogushi et al. J Clin Invest. 1987; 80(5): 1366-74, herein incorporated by reference in their entirety).

[0010] There are two disease phenotypes associated with the PiZZ genotype. A gain-of - function phenotype presents as the accumulation of polymerized AAT-Z protein in hepatocytes results in a gain-of-function cytotoxicity that can result in cellular stress, inflammation, fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and neonatal liver disease in 12% of patients. This accumulation may spontaneously remit but can be fatal in a small number of children. A loss-of- function phenotype results from the reduced systemic levels of AAT that lead to increased protease digestion of connective tissue in the lower airway. Excess protease-digestion of the connective tissues and alveolar linings deteriorates lung elasticity and pulmonary function, leading to emphysema, a hallmark of Chronic Obstructive Pulmonary Disease (COPD). This effect is severe in PiZZ individuals and typically manifests in middle age, resulting in a decline in quality of life and shortened lifespan (mean 68 years of age) (Tanash et al. I nt J Chron Obstruct Pulm Dis. 2016; 11:1663-9, herein incorporated by reference in its entirety). The effect is more pronounced in PiZZ individuals who smoke, resulting in an even further shortened lifespan (58 years) (Piitulainen and Tanash, COPD 2015; 12( l):36-41 , herein incorporated by reference in its entirety). PiZZ individuals account for the majority of patients with clinically relevant AATD lung disease.

[0011] A milder form of AATD is associated with the SZ genotype in which a patient has a Z allele and an S allele. The S allele is associated with somewhat reduced levels of circulating AAT but the AAT S-protein is not hepatotoxic. Accordingly, the SZ genotype is associated with clinically significant lung disease but not liver disease. Frcgoncsc and Stolk, Orphanct J Rare Dis. 2008; 33:16. As with the PiZZ genotype, the deficiency of circulating AAT in subjects with the SZ genotype results in dysregulated protease activity that degrades lung tissue over time and can result in emphysema, particularly in smokers.

[0012] While limited treatment options for AATD exist, there is currently no cure. A small fraction of newborn patients and patients having advanced stage liver disease undergo liver transplant. The current standard of care for AATD patients who have or show signs of significant or developing lung disease is augmentation therapy or protein replacement therapy. Augmentation therapy involves administration (weekly infuction) of a human AAT protein concentrate purified from pooled from healthy donor plasma. Although infusions of plasma protein have been shown to improve survival or slow the rate of emphysema progression, augmentation therapy is often insufficient under challenging conditions (e.g., active lung infection). Augmentation therapy also fails to restore the normal physiological regulation of AAT in patients and efficacy has been difficult to demonstrate. In addition, augmentation therapy does not remedy liver disease driven by the toxic gain-of-function of the Z allele. Accordingly, there is a need for new and more effective treatments for AATD.

[0013] SUMMARY OF THE INVENTION

[0014] This disclosure relates to novel compositions, systems, and methods for altering a genome at one or more locations in a host cell, tissue, or subject, in vivo or in vitro. The disclosure provides, for instance, gene modifying systems that comprise a gene modifying polypeptide comprising a reverse transcriptase (RT) domain and a StlCas9 domain, and a template RNA comprising a variant gRNA scaffold that has been engineered for improved performance, e.g., when used in concert with the StlCas9 domain. The disclosure also provides gene modifying systems that are capable of modulating (e.g., inserting, altering, or deleting sequences of interest) alpha- 1 antitrypsin (AAT) activity and methods of treating alpha- 1 antitrypsin deficiency (AATD) by administering one or more such systems to alter a genomic sequence at a single nucleotide to correct the SERPINA1 PiZ mutation that causes AATD.

[0015] In some embodiments, the present disclosure provides a system for modifying DNA to correct a human SERPINA1 gene mutation that causes AATD comprising (a) a nucleic acid encoding a gene modifying polypeptide capable of target primed reverse transcription, the polypeptide comprising (i) a reverse transcriptase domain and (ii) a St1Cas9 nickase that binds DNA and has endonuclease activity, and (b) a template RNA comprising (i) a gRNA spacer that is complementary to a first portion of the human SERPINA1 gene, (ii) a gRNA scaffold that binds the polypeptide, (iii) a heterologous object sequence comprising a mutation region to correct the SERPINA1 gene mutation, and (iv) a primer binding site (PBS) sequence comprising at least 3, 4, 5, 6, 7, or 8 bases of 100% homology to a target DNA strand at the 3' end of the template RNA. The SERPINA1 gene may comprise an E342K mutation (also referred to as a PiZ mutation). A template RNA sequence may comprise a sequence described herein, e.g., in Table 15, 16, 18-29, or 39-62.

[0016] A gRNA spacer may comprise at least 15 bases of 100% homology to a target DNA at the 5' end of the template RNA. A template RNA may further comprise a PBS sequence comprising at least 5 bases of at least 80% homology to a target DNA strand. A template RNA may comprise one or more chemical modifications.

[0017] Domains of a gene modifying polypeptide may be joined by a peptide linker. A polypeptide may comprise one or more peptide linkers. A gene modifying polypeptide may further comprise a nuclear localization signal. A polypeptide may comprise more than one nuclear localization signal, e.g., multiple adjacent nuclear localization signals or one or more nuclear localization signals in different regions of the polypeptide, e.g., one or more nuclear localization signals in the N-terminus of the polypeptide and one or more nuclear localization signals in the C-terminus of the polypeptide. A nucleic acid encoding a gene modifying polypeptide may encode one or more intein domains.

[0018] Introduction of a system of the present disclosure into a target cell may result in insertion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, or 1000 base pairs of exogenous DNA. Introduction of a system of the present disclosure into a target cell may result in a deletion, wherein the deletion is less than 2, 3, 4, 5, 10, 50, or 100 base pairs of genomic DNA upstream or downstream of an insertion. Introduction of a system of the present disclosure into a target cell may result in substitution, e.g., substitution of 1, 2, or 3 nucleotides, e.g., consecutive nucleotides.

[0019] A heterologous object sequence may be at least 5, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, or 700 base pairs. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a system described herein and a pharmaceutically acceptable excipient or carrier, wherein the pharmaceutically acceptable excipient or carrier is selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a system described herein and multiple pharmaceutically acceptable excipients or carriers, wherein the pharmaceutically acceptable excipients or carriers are selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle, e.g., where the system described above is delivered by two distinct excipients or carriers, e.g., two lipid nanoparticles, two viral vectors, or one lipid nanoparticle and one viral vector. A viral vector may be an adeno- associated virus (AAV).

[0020] In some embodiments, aspect, the disclosure relates to a host cell (e.g., a mammalian cell, e.g., a human cell) comprising the system described above.

[0021] In some emdodiments, the present disclosure provides a method of correcting a mutation in the human SERPINA1 gene in a cell, tissue or subject, the method comprising administering a system described herein to the cell, tissue or subject, wherein optionally the correction of the mutant SERPINA1 gene comprises an amino acid substitution of K342E (i.e., reversing the pathogenic E342K mutation). A system described herein may be introduced in vivo, in vitro, ex vivo, or in situ. A nucleic acid of a system described herein may be integrated into the genome of a host cell. In some embodiments, a nucleic acid of a system described herein is not integrated into the genome of a host cell. In some embodiments, a heterologous object sequence is inserted at only one target site in a host cell genome. A heterologous object sequence may be inserted at two or more target sites in a host cell genome, e.g., at the same corresponding site in two homologous chromosomes or at two different sites on the same or different chromosomes. A heterologous object sequence may encode a mammalian polypeptide, or a fragment or a variant thereof. Components of a system described herein may be delivered on 1, 2, 3, 4, or more distinct nucleic acid molecules. A system of the present disclosure may be introduced into a host cell by electroporation or by using at least one vehicle selected from a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle.

[0022] Features of the compositions or methods can include one or more of the following enumerated embodiments. Enumerated Embodiments

[0023] 1. A nucleic acid comprising a Repeat: anti-repeat (RAR) region which comprises a chemically modified nucleotide.

[0024] 2. The nucleic acid of embodiment 1, which further comprises a stem-loop 1 (SL1) region (e.g., wherein the SL1 region is 3’ of the RAR region), wherein optionally the SL1 region comprises a chemically modified nucleotide.

[0025] 3. The nucleic acid of embodiment 1 or 2, which further comprises a stem loop 2 (SL2) region (e.g., wherein the SL2 region is 3’ of the SL1 region), wherein optionally the SL2 region comprises a chemically modified nucleotide.

[0026] 4. A nucleic acid comprising a stem-loop 1 (SL1) region which comprises a chemically modified nucleotide.

[0027] 5. The nucleic acid of embodiment 4, which further comprises an RAR region (e.g., wherein the RAR region is 5’ of the SL1 region), wherein optionally the RAR region comprises a chemically modified nucleotide.

[0028] 6. A nucleic acid molecule comprising: an StlCas9 scaffold; wherein the StlCas9 scaffold comprises a chemically modified nucleotide.

[0029] 7. A nucleic acid molecule comprising: an StlCas9 scaffold; wherein the StlCas9 scaffold comprises a chemically modified nucleotide at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or all of) positions 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 relative to SEQ ID NO: 25999.

[0030] 8. A nucleic acid molecule comprising: an StlCas9 scaffold; wherein at least 15-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, or 70-75% of nucleotides in the StlCas9 scaffold are chemically modified.

[0031] 9. A nucleic acid molecule comprising: an StlCas9 scaffold comprising: a) a Repeat: anti-repeat (RAR) region, wherein optionally the RAR region comprises a RAR lower stem, a RAR upper stem, and an RAR loop (c.g., a tctraloop); b) a stem- loop 1 (SL1) region that is optionally 3’ of the RAR region, and c) optionally, a stem loop 2 (SL2) region that is optionally 3’ of the SL1 region; wherein the StlCas9 scaffold comprises a chemically modified nucleotide in one or both of the RAR region or the SL1 region.

[0032] 10. The nucleic acid of embodiment 7 or 9, wherein at least 15-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70, or 70-75% of nucleotides in the StlCas9 scaffold are chemically modified.

[0033] 11. The nucleic acid of embodiment 7 or 8, wherein the StlCas9 scaffold comprises: a) a Repeat: anti-repeat (RAR) region, wherein optionally the RAR region comprises a RAR lower stem, a RAR upper stem, and an RAR loop (e.g., a tetraloop); b) a stem-loop 1 (SL1) region that is 3’ of the RAR region, and c) optionally, a stem loop 2 (SL2) region that is 3’ of the SL1 region; wherein the StlCas9 scaffold comprises a chemically modified nucleotide in one or both of the RAR region or the SL1 region.

[0034] 12. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all of) positions 4, 5, 6, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 34, 35, 36, 37, 38, 39, 40, 41, and 42.

[0035] 13. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,

[0036] 11, 12, 13, 14, or all of) positions 7, 8, 9, 10, 11, 12, 25, 26, 27, 28, 29, 30, 31, 32, and 33.

[0037] 14. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a RAR region, wherein the RAR region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

[0038] 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 chemically modified nucleotides (e.g., wherein the chemically modified nucleotides have the same chemical modification).

[0039] 15. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a SL1 region, wherein the SL1 region comprises 1, 2, 3, 4, 5, 6, 7, or 8 chemically modified nucleotides (e.g., wherein the chemically modified nucleotides have the same chemical modification). 16. The nucleic acid of any of the preceding embodiments, wherein the St 1 Cas9 scaffold comprises a chemically modified nucleotide in the RAR region and a chemically modified nucleotide in the SL1 region.

[0040] 17. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises between 10-21 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21) chemically modified nucleotides in the RAR region.

[0041] 18. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises between 0-8 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, or 8) chemically modified nucleotides in the SL1 region.

[0042] 19. The nucleic acid of any of the preceding embodiments, wherein positions 1, 2, and 3 (if present) do not comprise a 2’-O-methyl chemically modified nucleotide.

[0043] 20. The nucleic acid of any of the preceding embodiments, wherein positions 1, 2, and 3 (if present) are not chemically modified.

[0044] 21. The nucleic acid of any of the preceding embodiments, wherein positions 43 through 54 (if present) do not comprise a 2’-O-methyl chemically modified nucleotide.

[0045] 22. The nucleic acid of any of the preceding embodiments, wherein positions 43 through 54 (if present) are not chemically modified.

[0046] 23. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 4 through 6 (if present).

[0047] 24. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 13 through 15 (if present).

[0048] 25. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 16 through 18 (if present).

[0049] 26. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 19 through 21 (if present).

[0050] 27. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 22 through 24 (if present).

[0051] 28. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 34 through 36 (if present).

[0052] 29. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 37 through 39 (if present). 30. The nucleic acid of any of the preceding embodiments, wherein the St 1 Cas9 scaffold comprises a chemically modified nucleotide at each of positions 40 through 42 (if present).

[0053] 31. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 13 through 24 (if present).

[0054] 32. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 34 through 42 (if present).

[0055] 33. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 12 through 21 (if present).

[0056] 34. The nucleic acid of embodiment 33, wherein the StlCas9 scaffold comprises a lengthened RAR upper stem.

[0057] 35. The nucleic acid of embodiment 33 or 34, wherein: the StlCas9 scaffold comprises a first insertion (e.g., of 4 nucleotides) between positions

[0058] 14 and 15, and a second insertion (e.g., of 4 nucleotides) between positions 18 and 19; or the StlCas9 scaffold comprises an insertion (e.g., of 10 nucleotides) between positions 15 and 18, and a deletion of positions 16 and 17, wherein optionally the insertion has a sequence according to GACUUCGGUC.

[0059] 36. The nucleic acid of any of embodiments 33-35, wherein the StlCas9 scaffold comprises a mutation in the tetraloop, e.g., wherein the tetraloop comprises a sequence of UUCG.

[0060] 37. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all of) positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25.

[0061] 38. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or all of) positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 51, 52, 53, and 54.

[0062] 39. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or all of) positions 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 51, 52, 53, and 54. 40. The nucleic acid of any of the preceding embodiments, wherein the St 1 Cas9 scaffold comprises a chemically modified nucleotide (c.g., 2’-O-mcthyl) at one or more of (c.g., 2, 3, 4, 5, 6, 7, 8, 9, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21.

[0063] 41. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all of) positions 4, 5, 6, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 34, 35, 36, 37, 38, 39, 40, 41, and 42.

[0064] 42. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 54.

[0065] 43. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 53.

[0066] 44. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 52.

[0067] 45. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 53, and 54.

[0068] 46. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or all of) positions 4,

[0069] 6, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42.

[0070] 47. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or all of) positions 5, 7, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and 41.

[0071] 48. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or all of) positions 5,

[0072] 7, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, and 42. 49. The nucleic acid of any of the preceding embodiments, wherein the St 1 Cas9 scaffold comprises a chemically modified nucleotide (c.g., 2’-O-mcthyl) at one or more of (c.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all of) positions 4, 5, 6, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21.

[0073] 50. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all of) positions 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24.

[0074] 51. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 26, 27, 28, 29, 30, 31, 32, and 33.

[0075] 52. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 35, 36, 37, 38, 39, 40, 41, and 42.

[0076] 53. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all of) positions 4, 5, 6, 12,

[0077] 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 35, 36, 37, 38, 39, 40, 41, and 42.

[0078] 54. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3,

[0079] 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or all of) positions 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 34, 35, 36, 37, 38, 39, 40, 41, and 42.

[0080] 55. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 43, and 44.

[0081] 56. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 43. 57. The nucleic acid of any of the preceding embodiments, wherein the St 1 Cas9 scaffold comprises a chemically modified nucleotide (c.g., 2’-O-mcthyl) at one or more of (c.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 44.

[0082] 58. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 45.

[0083] 59. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 46.

[0084] 60. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 47.

[0085] 61. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 49.

[0086] 62. The nucleic acid of any of the preceding embodiments, wherein; the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 50, or the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) positions 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 51.

[0087] 63. The nucleic acid of any of the preceding embodiments, wherein: a) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at each of positions 12 through 21; b) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-O-methyl) at each of positions 34 through 36; c) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at one or more of positions 37 through 42; d) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at each of positions 37 through 42; e) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at one or more of positions 45 through 47; f) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at each of positions 45 through 47; g) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at one or more of positions 52 through 54; h) the StlCas9 scaffold comprises a chemically modified nucleotide (e.g., 2’-Fluoro) at each of positions 52 through 54; i) the StlCas9 scaffold comprises a 2’-O-methyl chemically modified nucleotide at each of positions 12 through 21, a 2’-O-methyl chemically modified nucleotide at each of positions 34 through 36, and a 2’ -Fluoro chemically modified nucleotide at each of positions 37 through 42; j) the StlCas9 scaffold comprises a 2’-O-methyl chemically modified nucleotide at each of positions 12 through 21, a 2’-O-methyl chemically modified nucleotide at each of positions 34 through 36, a 2’ -Fluoro chemically modified nucleotide at each of positions 37 through 42, and a 2’ -Fluoro chemically modified nucleotide at each of positions 45 through 47 ; or k) the StlCas9 scaffold comprises a 2’-O-methyl chemically modified nucleotide at each of positions 12 through 21, a 2’-O-methyl chemically modified nucleotide at each of positions 34 through 36, a 2’ -Fluoro chemically modified nucleotide at each of positions 37 through 42, a 2’-Fluoro chemically modified nucleotide at each of positions 45 through 47 and a 2’ -Fluoro chemically modified nucleotide at each of positions 52 through 54.

[0088] 64. The nucleic acid of any of the preceding embodiments, which comprises a plurality of chemically modified nucleotides in the StlCas9 scaffold, wherein the plurality of chemically modified nucleotides in the StlCas9 scaffold have the same chemical modification.

[0089] 65. The nucleic acid of any of embodiments 1-63, which comprises a plurality of chemically modified nucleotides in the StlCas9 scaffold, wherein the plurality of chemically modified nucleotides in the StlCas9 scaffold have two or more different chemical modifications. 66. The nucleic acid of any of the preceding embodiments, wherein the St 1 Cas9 scaffold comprises a sequence having the chemically modified nucleotides set out in Table 22, 21, 20, E3, E7, E8, E9, E10, E12, or E13.

[0090] 67. The nucleic acid of any of the preceding embodiments, wherein the chemically modified nucleotide is a modification to a sugar group, e.g., a modification to the 2’-0 of ribose, e.g., a 2’-O-Methyl chemically modified nucleotide.

[0091] 68. The nucleic acid of any of the preceding embodiments, wherein the chemically modified nucleotide is a modification to a sugar group, e.g., the ribose ring contains a bridging moiety, e.g., a “locked” nucleic acid (LNA).

[0092] 69. The nucleic acid of any of the preceding embodiments, which further comprises a second chemically modified nucleotide.

[0093] 70. The nucleic acid of any of the preceding embodiments, which comprises a sequence according to SEQ ID NO: 26000, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0094] 71. The nucleic acid of any of the preceding embodiments, which comprises a sequence of Tables 42, 43, or 44, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0095] 72. The nucleic acid of any of the preceding embodiments, which comprises a sequence according to SEQ ID NO: 25999, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0096] 73. The nucleic acid of any of the preceding embodiments, which comprises one or more (e.g., at least 1, 2, or 3, and optionally no more than 10, 20, or 30) sequence differences (e.g., insertions, deletions, or substitutions) relative to SEQ ID NO: 25999.

[0097] 74. The nucleic acid of any of the preceding embodiments, wherein the scaffold has a length of 50-60, 60-70, 70-80, or 80-90 nucleotides.

[0098] 75. The nucleic acid of any of the preceding embodiments, wherein the RAR region has a length of 30-40 nucleotides (e.g. 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides).

[0099] 76. The nucleic acid of any of the preceding embodiments, wherein the SL1 region has a length of 6-15 nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 nucleotides).

[0100] 77. The nucleic acid of any of the preceding embodiments, wherein the SL2 region has a length of 20-30 nucleotides (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). 78. The nucleic acid of any of embodiments 1-69, which comprises an SL2 region.

[0101] 79. The nucleic acid of any of embodiments 1-69, which docs not comprise an SL2 region.

[0102] 80. The nucleic acid of any of the preceding embodiments, wherein the StlCas9 scaffold binds to an StlCas9 protein having an amino acid sequence of SEQ ID NO: 23818.

[0103] 81. The nucleic acid of any of the preceding embodiments, which further comprises a gRNA spacer situated 5’ of the StlCas9 scaffold region.

[0104] 82. The nucleic acid of embodiment 81, wherein the gRNA spacer is complementary to a first portion of the human SERPINA1 gene.

[0105] 83. The nucleic acid of any of the preceding embodiments, which further comprises a heterologous object sequence situated 3’ of the StlCas9 scaffold region.

[0106] 84. The nucleic acid of any of the preceding embodiments, which further comprises a primer binding site (PBS) sequence situated 3’ of the StlCas9 scaffold region, e.g., wherein the PBS sequence is situated 3’ of the heterologous object sequence.

[0107] 85. A template RNA comprising (tgRNA) comprising, from 5’ to 3’:

[0108] (1) a gRNA spacer;

[0109] (2) a chemically modified StlCas9 scaffold comprising a nucleic acid of any of claims 1-

[0110] 80;

[0111] (3) a heterologous object sequence; and

[0112] (4) a primer binding site (PBS) sequence.

[0113] 86. The template RNA of embodiment 85, which further comprises a chemical modification outside of the chemically modified StlCas9 scaffold.

[0114] 87. The template RNA of embodiment 86, wherein the chemical modification outside of the chemically modified StlCas9 scaffold is situated in the gRNA spacer, optionally wherein the chemical modification is a phosphorothioate linkage or a 2’-O-Methyl nucleotide.

[0115] 88. The template RNA of embodiment 86, wherein the chemical modification outside of the chemically modified StlCas9 scaffold is situated in the PBS sequence, optionally wherein the chemical modification is a phosphorothioate linkage or a 2’-O-Methyl nucleotide.

[0116] 89. The template RNA of embodiment 86, wherein the chemical modification outside of the chemically modified StlCas9 scaffold is situated in the heterologous object sequence, optionally wherein the chemical modification is a 2’-fluoro nucleotide.

[0117] 90. A template RNA comprising (tgRNA) comprising, from 5’ to 3’: (1) a gRNA spacer;

[0118] (2) a gRNA scaffold, c.g., a StlCas9 scaffold, c.g., a chemically modified StlCas9 scaffold of any of claims 1-80;

[0119] (3) a heterologous object sequence comprising a chemically modified nucleotide; and

[0120] (4) a primer binding site (PBS) sequence.

[0121] 91. The template RNA of embodiment 90, wherein the chemically modified nucleotide in the heterologous object sequence is a 2’-Fluoro nucleotide.

[0122] 92. The template RNA of embodiment 90 of 91, wherein the heterologous object sequence further comprises one or more (e.g., 2) additional chemically modified nucleotides, e.g., 2’- Fluoro nucleotides.

[0123] 93. The template RNA of any of embodiments 90-92, wherein: position +4 of the heterologous object sequence is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); position +5 of the heterologous object sequence is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); position +6 of the heterologous object sequence is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); positions +4, +5, and +6 of the heterologous object sequence are each a chemically modified nucleotide (e.g., each is a 2’-Fluoro nucleotide);

[0124] 94. The template RNA of any of the preceding embodiments, wherein: the 3’ most nucleotide in the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the second nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the third nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the 3’ most nucleotide and the second and third nucleotides from the 3’ end of the template RNA are each a chemically modified nucleotide (e.g., each is a 2’-O-methyl nucleotide); the twelfth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the thirteenth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the fourteenth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2 ’-Fluoro nucleotide); or the twelth, thirteenth, and fourteenth nucleotides from the 3’ end of the template RNA are each a chemically modified nucleotide (e.g., each is a 2’-Fluoro nucleotide); the nineteenth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the twentieth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the twenty-first nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the nineteenth, twentieth, and twenty-first nucleotides from the 3’ end of the template RNA are each a chemically modified nucleotide (e.g., each is a 2’-Fluoro nucleotide); the twenty-sixth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the twenty- seventh nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the twenty-eighth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the twenty-sixth, twenty- seventh, and twenty-eighth nucleotides from the 3’ end of the template RNA are each a chemically modified nucleotide (e.g., each is a 2’ -Fluoro nucleotide); the thirty-first nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the thirty-first nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the thirty-second nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the thirty-third nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the thirty-fourth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (c.g., a 2’-Fluoro nucleotide); the thirty-fifth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2 ’-Fluoro nucleotide); the thirty-sixth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-Fluoro nucleotide); the thirty-first, thirty- second, thirty-third, thirty-fourth, thirty-fifth, and thirty-sixth nucleotide from the 3’ end of the template RNA each each a chemically modified nucleotide (e.g., each is a 2’ -Fluoro nucleotide); the thirty-seventh nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty- second nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty-third nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty-fourth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty-fifth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty- sixth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty-seventh nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty-eighth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the fifty-ninth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixtieth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-first nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-second nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-mcthyl nucleotide); the sixty-third nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-fourth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-fifth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-sixth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-seventh nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-eighth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); the sixty-ninth nucleotide from the 3’ end of the template RNA is a chemically modified nucleotide (e.g., a 2’-O-methyl nucleotide); or the fifty- second through the sixty-first nucleotides from the 3’ end of the template RNA are each a chemically modified nucleotide (e.g., each is a 2’-O-methyl nucleotide).

[0125] 95. The template RNA or the nucleic acid of any of the preceding embodiments, wherein the chemically modified StlCas9 scaffold comprises a variant StlCas9 scaffold having a deletion of part or all of Stem loop 2.

[0126] 96. The template RNA or nucleic acid of embodiment 95, wherein the deletion is between 1-32 (e.g., 2-29, 2-20, 2-10, or 10-20) nucleotides in length.

[0127] 97. The template RNA or nucleic acid of embodiment 95, wherein the deletion is of all of Stem loop 2.

[0128] 98. The template RNA or nucleic acid of embodiment 95, wherein the deletion is of positions 55 through 84.

[0129] 99. The template RNA or nucleic acid of embodiment 95, wherein the StlCas9 scaffold comprises a deletion of part of the second single stranded region (e.g., 1, 2, 3, or 4 nucleotides at the 3’ end of the single stranded region). 100. The template RNA or the nucleic acid of any of the preceding embodiments, wherein the chemically modified StlCas9 scaffold comprises a variant StlCas9 scaffold having one or both of a lengthened RAR upper stem or a substitution resulting in a G-C base pair in the RAR upper stem.

[0130] 101. The template RNA or nucleic acid of any of the preceding embodiments, wherein the RAR upper stem is lengthened by 1-8 base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 base pairs) relative to the wild-type sequence of SEQ ID NO: 25999.

[0131] 102. The template RNA or nucleic acid of embodiment 101, wherein at least 50%, 60%, 70%, 80%, or 90% of the base pairs that are new relative to SEQ ID NO: 25999 are G-C base pairs.

[0132] 103. The template RNA or nucleic acid of embodiment 101 or 102, wherein the lengthened RAR upper stem comprises one or more base pairs that are new relative to SEQ ID NO: 25999, wherein the one or more new base pairs comprise one or more chemically modified nucleotides.

[0133] 104. The template RNA or the nucleic acid of any of the preceding embodiments, wherein the chemically modified StlCas9 scaffold comprises a variant StlCas9 scaffold having a mutation in the tetraloop.

[0134] 105. The template RNA or nucleic acid of any of the preceding embodiments, wherein one or more nucleotides in the tetraloop are substituted.

[0135] 106. The template RNA or nucleic acid of any of the preceding embodiments, wherein the tetraloop comprises a sequence chosen from: AACA, AAUA, ACCA, ACUA, AGUA, AGCA, AUCA, AUUA, CAAC, CUCG, CUUG, GAAA, GAGA, GCAA, GCGA, GGAA, GGAG, GGGA, GUAA, GUGA, UAAC, UACG, UCAC, UCCG, UGAA, UGAC, UGCG, UUAC, or UUCG.

[0136] 107. The template RNA or nucleic acid of any of the preceding embodiments, wherein the tetraloop is lengthened, e.g., to 5 nucleotides.

[0137] 108. The template RNA or nucleic acid of embodiment On, wherein the lengthened tetraloop comprises a sequence chosen from: GAAGA or GACAA.

[0138] 109. The template RNA or nucleic acid of embodiment 107 or 108, wherein the lengthened tetraloop comprises one or more chemically modified nucleotides (e.g., comprises 1, 2, 3, 4, or 5 chemically modified nucleotides). 110. The template RNA or nucleic acid of any of the preceding embodiments, wherein the variant gRNA scaffold comprises a sequence according to Tabic 23, or a sequence having no more than 1, 2, or 3 sequence alterations (e.g., substitutions) relative thereto.

[0139] 111. The template RNA or nucleic acid of any of the preceding embodiments, which comprises a sequence according to any of Tables 39, 24, 40, 57, 41, 42, 45, 56, or 12-62 or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0140] 112. The template RNA or nucleic acid of any of the preceding embodiments, which comprises a sequence according to SEQ ID NO: 27131, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0141] 113. The template RNA or nucleic acid of any of the preceding embodiments, which comprises a sequence according to SEQ ID NO: 27132, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0142] 114. The template RNA or nucleic acid of any of the preceding embodiments, which comprises a sequence according to SEQ ID NO: 27133, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0143] 115. The template RNA or nucleic acid of any of the preceding embodiments, which comprises a sequence according to SEQ ID NO: 27134, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0144] 116. The template RNA or nucleic acid of any of the preceding embodiments, wherein the variant StlCas9 scaffold has a length of 50-60, 60-70, 70-80, or 80-84 nucleotides.

[0145] 117. The template RNA or the nucleic acid of any of the preceding embodiments, wherein the chemically modified StlCas9 scaffold comprises a variant gRNA scaffold comprising a sequence according to Table 23, or a sequence having no more than 1, 2, or 3 sequence alterations (e.g., substitutions) relative thereto.

[0146] 118. A system comprising: a nucleic acid or template RNA of any of the preceding embodiments; and a polypeptide comprising a StlCas9 domain, or a nucleic acid encoding the polypeptide.

[0147] 119. The system of embodiment 118, wherein the polypeptide further comprises a RT domain, and optionally comprises a linker situated between the RT domain and the StlCas9 domain.

[0148] 120. A gene modifying system comprising: a template RNA of any of claims 85-117; and a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, the gene modifying polypeptide comprising:

[0149] (1) a Cas9 domain, e.g., a StlCas9 domain;

[0150] (2) a linker; and

[0151] (3) a reverse transcriptase (RT) domain.

[0152] 121. The gene modifying system of embodiment 120, wherein the Cas9 nickase domain comprises an StlCas9 nickase domain.

[0153] 122. The gene modifying system of embodiment 120 or 121, wherein the gene modifying polypeptide comprises, in an N-terminal to C-terminal direction, a first NLS, the StlCas9 nickase domain, a linker, an RT domain, and a second NLS.

[0154] 123. The gene modifying system of embodiment 122, wherein one or both of: the first NLS comprises a sequence of SEQ ID NO: 11,095, and the second NLS comprises a sequence of SEQ ID NO: 11,099.

[0155] 124. The gene modifying system of any of embodiments 120-123, wherein the linker comprises a sequence according to SEQ ID NO: 5006.

[0156] 125. The gene modifying system of embodiment 120, wherein the StlCas9 domain has a sequence according to SEQ ID NO: 23818, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0157] 126. The gene modifying system of embodiment 120, wherein the linker has a sequence according to SEQ ID NO: 5006, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0158] 127. The gene modifying system of embodiment 120, wherein the RT domain has a sequence according to SEQ ID NO: 26006, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0159] 128. The gene modifying system of embodiment 120, wherein the gene modifying polypeptide has a sequence according to SEQ ID NO: 26002, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0160] 129. The system of any of embodiments 118-128, which is capable of specifically cleaving a target nucleic acid.

[0161] 130. The system of any of embodiments 118-128, which is capable of producing indels in a target nucleic acid. 131 . The system of any of embodiments 118-128, which is capable of introducing an edit specified by the heterologous object sequence in a target nucleic acid.

[0162] 132. A pharmaceutical composition, comprising the template RNA of nucleic acid of any one of embodiments 1-117 or the system of any one of embodiments 118-131, and a pharmaceutically acceptable excipient or carrier.

[0163] 133. The pharmaceutical composition of embodiment 132, wherein the pharmaceutically acceptable excipient or carrier is selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle (LNP).

[0164] 134. A host cell (e.g., a mammalian cell, e.g., a human cell) comprising the gene modifying system, template RNA, or nucleic acid of any one of the preceding claims.

[0165] 135. A method of making the nucleic acid or template RNA of any one of embodiments 118- 131, the method comprising synthesizing the template RNA in vitro (e.g., by in vitro transcription or solid state synthesis).

[0166] 136. A method for modifying a target site (e.g., a target site in the human SERPINA1 gene) in a cell, the method comprising contacting the cell with the gene modifying system of any one of embodiments 118-131, or DNA encoding the same, or the pharmaceutical composition of embodiment 132 or 133, thereby modifying the target site.

[0167] 137. A method for treating a subject having a disease or condition associated with a mutation in a gene (e.g., the human SERPINA1 gene), the method comprising administering to the subject the gene modifying system of any one of embodiments 118-131, or DNA encoding the same, or the pharmaceutical composition of embodiment 132 or 133, thereby treating the subject having a disease or condition.

[0168] 138. The method of claim 137, wherein the disease or condition is alpha-1 antitrypsin deficiency (AATD).

[0169] 139. The method of claim 137 or 138, wherein the subject has a E342K mutation.

[0170] 140. A method for treating a subject having AATD, the method comprising administering to the subject the gene modifying system of any one of embodiments 118-131, or DNA encoding the same, or the pharmaceutical composition of embodiment 132 or 133, thereby treating the subject having AATD. BRIEF DESCRIPTION OF THE DRAWINGS

[0171] FIG. 1 is a diagram depicting components of a gene modifying system as described herein. FIG. 1A is a diagram showing a gene modifying polypeptide comprising a Cas nickase domain (e.g., spCas9 N863A) and a reverse transcriptase domain (RT domain) which are linked by a linker. FIG. IB is a diagram showing a template RNA comprising, from 5’ to 3’, a gRNA spacer, a gRNA scaffold, a heterologous object sequence, and a primer binding site sequence (PBS sequence). A heterologous object sequence can comprise a mutation region that comprises one or more sequence differences relative to a target site. A heterologous object sequence can also comprise a pre-edit homology region and a post-edit homology region, which flank a mutation region. Without wishing to be bound by theory, it is thought that a gRNA spacer of a template RNA binds to a second strand of a target site in the genome, and a gRNA scaffold of the template RNA binds to a gene modifying polypeptide, e.g., localizing the gene modifying polypeptide to a target site in the genome. It is thought that a Cas domain of a gene modifying polypeptide nicks a target site (e.g., a first strand of the target site), e.g., allowing a PBS sequence to bind to a sequence adjacent to the target site to be altered on the first strand of the target site. It is thought that an RT domain of a gene modifying polypeptide uses a first strand of a target site that is bound to a complementary sequence comprising a PBS sequence of a template RNA as a primer and a heterologous object sequence of the template RNA as a template to, e.g., polymerize a sequence complementary to the heterologous object sequence. Without wishing to be bound by theory, it is thought that reverse transcription can then proceed through a pre-edit homology region, then through a mutation region, and then through a post-edit homology region, thereby producing a DNA strand comprising a mutation specified by a heterologous object sequence.

[0172] FIG. 2 is a diagram illustrating the hypothesized secondary structure of a wild-type StlCas9 gRNA scaffold and is overlaid with description of valiants described herein.

[0173] FIG. 3A is a bar graph showing the rewriting performance of exemplary StlCas9-based gene modifying systems comprising exemplary template RNAs comprising various scaffolds truncated in the stem loop 2 region as depicted in FIG. 2.

[0174] FIG. 3B is a bar graph showing the rewriting performance of exemplary StlCas9-based gene modifying systems comprising exemplary template RNAs comprising various scaffolds further engineered in the tetraloop (TL) region as depicted in FIG. 2. Bars without asterisk = 1 pmol of exemplary gene modifying system, bars with asterisk = 0.01 pmol of exemplary gene modifying system.

[0175] FIG. 3C is a bar graph showing the rewriting performance of exemplary StlCas9-based gene modifying systems comprising exemplary template RNAs comprising various scaffolds further engineered to elongate and / or stabilize the TL and / or RAR regions as depicted in FIG. 2. Bars without asterisk = 1 pmol of exemplary gene modifying system, bars with asterisk = 0.01 pmol of exemplary gene modifying system.

[0176] FIG. 3D is a bar graph showing the rewriting performance of exemplary StlCas9-based gene modifying systems comprising exemplary template RNAs comprising various lengths of spacers. Bars without asterisk = 1 pmol of exemplary gene modifying system, bars with asterisk = 0.01 pmol of exemplary gene modifying system.

[0177] FIG. 4A is a bar graph showing the rewriting efficiency of exemplary gene modifying systems comprising different StlCas9-compatible template RNAs comprising modified scaffold sequences.

[0178] FIG. 4B is a bar graph showing the % indel levels introduced by exemplary gene modifying systems evaluated in FIG. 4A.

[0179] FIG. 4C is a bar graph showing the rewriting efficiency of additional exemplary gene modifying systems comprising different StlCas9-compatible template RNAs comprising modified scaffold sequences.

[0180] FIG. 5 is a bar graph showin the rewriting efficiency of exemplary gene modifying systems comprising StlCas9-based gene modifying polypeptide.

[0181] FIG. 6A is a bar graph showing the rewriting efficiency of exemplary gene modifying systems comprising StlCas9-based gene modifying polypeptide, with and without ngRNA.

[0182] FIG. 6B is a bar graph showing the % indel levels introduced by exemplary gene modifying systems comprising StlCas9-based gene modifying polypeptide, with (open bars) and without ngRNA (closed bars).

[0183] FIG. 7 A is a bar graph showing the rewriting efficiency of exemplary StlCas9-based gene modifying systems comprising exemplary template RNAs containing a dSL2 variant gRNA scaffold, various lengths of PBS sequences and heterologous object sequences in primary hepatocytes. FIG. 7B is a bar graph showing the % indel levels introduced by exemplary StlCas9-based gene modifying systems comprising exemplary template RNAs containing a dSL2 variant gRNA scaffold, various lengths of PBS sequences and heterologous object sequences in primary hepatocytes.

[0184] FIG. 8A is a bar graph showing percent rewriting achieved using an exemplary gene modifying system comprising different StlCas9-compatible template RNAs comprising variant scaffolds containing various exemplary variant tetraloop structures in primary hepatocytes.

[0185] FIG. 8B is a bar graph showing percent rewriting achieved using an exemplary gene modifying system comprising different StlCas9-compatible template RNAs comprising variant scaffolds containing various exemplary variant tetraloop structures in HEK293T cells treated with a high dose of exemplary gene modifying system.

[0186] FIG. 8C is a bar graph showing percent rewriting achieved using an exemplary gene modifying system comprising different StlCas9-compatible template RNAs comprising variant scaffolds containing various exemplary variant tetraloop structures in HEK293T cells treated with a low dose of exemplary gene modifying system.

[0187] FIG. 8D is a diagram illustrating the hypothesized secondary structure of a dSL2 truncated StlCas9 gRNA scaffold and is overlaid with description of valiants described herein.

[0188] FIG. 9A is a bar graph showing the rewriting activity of exemplary StlCas9-based gene modifying systems comprising variant template RNAs having the nucleotide sequence of exemplary template RNA RNACS6681 with various 2-O'-methyl chemical modifications in the gRNA scaffold region.

[0189] FIGs. 9B-9K are schematic diagrams of tested chemical modification pattern designs of exemplary StlCas9-based gene modifying systems comprising variant template RNAs having the nucleotide sequence of exemplary template RNA RNACS6681, with 2’-O-methyl chemical modifications shown in bold.

[0190] FIG. 10A is a bar graph showing the rewriting activity of exemplary StlCas9-based gene modifying systems comprising variant template RNAs having the nucleotide sequence of exemplary template RNA RNACS9201 (containing a dSL2 variant gRNA scaffold) with various 2-O'-methyl chemical modifications in the gRNA scaffold region. FIG. 10B is a bar graph showing the rewriting activity of exemplary StlCas9-based gene modifying systems comprising variant template RNAs having a scaffold comprising three modified nucleotides at a time with 2’-O-methyl chemical modifications.

[0191] FIG. IOC is a diagram illustrating the positions of poorly tolerated (lowercase,), somewhat tolerated positions (capital), and tolerated positions (italic) to 2’-O-methyl chemical modification in the dSL2 StlCas9 scaffold sequence.

[0192] FIGs. 10D-10L are diagrams illustrating exemplary design patterns of 2’-O-methyl chemical modified nucleotides in the dSL2 StlCas9 scaffold sequence in FIG. 10A. Bold bases represent2'-O-methyl modified nucleotide positions.

[0193] FIG. 11 is a bar graph showing the rewriting efficiency of exemplary gene modifying systems comprising different StlCas9-compatible template RNAs comprising different patterns of 2’-O-methyl chemical modifications in the dSL2 StlCas9 scaffold in primary hepatocytes.

[0194] FIG. 12A is a bar graph showing the rewriting activity in the livers of mice administered with exemplary gene modifying systems comprising different StlCas9-compatible template RNAscontaining a dSL2 variant gRNA scaffold and formulated in lipid nanoparticles (LNP).

[0195] FIG. 12B is a bar graph showing the % indel levels in the livers of mice administered with exemplary gene modifying systems comprising different StlCas9-compatible template RNAscontaining a dSL2 variant gRNA scaffold and formulated in LNP.

[0196] FIG. 12C is a bar graph showing the the concentration of hAlAT in the serum of mice administered with exemplary gene modifying systems comprising different StlCas9-compatible template RNAscontaining a dSL2 variant gRNA scaffold and formulated in LNP.

[0197] FIG. 13A is a bar graph showing of the percent of perfect rewriting in the liver of mice administered with exemplary gene modifying systems that comprises different StlCas9- compatible template RNAs comprising different patterns of 2’-O-methyl chemical modifications in the dSL2 StlCas9 scaffold and are formulated in LNP.

[0198] FIG. 13B is a bar graph showing of the % indel levels in the liver of mice administered with exemplary gene modifying systems that comprises different StlCas9-compatible template RNAs comprising different patterns of 2’-O-methyl chemical modifications in the dSL2 StlCas9 scaffold and are formulated in LNP.

[0199] FIG. 13C is a bar graph showing the concentration of hAlAT in the serum of mice administered with exemplary gene modifying systems that comprises different StlCas9- compatible template RNAs comprising different patterns of 2’-O-methyl chemical modifications in the dSL2 StlCas9 scaffold and arc formulated in LNP.

[0200] FIG. 14A is a diagram illustrating the positions of the reference dSL2 StlCas9 scaffold sequence.

[0201] FIG. 14B is a diagram illustrating the positions of the reference wild-type StlCas9 scaffold sequence.

[0202] FIG. 14C is a diagram illustrating the hypothesized structure of RNACS 13597, having RAR+4_UUCG mutations relative to dSL2.

[0203] FIG. 14D is a diagram illustrating the hypothesized structure of RNACS17210, having RAR+4_AGCA mutations relative to dSL2.

[0204] FIG. 15 are schematics of three exemplary template RNAs comprising chemical modifications. Closed circles are 2’F modifications, open circles are 2’0Me modifications, diamonds are phosphorothioate modifications.

[0205] FIG. 16A is a bar graph showing the rewriting efficiency in the livers of mice administered with exemplary gene modifying systems comprising different template RNAs comprising scaffold chemical modifications.

[0206] FIG. 16B is a bar graph showing the % indel levels in the livers of mice administered with the exemplary gene modifying systems evaluated in FIG. 16A.

[0207] FIG. 17A is a bar graph showing the rewriting efficiency in the livers of mice administered with exemplary gene modifying systems comprising different template RNAs comprising scaffold chemical modifications in combination with fluoro modifications at the heterologous object sequence.

[0208] FIG. 17B is a bar graph showing the % indel levels in the livers of mice administered with the exemplary gene modifying systems evaluated in FIG. 17A.

[0209] FIG. 18A is a bar graph showing the rewriting efficiency in the livers of mice administered with exemplary gene modifying systems comprising different gene modifying polypeptides and different template RNAs comprising scaffold chemical modifications in combination with fluoro modifications at the heterologous object sequence.

[0210] FIG. 18B is a bar graph showing the % indel levels in the livers of mice administered with the exemplary gene modifying systems evaluated in FIG. 18A. FIG. 19A is a bar graph showing the % corrected genomic DNA in the livers of hSERPINAl E342K mice by administered with exemplary gene modifying systems comprising RNAIVT6241 or RNAIVT6898 polypeptides over evaluated dosages.

[0211] FIG. 19B is a bar graph showing the % corrected genomic DNA in the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 7 days post-administration.

[0212] FIG. 19C is a bar graph showing the % corrected genomic DNA in the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 21 days postadministration.

[0213] FIG. 19D is a bar graph showing the % edited mRNA in the livers of hSERPINAl E342K and NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 21 days postadministration.

[0214] FIG. 19E is a bar graph showing the % indels introduced into the livers of hSERPINAl E342K by exemplary gene modifying polypeptides RNAVT6241 and RNAIVT6838 over evaluated dosages.

[0215] FIG. 19F is a bar graph showing the % indels introduced into the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 7 days post-administration.

[0216] FIG. 19G is a bar graph showing the % indels introduced into the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 21 days post-administration.

[0217] FIG. 20A is a bar graph showing the serum concentration of human Al AT in hSERPINAl E342K mice administered with exemplary gene modifying systems comprising RNAIVT6241 or RNAIVT6898 polypeptides over evaluated dosages.

[0218] FIG. 20B is a bar graph showing the serum concentration of human Al AT in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 7 days postadministration.

[0219] FIG. 20C is a bar graph showing the serum concentration of human Al AT in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 21 days postadministration.

[0220] FIG. 21A is a bar graph showing the % liver area occupied by globules in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 7 days postadministration. FIG. 21B is a bar graph showing the % liver area occupied by globules in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 21 days postadministration.

[0221] FIG. 22A is a bar graph showing the % corrected genomic DNA in the livers of NSG-PiZ mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.

[0222] FIG. 22B is a bar graph showing the % edited mRNA in the livers of NSG-PiZ mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.

[0223] FIG. 22C is a bar graph showing the % indels introduced into the livers of NSG-PiZ mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.

[0224] FIG. 23A is a bar graph showing the serum concentration of human Al AT in NSG-PiZ mice administered with exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.

[0225] FIG. 23B is a line graph showing the serum concentration of human Al AT as a function of % genomic DNA rewriting in NSG-PiZ mice administered with exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages.

[0226] FIG. 24A is a bar graph showing the % corrected genomic DNA in the livers of hSERPINAl E342K mice by exemplary gene modifying systems comprising RNAIVT9315 or RNAIVT9318 gene modifying polypeptides and RNACS22230 template RNA over evaluated dosages.

[0227] FIG. 24B is a bar graph showing the % indels introduced in the livers of hSERPINAl E342K mice by exemplary gene modifying systems comprising RNAIVT9315 or RNAIVT9318 gene modifying polypeptides and RNACS22230 template RNA over evaluated dosages.

[0228] FIGs. 25A to 25D show bar graphs of the rewriting performance in the livers of hSERPINAl E342K mice administered with exemplary StlCas9-based gene modifying systems comprising exemplary RNAIVT9315 gene modifying polypeptide and exemplary template RNAs RNACS24756 or RNACS24757 at 0.012 mg / kg (FIG. 25A), 0.025 mg / kg (FIG. 25B), 0.05 mg / kg (FIG. 25C) and 0.1 mg / kg (FIG. 25D). FIGs. 26A to 26D show bar graphs of the % indel levels in the livers of hSERPINA l E342K mice administered with exemplary StlCas9-bascd gene modifying systems comprising exemplary RNAIVT9315 gene modifying polypeptide and exemplary template RNAs RNACS24756 or RNACS24757 at 0.012 mg / kg (FIG. 26A), 0.025 mg / kg (FIG. 26B), 0.05 mg / kg (FIG. 26C) and 0.1 mg / kg (FIG. 26D).

[0229] FIG. 27A is a bar graph showing the % corrected genomic DNA in the livers of hSERPINAl E342K mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg / kg) and formulated in LNP.

[0230] FIG. 27B is a bar graph showing the % edited mRNA in the livers of hSERPINAl E342K mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg / kg) and formulated in LNP.

[0231] FIG. 27C is a bar graph showing the % indels introduced in the livers of hSERPINAl E342K mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg / kg) and formulated in LNP.

[0232] FIG. 28A is a bar graph showing the serum concentration of human Al AT in hSERPINAl E342K mice administered with exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg / kg) and formulated in LNP.

[0233] FIG. 28B is a line graph showing the serum concentration of human Al AT as a function of % genomic DNA rewriting in hSERPINAl E342K mice administered with exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg / kg) and formulated in LNP.

[0234] Definitions

[0235] The term '‘expression cassette,” as used herein, refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of the nucleic acid molecule of the instant invention. A “gRNA spacer,” as used herein, refers to a portion of a nucleic acid that has complementarity to a target nucleic acid and can, together with a gRNA scaffold, target a Cas protein to the target nucleic acid.

[0236] A “gRNA scaffold,” as used herein, refers to a portion of a nucleic acid that can bind a Cas protein and can, together with a gRNA spacer, target the Cas protein to the target nucleic acid. In some embodiments, the gRNA scaffold comprises a crRNA sequence, tetraloop, and tracrRNA sequence.

[0237] The term “StlCas9 scaffold,” as used herein, refers to a gRNA scaffold that can bind an StlCas9 protein and can, together with a gRNA spacer, target the StlCas9 protein to the target nucleic acid. In some embodiments, an StlCas9 scaffold comprises a crRNA sequence, tetraloop, and tracerRNA sequence. An exemplary position of StlCas9 scaffold within an exemplary template RNA is illustrated in FIG. 1.

[0238] In some embodiments, an StlCas9 scaffold comprises a full length wild type sequence. In some embodiments, an StlCas9 scaffold comprises a sequence with at least 80%, 85%. 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of embodiments, an StlCas9 scaffold comprises a sequence identical to SEQ ID NO: 25999. In some embodiments, an StlCas9 scaffold comprises a truncation mutant. In some embodiments, an StlCas9 scaffold comprises a sequence with at least 80%, 85%. 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of

[0239] A “variant gRNA scaffold,” as used herein, refers to gRNA scaffold having a non- naturally occurring sequence. In some embodiments, a variant gRNA scaffold sequence comprises one or more substitutions relative to the closest naturally occurring sequence. In some embodiments, a variant gRNA scaffold sequence comprises one or more insertions relative to the closest naturally occurring sequence. In some embodiments, a variant gRNA scaffold sequence comprises one or more deletions relative to the closest naturally occurring sequence.

[0240] A “gene modifying polypeptide,” as used herein, refers to a polypeptide comprising a retroviral reverse transcriptase, or a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to a retroviral reverse transcriptase, which is capable of integrating a nucleic acid sequence (e.g., a sequence provided on a template nucleic acid) into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell). In some embodiments, a gene modifying polypeptide is capable of integrating a sequence substantially without relying on host machinery. In some embodiments, a gene modifying polypeptide integrates a sequence into a random position in a genome. In some embodiments, a gene modifying polypeptide integrates a sequence into a specific target site. In some embodiments, a gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding a template nucleic acid, 2) binding a target DNA molecule, and 3) integration of at least a portion of the template nucleic acid into the target DNA. Gene modifying polypeptides include both naturally occurring polypeptides as well as engineered variants of the foregoing, e.g., having one or more amino acid substitutions to a naturally occurring sequence. Gene modifying polypeptides also include heterologous constructs, e.g., where one or more of the domains are heterologous to each other, whether through a heterologous fusion (or other conjugate) of otherwise wild-type domains, as well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous subdomain or other substituted domain. Exemplary gene modifying polypeptides, and systems comprising the same can be used in methods provided herein and described, e.g., in PCT / US2021 / 020948, which is incorporated herein by reference with respect to gene modifying polypeptides that comprise a retroviral reverse transcriptase domain. In some embodiments, a gene modifying polypeptide integrates a sequence into a gene. In some embodiments, a gene modifying polypeptide integrates a sequence into a sequence outside of a gene. A “gene modifying system,” as used herein, refers to a system comprising a gene modifying polypeptide and a template nucleic acid.

[0241] The term “domain,” as used herein, refers to a structure of a biomolecule that contributes to a specified function of the biomolecule. A domain may comprise a contiguous region (e.g., a contiguous sequence) or distinct, non-contiguous regions (e.g., non-contiguous sequences) of a biomolcculc. Examples of protein domains include, but arc not limited to, an endonuclease domain, a DNA binding domain, a reverse transcription domain; an example of a domain of a nucleic acid is a regulatory domain, such as a transcription factor binding domain. In some embodiments, a domain (e.g., a Cas domain) can comprise two or more smaller domains (e.g., a DNA binding domain and an endonuclease domain).

[0242] As used herein, the term “exogenous,” when used with reference to a biomolecule (such as a nucleic acid sequence or polypeptide) means that the biomolecule was introduced into a host genome, cell or organism by the hand of man. For example, a nucleic acid that is as added into an existing genome, cell, tissue or subject using recombinant DNA techniques or other methods is exogenous to the existing nucleic acid sequence, cell, tissue or subject.

[0243] As used herein, “first strand” and “second strand,” as used to describe the individual DNA strands of target DNA, distinguish the two DNA strands based upon which strand a reverse transcriptase domain initiates polymerization, e.g., based upon where target primed synthesis initiates. A “first strand” refers to the strand of a target DNA upon which a reverse transcriptase domain initiates polymerization, e.g., where target primed synthesis initiates. A “second strand” refers to the other strand of the target DNA. First and second strand designations do not describe a target site DNA strands in other respects; for example, in some embodiments the first and second strands are nicked by a polypeptide described herein, but the designations ‘first’ and ‘second’ strand have no bearing on the order in which such nicks occur.

[0244] The term “heterologous,” as used herein to describe a first element in reference to a second element means that a first element and second element do not exist in nature disposed as described. For example, a heterologous polypeptide, nucleic acid molecule, construct or sequence refers to (a) a polypeptide, nucleic acid molecule or portion of a polypeptide or nucleic acid molecule sequence that is not native to a cell in which it is expressed, (b) a polypeptide or nucleic acid molecule or portion of a polypeptide or nucleic acid molecule that has been altered or mutated relative to its native state, or (c) a polypeptide or nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions. For example, a heterologous regulatory sequence (e.g., promoter, enhancer) may be used to regulate expression of a gene or a nucleic acid molecule in a way that is different than the gene or a nucleic acid molecule is normally expressed in nature. In another example, a heterologous domain of a polypeptide or nucleic acid sequence (e.g., a DNA binding domain of a polypeptide or nucleic acid encoding a DNA binding domain of a polypeptide) may be disposed relative to other domains or may be a different sequence or from a different source, relative to other domains or portions of a polypeptide or its encoding nucleic acid. In certain embodiments, a heterologous nucleic acid molecule may exist in a native host cell genome but may have an altered expression level or have a different sequence or both. In other embodiments, heterologous nucleic acid molecules may not be endogenous to a host cell or host genome but instead may have been introduced into a host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector).

[0245] As used herein, “insertion” of a sequence into a target site refers to the net addition of DNA sequence at a target site, e.g., where there are new nucleotides in a heterologous object sequence with no cognate positions in the unedited target site. In some embodiments, a nucleotide alignment of a PBS sequence and heterologous object sequence to a target nucleic acid sequence would result in an alignment gap in the target nucleic acid sequence.

[0246] As used herein, a “deletion” generated by a heterologous object sequence in a target site refers to the net deletion of DNA sequence at the target site, e.g., where there are nucleotides in the unedited target site with no cognate positions in the heterologous object sequence. In some embodiments, a nucleotide alignment of the PBS sequence and heterologous object sequence to the target nucleic acid sequence would result in an alignment gap in the molecule comprising the PBS sequence and heterologous object sequence.

[0247] The term “inverted terminal repeats” or “ITRs” as used herein refers to AAV viral ciselements named so because of their symmetry. These elements promote efficient multiplication of an AAV genome. It is hypothesized that the minimal elements for ITR function are a Repbinding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' for AAV2; SEQ ID NO: 4601) and a terminal resolution site (TRS; 5'-AGTTGG-3' for AAV2; SEQ ID NO: 4602) plus a variable palindromic sequence allowing for hairpin formation. According to the present invention, an ITR comprises at least these three elements (RBS, TRS, and sequences allowing the formation of a hairpin). In addition, in the present invention, the term “ITR” refers to ITRs of known natural AAV serotypes (e.g., ITR of a serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 AAV), to chimeric ITRs formed by the fusion of ITR elements derived from different serotypes, and to functional variants thereof. “Functional variant” refers to a sequence presenting a sequence identity of at least 80%, 85%, 90%, preferably of at least 95% with a known ITR and allowing multiplication of the sequence that includes said ITR in the presence of Rep proteins.

[0248] The term “mutation region,” as used herein, refers to a region in a template RNA having one or more sequence difference relative to the corresponding sequence in a target nucleic acid. The one or more sequence difference may comprise, for example, a substitution, insertion, frameshift, or deletion.

[0249] The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence are inserted, deleted, or changed compared to a reference (e.g., native) nucleic acid sequence. A single alteration may be made at a locus (a point mutation), or multiple nucleotides may be inserted, deleted, or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the ail.

[0250] “Nucleic acid molecule” refers to both RNA and DNA molecules including, without limitation, complementary DNA (“cDNA”), genomic DNA (“gDNA”), and messenger RNA (“mRNA”), and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced, such as RNA templates, as described herein. A nucleic acid molecule can be double- stranded or single-stranded, circular’, or linear. If single- stranded, a nucleic acid molecule can be a sense strand or an antisense strand. Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:,” or “nucleic acid comprising SEQ ID NO: 1” refers to a nucleic acid, at least a portion which has either (i) the sequence of SEQ ID NO: 1, or (ii) a sequence complimentary to SEQ ID NO: 1. The choice between the two is dictated by the context in which SEQ ID NO: 1 is used. For instance, if a nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to a desired target. Nucleic acid sequences of the present disclosure may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with an analog, inter-nucleotide modifications such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendant moieties, (for example, polypeptides), intcrcalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.). Also included are chemically modified bases (see, for example, Table 32), backbones (see, for example, Table 33), and modified caps (see, for example, Table 34). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of a molecule, e.g., peptide nucleic acids (PNAs). Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as modifications found in “locked” nucleic acids (LNAs). In some embodiments, nucleic acids are in operative association with additional genetic elements, such as tissue-specific expressioncontrol sequence(s) (e.g., tissue- specific promoters and tissue-specific microRNA recognition sequences), as well as additional elements, such as inverted repeats (e.g., inverted terminal repeats, such as elements from or derived from viruses, e.g., AAV ITRs) and tandem repeats, inverted repeats / direct repeats, homology regions (segments with various degrees of homology to a target DNA), untranslated regions (UTRs) (5k 3k or both 5' and 3" UTRs), and various combinations of the foregoing. Nucleic acid elements of systems disclosed in the present application may be provided in a variety of topologies, including single-stranded, doublestranded, circular, linear, linear with open ends, linear with closed ends, and particular versions of these, such as doggybone DNA (dbDNA), and closed-ended DNA (ceDNA).

[0251] The term “chemically modified nucleotide,” as used herein, refers to a nucleotide comprising one or more structural differences relative to the canonical ribonucleotides (i.e., G, U, C, and .A). A chemically modified nucleotide may have (relative to a canonical nucleotide) a chemically modified nudeobase, a chemically modified sugar, a chemically modified phosphodiester linkage, or a combination thereof. In some embodiments, a chemically modified nucleotide is a 2'-O-methyl nucleotide, e.g., 2'-O-methyl- Adenosine, 2'-O-methyl-Cytidine, 2'-O- methyl-Guanosine, or 2'-O-methyl-Uridine. No particular process of making is implied: for instance, a chemically modified nucleotide can be produced directly by chemical synthesis, or by covalently modifying a canonical nucleotide. The term “chemical modification,” as used herein, refers to a structural difference of a chemical modified nucleotide relative to die canonical ribonucleotides (i.c., G, U, C. and A). A chemical modification may comprise a modification resulting in a chemically modified nucleobase, a chemically modified sugar, a chemically modified phosphodiester linkage, or a combination thereof. In some embodiments, a chemical modification is 2'-O-methylation or 2’- fluoro modification. No particular process of making is implied; for instance, a chemical modification can be produced directly by chemical synthesis, or by covalently modifying a canonical nucleotide.

[0252] As used herein, the term “position” with respect to an StlCas9 scaffold refers to anucleotide of the StlCas9 scaffold that aligns with a corresponding nucleotide of a reference sequence of SEQ ID NO: 25999. The positions of the reference sequence are illustrated in FIG, 14. Alignments of nucleic acid or polypeptide sequences can be performed by using a routine sequence analysis tool such as Basic Local Alignment Search Tool (BLAST), for instance NIH megablast using default parameters.

[0253] In some embodiments, a position of an StlCas9 scaffold can be identified by providing an alignment of an StlCas9 scaffold (query sequence) to a reference sequence of SEQ ID NO: 25999 (a full length wild-type sequence, see e.g., FIG 14B) or SEQ ID NO: 26000 (a truncation mutant, see e.g., FIG. 14A), and identifying the position in the query sequence that corresponds to the position in the reference sequence. For example, in an StlCas9 scaffold consisting of the sequence of SEQ ID NO: 25999 except that the 5’ most G is substituted with a single nucleotide other than G, the substituted position is position 1.

[0254] As another example, in an StlCas9 scaffold consisting of the sequence of SEQ ID NO: 25999 except that a single new nucleotide is inserted just 5’ of the 5’ most G, the G is still position 1.

[0255] As yet another example, in an StICas9 scaffold consisting of the sequence of SEQ ID NO: 25999 except that a sequence of n nucleotides is inserted between the G of position 1 and the U of position 2, nucleotides 3’ of the insert maintain their original position number. For example, the U of position 2 is still position 2 rather than position n+2. A nucleotide that is inserted relative to the reference sequence need not be assigned a position number. A range of nucleotides includes all nucleotides in that range regardless of whether they are assigned a number; for example, if a scaffold comprises a chemically modified nucleotide at each of positions 12 through 21 , and the scaffold comprises inserted nucleotides anywhere between positions 12 and 21, then the scaffold comprises chemically modified nucleotides at each of the inserted nucleotides situated anywhere between positions 12 and 21 (which inserted nucleotides do not have a position number in this example), as well as chemically modified nucleotides at positions 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21.

[0256] As used herein, a “gene expression unit” is a nucleic acid sequence comprising at least one regulatory nucleic acid sequence operably linked to at least one effector sequence. A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be contiguous or non-contiguous. Where necessary to join two protein-coding regions, operably linked sequences may be in the same reading frame.

[0257] The terms “host genome” or “host cell,” as used herein, refer to a cell and / or its genome into which protein and / or genetic material has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell and / or genome, but to the progeny of such a cell and / or the genome of the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A host genome or host cell may be an isolated cell or cell line grown in culture, or genomic material isolated from such a cell or cell line, or may be a host cell or host genome which composing living tissue or an organism. In some embodiments, a host cell may be an animal cell or a plant cell, e.g., as described herein. In some embodiments, a host cell may be a mammalian cell, a human cell, avian cell, reptilian cell, bovine cell, horse cell, pig cell, goat cell, sheep cell, chicken cell, or turkey cell. In some embodiments, a host cell may be a corn cell, soy cell, wheat cell, or rice cell.

[0258] As used herein, “operative association” describes a functional relationship between two nucleic acid sequences, such as a 1) promoter and 2) a heterologous object sequence, and means, in such example, the promoter and heterologous object sequence (e.g., a gene of interest) are oriented such that, under suitable conditions, the promoter drives expression of the heterologous object sequence. For instance, a template nucleic acid carrying a promoter and a heterologous object sequence may be single-stranded, e.g., either the (+) or (-) orientation. An “operative association” between the promoter and the heterologous object sequence in this template means that, regardless of whether the template nucleic acid will be transcribed in a particular state, when it is in the suitable state (e.g., is in the (+) orientation, in the presence of required catalytic factors, and NTPs, etc.), it is accurately transcribed. Operative association applies analogously to other pairs of nucleic acids, including other tissue- specific expression control sequences (such as enhancers, repressors and microRNA recognition sequences), IR / DR, ITRs, UTRs, or homology regions and heterologous object sequences or sequences encoding a retroviral RT domain.

[0259] The term “primer binding site sequence” or “PBS sequence,” as used herein, refers to a portion of a template RNA capable of binding to a region in a target nucleic acid sequence, hi some embodiments, a PBS sequence is a nucleic acid sequence comprising at least 3, 4, 5, 6, 7, or 8 bases with 100% identity to a region in a target nucleic acid sequence. In some embodiments, a primer region comprises at least 5, 6, 7, 8 bases with 100% identity to a region in a target nucleic acid sequence. Without wishing to be bound by theory, in some embodiments, when a template RNA comprises a PBS sequence and a heterologous object sequence, the PBS sequence binds to a region in a target nucleic acid sequence, allowing a reverse transcriptase domain to use that region as a primer for reverse transcription, and to use the heterologous object sequence as a template for reverse transcription.

[0260] As used herein, a “stem-loop sequence” refers to a nucleic acid sequence (e.g., RNA sequence) with sufficient self-complementarity to form a stem-loop, e.g., having a stem comprising at least two (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) base pairs, and a loop with at least three (e.g., four) base pairs. The stem may comprise mismatches or bulges.

[0261] As used herein, a “tissue- specific expression-control sequence” means nucleic acid elements that increase or decrease the level of a transcript comprising the heterologous object sequence in a target tissue in a tissue-specific manner, e.g., preferentially in on-target tissue(s), relative to off-target tissue(s). In some embodiments, a tissue- specific expression-control sequence preferentially drives or represses transcription, activity, or the half-life of a transcript comprising the heterologous object sequence in the target tissue in a tissue- specific manner, e.g., preferentially in an on-target tissue(s), relative to an off-target tissue(s). Exemplary tissuespecific expression-control sequences include tissue-specific promoters, repressors, enhancers, or combinations thereof, as well as tissue-specific microRNA recognition sequences. Tissue specificity refers to on-target (tissuc(s) where expression or activity of the template nucleic acid is desired or tolerable) and off-target (tissue(s) where expression or activity of the template nucleic acid is not desired or is not tolerable). For example, a tissue-specific promoter drives expression preferentially in on-target tissues, relative to off-target tissues. In contrast, a microRNA that binds the tissue- specific microRNA recognition sequences is preferentially expressed in off-target tissues, relative to on-target tissues, thereby reducing expression of a template nucleic acid in off-target tissues. Accordingly, a promoter and a microRNA recognition sequence that are specific for the same tissue, such as the target tissue, have contrasting functions (promote and repress, respectively, with concordant expression levels, i.e., high levels of the microRNA in off-target tissues and low levels in on-target tissues, while promoters drive high expression in on-target tissues and low expression in off-target tissues) with regard to the transcription, activity, or half-life of an associated sequence in that tissue.

[0262] As used herein, “indel” refers to a mutation resulting from an insertion, deletion, or a combination thereof. As will be appreciated by those skilled in the art, an indel in a coding region of a genomic sequence will result in a frameshift mutation, unless the length of the indel is a multiple of three. In some embodiments, a genetic modification is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one of the nucleotides. A system of the present disclosure can be used to induce an indel of any length or a point mutation in a target polynucleotide sequence.

[0263] DETAILED DESCRIPTION

[0264] This disclosure provides methods for treating alpha- 1 antitrypsin deficiency (AATD) and compositions for targeting, editing, modifying or manipulating a DNA sequence (e.g., inserting a heterologous object sequence into a target site of a mammalian genome) at one or more locations in a DNA sequence in a cell, tissue or subject, e.g., in vivo or in vitro. A heterologous object DNA sequence may include, e.g., a substitution.

[0265] In some embdoients, the disclosure provides methods for treating AATD using reverse transcriptase-based systems for altering a genomic DNA sequence of interest, e.g., by inserting, deleting, or substituting one or more nucleotides into / from a sequence of interest.

[0266] In some embodiments, the disclosure provides methods for treating AATD using a gene modifying system comprising a gene modifying polypeptide component and a template nucleic acid (e.g., template RNA) component. Tn some embodiments, a gene modifying system can be used to introduce an alteration into a target site in a genome. In some embodiments, a gene modifying polypeptide component comprises a writing domain (e.g., a reverse transcriptase domain), a DNA-binding domain, and an endonuclease domain (e.g., a nickase domain). In some embodiments, a template nucleic acid (e.g., template RNA) comprises a sequence (e.g., a gRNA spacer) that binds a target site in the genome (e.g., that binds to a second strand of a target site), a sequence (e.g., a gRNA scaffold) that binds a gene modifying polypeptide component, a heterologous object sequence, and a PBS sequence. Without wishing to be bound by theory, it is thought that a template nucleic acid (e.g., template RNA) binds to the second strand of a target site in the genome and binds to a gene modifying polypeptide component (e.g., localizing the gene modifying polypeptide component to the target site in the genome). It is thought that the endonuclease (e.g., nickase) of a gene modifying polypeptide component cuts a target site (e.g., the first strand of the target site), e.g., allowing a PBS sequence to bind to a sequence adjacent to a site to be altered on the first strand of the target site. It is thought that a writing domain (e.g., reverse transcriptase domain) of a gene modifying polypeptide component uses the first strand of a target site that is bound to a complementary sequence comprising a PBS sequence of a template nucleic acid as a primer and a heterologous object sequence of the template nucleic acid as a template to, e.g., polymerize a sequence complementary to the heterologous object sequence. Without wishing to be bound by theory, it is thought that selection of an appropriate heterologous object sequence can result in substitution, deletion, and / or insertion of one or more nucleotides at the target site.

[0267] Gene modifying systems

[0268] In some embodiments, a gene modifying system described herein comprises: (A) a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide, wherein the gene modifying polypeptide comprises (i) a reverse transcriptase domain, and either (x) an endonuclease domain that contains DNA binding functionality or (y) an endonuclease domain and separate DNA binding domain; and (B) a template RNA. A gene modifying polypeptide, in some embodiments, acts as a substantially autonomous protein machine capable of integrating a template nucleic acid sequence into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell), substantially without relying on host machinery. For example, a gene modifying polypeptide may comprise a DNA-binding domain, a reverse transcriptase domain, and an endonuclease domain. In some embodiments, a DNA-binding function may involve an RNA component that directs a gene modifying polypeptide to a DNA sequence, e.g., a gRNA spacer. In other embodiments, a gene modifying polypeptide may comprise a reverse transcriptase domain and an endonuclease domain. An RNA template element of a gene modifying system may be heterologous to a gene modifying polypeptide element and provides an object sequence to be inserted (reverse transcribed) into a host genome. In some embodiments, a gene modifying polypeptide is capable of target primed reverse transcription. In some embodiments, a gene modifying polypeptide is capable of second-strand synthesis.

[0269] In some embodiments, a gene modifying system is combined with a second polypeptide. In some embodiments, a second polypeptide may comprise an endonuclease domain. In some embodiments, a second polypeptide may comprise a polymerase domain, e.g., a reverse transcriptase domain. In some embodiments, a second polypeptide may comprise a DNA- dependent DNA polymerase domain. In some embodiments, a second polypeptide aids in completion of a genome edit, e.g., by contributing to second-strand synthesis or DNA repair resolution.

[0270] A functional gene modifying polypeptide can be made up of unrelated DNA binding, reverse transcription, and endonuclease domains. This modular structure allows combining of functional domains, e.g., dCas9 (DNA binding), MMLV reverse transcriptase (reverse transcription), FokI (endonuclease). In some embodiments, multiple functional domains may arise from a single protein, e.g., Cas9 or Cas9 nickase (DNA binding, endonuclease).

[0271] In some embodiments, a gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding a template nucleic acid, 2) binding a target DNA molecule, and 3) integration of at least a portion of the template nucleic acid into the target DNA. In some embodiments, a gene modifying polypeptide is an engineered polypeptide that comprises one or more amino acid substitutions to a corresponding naturally occurring sequence. In some embodiments, a gene modifying polypeptide comprises two or more domains that are heterologous relative to each other, e.g., through a heterologous fusion (or other conjugate) of otherwise wild-type domains, or well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain. For instance, in some embodiments, one or more of: an RT domain is heterologous to a DNA-binding domain (DBD); a DBD is heterologous to an endonuclease domain; or an RT domain is heterologous to an endonuclease domain.

[0272] In some embodiments, a template RNA molecule for use in a system of the present disclosure comprises, from 5' to 3' (1) a gRNA spacer; (2) a gRNA scaffold; (3) a heterologous object sequence; and (4) a primer binding site (PBS) sequence. In some embodiments, a gRNA spacer is about!8 to -22 nucleotides in length (e.g., about 20 nucleotides in length). In some embodiments, a gRNA scaffold comprises one or more hairpin loops, e.g., 1, 2, or 3 loops for associating a template RNA with a Cas domain, e.g., a nickase Cas9 domain. In some embodiments, a gRNA scaffold comprises the sequence, from 5' to 3', GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGGACCGAGTCGGTCC (SEQ ID NO: 5008). In some embodiments, a heterologous object sequence is, e.g., 7-74, e.g., 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, or 70-80 nucleotides or, 80-90 nucleotdies in length. In some embodiments, a first (i.e., 5 '-most) base of a heterologous object sequence is not C. In some embodiments, a PBS sequence that binds a target priming sequence after nicking occurs is e.g., 3-20 nucleotides, e.g., 7-15 nucleotides, e.g., 12-14 nucleotides in length. In some embodiments, a PBS sequence has 40-60% GC content.

[0273] In some embodiments, a second gRNA associated with a system of the present disclosure may help drive complete integration. In some embodiments, a second gRNA may target a location that is 0-200 nucleotides away from a first-strand nick, e.g., 0-50, 50-100, 100-200 nucleotides away from the first-strand nick. In some embodiments, a second gRNA can only bind its target sequence after an edit is made, e.g., the gRNA binds a sequence present in a heterologous object sequence, but not in the initial target sequence.

[0274] In some embodiments, a gene modifying system described herein is used to make an edit in HEK293, K562, U2OS, or HeLa cells. In some embodiment, a gene modifying system is used to make an edit in primary cells, e.g., primary liver cells or primary lung cells.

[0275] In some embodiments, a gene modifying polypeptide as described herein comprises a reverse transcriptase or RT domain (e.g., as described herein) that comprises a MoMLV RT sequence or variant thereof. In embodiments, a MoMLV RT sequence comprises one or more mutations selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, and K103L. In some embodiments, a MoMLV RT sequence comprises a combination of mutations, such as D200N, L603W, and T33OP, optionally further including T306K and / or W313F.

[0276] In some embodiments, an endonuclease domain (e.g., as described herein) is Cas9. In some embodiments, an endonuclease domain is nCas9. In some embodiments, an endonuclease domain comprises an N863A mutation (e.g., in spCas9). In some embodiments, an endonuclease domain comprises a H840A mutation.

[0277] In some embodiments, a heterologous object sequence (e.g., of a system as described herein) is about 1-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, or more, nucleotides in length.

[0278] In some embodiments, RT and endonuclease domains are joined by a flexible linker. In some embdoiments, a linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 5006).

[0279] In some embodiments, an endonuclease domain is N-terminal relative to an RT domain. In some embodiments, an endonuclease domain is C-terminal relative to an RT domain.

[0280] In some embodiments, a system of the present disclosure incorporates a heterologous object sequence into a target site by target primed reverse transcription (TPRT), e.g., as described herein.

[0281] In some embodiments, a gene modifying polypeptide comprises a DNA binding domain (DBD). In some embodiments, a gene modifying polypeptide comprises an RNA binding domain. In some embodiments, an RNA binding domain comprises an RNA binding domain of B-box protein, MS2 coat protein, dCas, or an element of a sequence of a Table herein. In some embodiments, an RNA binding domain is capable of binding to a template RNA with greater affinity than a reference RNA binding domain.

[0282] In some embodiments, a gene modifying system is capable of producing an insertion of at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides (and optionally no more than 500, no more than 400, no more than 300, no more than 200, or no more than 100 nucleotides) in a target site. In some embodiments, a gene modifying system is capable of producing an insertion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides (and optionally no more than 500, no more than 400, no more than 300, no more than 200, or no more than 100 nucleotides) in a target site. In some embodiments, a gene modifying system is capable of producing an insertion of at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5 or at least 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases) into a target site. In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides (and optionally no more than 500, no more than 400, no more than 300, or no more than 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides (and optionally no more than 500, no more than 400, no more than 300, no more than 200, or no more than 100 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5 or at least 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases). In some embodiments, a gene modifying system is capable of producing a substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100, or more nucleotides in a target site. In some embodiments, a gene modifying system is capable of producing a substitution of 1 -2, 2-3, 3-4, 4-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40- 50, 50-60, 60-70, 70-80, 80-90, or 90-100 nucleotides in a target site.

[0283] In some embodiments, a substitution is a transition mutation. In some embodiments, a substitution is a transversion mutation. In some embodiments, a substitution converts an adenine to a thymine, an adenine to a guanine, an adenine to a cytosine, a guanine to a thymine, a guanine to a cytosine, a guanine to an adenine, a thymine to a cytosine, a thymine to an adenine, a thymine to a guanine, a cytosine to an adenine, a cytosine to a guanine, or a cytosine to a thymine.

[0284] In some embodiments, an insertion, deletion, substitution, or combination thereof, increases or decreases expression (e.g., transcription or translation) of a gene. In some embodiments, an insertion, deletion, substitution, or combination thereof, increases or decreases expression (e.g,. transcription or translation) of a gene by altering, adding, or deleting sequences in a promoter or enhancer, e.g., sequences that bind transcription factors. In some embodiments, an insertion, deletion, substitution, or combination thereof alters translation of a gene (e.g., alters an amino acid sequence), inserts or deletes a start or stop codon, or alters or fixes the translation frame of a gene. In some embodiments, an insertion, deletion, substitution, or combination thereof alters splicing of a gene, e.g., by inserting, deleting, or altering a splice acceptor or donor site. In some embodiments, an insertion, deletion, substitution, or combination thereof alters transcript or protein half-life. In some embodiments, an insertion, deletion, substitution, or combination thereof alters protein localization in the cell (e.g., from the cytoplasm to a mitochondria, from the cytoplasm into the extracellular space (e.g., adds a secretion tag)). In some embodiments, an insertion, deletion, substitution, or combination thereof alters (e.g., improves) protein folding (e.g., to prevent accumulation of misfolded proteins). In some embodiments, an insertion, deletion, substitution, or combination thereof, alters, increases, decreases the activity of a gene, e.g., a protein encoded by the gene.

[0285] Exemplary gene modifying polypeptides, systems comprising the same, and methods of using the same are described, e.g., in PCT / US2021 / 020948, which is incorporated herein by reference with respect to retroviral RT domains, including the amino acid and nucleic acid sequences therein.

[0286] Exemplary gene modifying polypeptides and retroviral RT domain sequences are also described, e.g., in International Application No. PCT / US21 / 20948, filed March 4, 2021, e.g., at Table 30, Table 31 , and Table 44 therein; the entire application is incorporated by reference herein with respect to retroviral RTs, e.g., in said sequences and Tables. Accordingly, a gene modifying polypeptide described herein may comprise an amino acid sequence according to any of the Tables mentioned in this paragraph, or a domain thereof (e.g., a retroviral RT domain), or a functional fragment or variant of any of the foregoing, or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0287] In some embodiments, a polypeptide for use in any of the systems described herein can be a molecular reconstruction or ancestral reconstruction based upon the aligned polypeptide sequence of multiple homologous proteins. In some embodiments, a reverse transcriptase domain for use in any of the systems described herein can be a molecular reconstruction or an ancestral reconstruction, or can be modified at particular residues, based upon alignments of reverse transcriptase domains from the same or different sources. A skilled artisan can, based on the Accession numbers provided herein, align polypeptides or nucleic acid sequences, e.g., by using routine sequence analysis tools as Basic Local Alignment Search Tool (BLAST) or CD- Search for conserved domain analysis. Molecular reconstructions can be created based upon sequence consensus, e.g., using approaches described in Ivies et al., Cell 1997, 501 - 510; Wagstaff et al., Molecular Biology and Evolution 2013, 88-99.

[0288] Polypeptide components of gene modifying systems

[0289] In some embodiments, a gene modifying polypeptide possesses the functions of DNA target site binding, template nucleic acid (e.g., template RNA) binding, DNA target site cleavage, and template nucleic acid (e.g., template RNA) writing (e.g., reverse transcription). In some embodiments, each function is contained within a distinct domain. In some embodiments, a function may be attributed to two or more domains (e.g., two or more domains, together, exhibit the functionality). In some embodiments, two or more domains may have the same or similar function (e.g., two or more domains each independently have DNA-binding functionality, e.g., for two different DNA sequences). In some embodiments, one or more domains may be capable of enabling one or more functions, e.g., a Cas9 domain enabling both DNA binding and target site cleavage. In some embodiments, domains are all located within a single polypeptide. In some embodiments, a first domain is in one polypeptide and a second domain is in a second polypeptide. For example, in some embodiments, sequences may be split between a first polypeptide and a second polypeptide, e.g., wherein the first polypeptide comprises a reverse transcriptase (RT) domain and wherein the second polypeptide comprises a DNA-binding domain and an endonuclease domain, c.g., a nickase domain. As a further example, in some embodiments, a first polypeptide and a second polypeptide each comprise a DNA binding domain (e.g., a first DNA binding domain and a second DNA binding domain). In some embodiments, a first polypeptides and a second polypeptide may be brought together post- translationally via a split-intein to form a single gene modifying polypeptide.

[0290] In some embodiments, a gene modifying polypeptide described herein comprises an StlCas9 domain. An StlCas9 domain can comprise a naturally occurring StlCas9 amino acid sequence, or a variant thereof. In some embodiments, an StlCas9 domain is a nickase. In some embodiments, an StlCas9 domain comprises a sequence according to SEQ ID NO: 23818, or a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprising an StlCas9 domain is used together with a compatible template RNA comprising a variant gRNA scaffold described herein.

[0291] In some embodiments, a gene modifying polypeptide described herein comprises (e.g., a system described herein comprises a gene modifying polypeptide that comprises): 1) a Cas domain (e.g., a Cas nickase domain, e.g., a Cas9 nickase domain); 2) a reverse transcriptase (RT) domain of Table 7 or Table 8, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, wherein the RT domain is C-terminal of the Cas domain; and a linker is disposed between the RT domain and the Cas domain, wherein the linker has a sequence from the same row of Table 7 or Table 8 as the RT domain, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0292] In some embodiments, an RT domain has a sequence with 100% identity to an RT domain of Table 7 or Table 8and a linker has a sequence with 100% identity to the linker sequence from the same row of Table 7 or Table 8as the RT domain. In some embodiments, a Cas domain comprises a sequence of Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-3332 in the sequence listing, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0293] In some embodiments, a gene modifying polypeptide comprises a GG amino acid sequence between a Cas domain and a linker, an AG amino acid sequence between an RT domain and a second nuclear localization seqeuence (NLS), and / or a GG amino acid sequence between the linker and the RT domain. In some embodiments, a gene modifying polypeptide comprises a sequence of SEQ ID NO: 4000 which comprises a first NLS and a Cas domain, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a sequence of SEQ ID NO: 4001 which comprises a second NLS, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0294] Writing domain (RT Domain)

[0295] In some embodiments, a writing domain of a gene modifying system of the present disclosure possesses reverse transcriptase activity and is also referred to as a reverse transcriptase domain (an RT domain). In some embodiments, an RT domain comprises an RT catalytic portion and an RNA-binding region (c.g., a region that binds a template RNA).

[0296] In some embodiments, a nucleic acid encoding a reverse transcriptase is altered from its natural sequence to have altered codon usage, e.g., improved for human cells. In some embodiments a reverse transcriptase domain is a heterologous reverse transcriptase from a retrovirus. In some embodiments, an RT domain has been mutated from its original amino acid sequence, e.g., has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 substitutions. In some embodiments, an RT domain is derived from an RT of a retrovirus, e.g., HIV-1 RT, Moloney Murine Leukemia Virus (MMLV) RT, avian myeloblastosis virus (AMV) RT, or Rous Sarcoma Virus (RSV) RT.

[0297] In some embodiments, a retroviral reverse transcriptase (RT) domain exhibits enhanced stringency of target-primed reverse transcription (TPRT) initiation, e.g., relative to an endogenous RT domain. In some embodiments, an RT domain initiates TPRT when the 3 nucleotides in a target site immediately upstream of a first strand nick, e.g., genomic DNA priming of an RNA template, have at least 66% or 100% complementarity to 3 nucleotides of homology in the RNA template. In some embodiments, an RT domain initiates TPRT when there are less than 5 nucleotides mismatched (e.g., less than 1, less than 2, less than 3, less than 4, or less than 5 nt mismatched) between an RNA template and a target DNA priming reverse transcription. In some embodiments, an RT domain is modified such that the stringency for mismatches in priming a TPRT reaction is increased, e.g., wherein the RT domain does not tolerate any mismatches or tolerates fewer mismatches in a priming region relative to a wild-type (e.g., unmodified) RT domain. In some embodiments, an RT domain comprises a HIV-1 RT domain. In embodiments, an HIV-1 RT domain initiates lower levels of synthesis even with three nucleotide mismatches relative to an alternative RT domain (e.g., as described by Jamburuthugoda and Eickbush J Mol Biol 407(5):661-672 (2011); incorporated herein by reference in its entirety). In some embodiments, an RT domain forms a dimer (e.g., a heterodimer or homodimer). In some embodiments, an RT domain is monomeric. In some embodiments, an RT domain naturally functions as a monomer or as a dimer (e.g., heterodimer or homodimer). In some embodiments, an RT domain naturally functions as a monomer, e.g., is derived from a virus wherein it functions as a monomer. In embodiments, an RT domain is selected from an RT domain from murine leukemia virus (MLV ; sometimes referred to as MoMLV) (e.g., P03355), porcine endogenous retrovirus (PERV) (e.g., UniProt Q4VFZ2), mouse mammary tumor virus (MMTV) (e.g., UniProt P03365), Avian reticuloendotheliosis vims (AVIRE) (e.g., UniProtKB accession: P03360); Feline leukemia virus (FLV or FeLV) (e.g., e.g., UniProtKB accession: P10273); Mason-Pfizer monkey vims (MPMV) (e.g., UniProt P07572), bovine leukemia virus (BLV) (e.g., UniProt P03361), human T-cell leukemia virus-1 (HTLV-1) (e.g., UniProt P03362), human foamy virus (HFV) (e.g., UniProt P14350), simian foamy vims (SFV) (e.g., SFV3L) (e.g., UniProt P23074 or P27401), or bovine foamy / syncytial virus (BFV / BSV) (e.g., UniProt 041894), or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity thereto). In some embodiments, an RT domain is dimeric in its natural functioning. In some embodiments, an RT domain is derived from a virus wherein it functions as a dimer. In embodiments, an RT domain is selected from an RT domain from avian sarcoma / leukemia vims (ASLV) (e.g., UniProt A0A142BKH1), Rous sarcoma virus (RSV) (e.g., UniProt P03354), avian myeloblastosis vims (AMV) (e.g., UniProt Q83133), human immunodeficiency vims type I (HIV-1) (e.g., UniProt P03369), human immunodeficiency virus type II (HIV-2) (e.g., UniProt P15833), simian immunodeficiency virus (SIV) (e.g., UniProt P05896), bovine immunodeficiency vims (BIV) (e.g., UniProt P19560), equine infectious anemia virus (EIAV) (e.g., UniProt P03371), or feline immunodeficiency vims (FIV) (e.g., UniProt P16088) (Herschhom and Hizi Cell Mol Life Sci 67(16):2717-2747 (2010)), or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity thereto). Naturally heterodimeric RT domains may, in some embodiments, also be functional as homodimers. In some embodiments, dimeric RT domains are expressed as fusion proteins, e.g., as homodimeric fusion proteins or heterodimeric fusion proteins. In some embodiments, an RT function ofa system of the present disclosure is fulfilled by multiple RT domains (e.g., as described herein). In further embodiments, multiple RT domains are fused or separate, e.g., may be on the same polypeptide or on different polypeptides.

[0298] In some embodiments, a gene modifying system described herein comprises an integrase domain, e.g., wherein the integrase domain may be part of an RT domain. In some embodiments, an RT domain (e.g., as described herein) comprises an integrase domain. In some embodiments, an RT domain (c.g., as described herein) lacks an integrase domain, or comprises an integrase domain that has been inactivated by mutation or deleted. In some embodiment, a gene modifying system described herein comprises an RNase H domain, e.g., wherein the RNase H domain may be part of an RT domain. In some embodiments, an RNase H domain is not part of an RT domain and is covalently linked via a flexible linker. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain, e.g., an endogenous RNAse H domain or a heterologous RNase H domain. In some embodiments, an RT domain (e.g., as described herein) lacks an RNase H domain. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain that has been added, deleted, mutated, or swapped for a heterologous RNase H domain. In some embodiments, a gene modifying polypeptide comprises an inactivated endogenous RNase H domain. In some embodiments, an endogenous RNase H domain of a polypeptide is genetically removed such that it is not included in the polypeptide, e.g., the endogenous RNase H domain is partially or completely truncated from the polypeptide. In some embodiments, one or more mutations of an RNase H domain yields a polypeptide exhibiting lower RNase activity, e.g., as determined by the methods described in Kotewicz et al. Nucleic Acids Res 16( 1 ) :265-277 (1988) (incorporated herein by reference in its entirety), e.g., lower by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to an otherwise similar domain without the one or more mutations. In some embodiments, RNase H activity of a gene modifying polypeptide is abolished.

[0299] In some embodiments, an RT domain is mutated to increase fidelity compared to an otherwise similar domain without the mutation. In some embodiments, a YADD or YMDD motif in an RT domain (e.g., in a reverse transcriptase) is replaced with YVDD. In some embodiments, replacement of a YADD, YMDD, or YVDD motif results in higher fidelity in retroviral reverse transcriptase activity (e.g., as described in Jamburuthugoda and Eickbush J Mol Biol 2011; incorporated herein by reference in its entirety).

[0300] In some embodiments, a gene modifying polypeptide described herein comprises an RT domain having an amino acid sequence according to any RT domain described in Table 1, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, a nucleic acid described herein encodes an RT domain having an amino acid sequence according to any RT domain described in Table 1, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0301] Table 1: Exemplary reverse transcriptase domains from retroviruses

[0302] In some embodiments, an RT domain described herein is modified, for example, by sitespecific mutation. In some embodiments, an RT domain is engineered to have improved properties, e.g. SuperScript IV (SSIV) RT derived from the MMLV RT. In some embodiments, an RT domain may be engineered to have lower error rates as compared to a reference RT domain, e.g., as described in W02001068895, incorporated herein by reference. In some embodiments, an RT domain may be engineered to be more thermostable as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to be more processive as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to have improved tolerance to inhibitors as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to be faster as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to better tolerate modified nucleotides in an RNA template as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to be capable of inserting modified DNA nucleotides. In some embodiments, an RT domain is engineered to bind a template RNA. In some embodiments, one or more mutations are chosen from D200N, L603W, T33OP, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L671P, E69K, H8Y, T306K, or D653N in an RT domain of murine leukemia virus reverse transcriptase or a corresponding mutation at a corresponding position of another RT domain.

[0303] In some embodiments, a gene modifying polypeptide comprises an RT domain from a retroviral reverse transcriptase, e.g., a wild-type M-MLV RT, e.g., comprising the following sequence:

[0304] In some embodiments, a gene modifying polypeptide comprises an RT domain from a retroviral reverse transcriptase comprising the sequence of amino acids 659-1329 of NP_057933. In some embodiments, a gene modifying polypeptide further comprises one additional amino acid at the N-terminus of the sequence of amino acids 659-1329 of NP_057933, e.g., as shown below:

[0305] In some embodiments, a gene modifying polypeptide further comprises one additional amino acid at the C-terminus of the sequence of amino acids 659-1329 of NP_057933. In embodiments, a gene modifying polypeptide comprises an RNaseHl domain (e.g., amino acids 1178-1318 of NP_057933).

[0306] In some embodiments, a retroviral reverse transcriptase domain, e.g., M-MLV RT, may comprise one or more mutations from a wild-type sequence that may improve features of the RT, e.g., thermostability, processivity, and / or template binding. In some embodiments, an M-MLV RT domain comprises, relative to the M-MLV (WT) sequence above, one or more mutations, e.g., selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, K103L, or a combination thereof. In some embodiments, an M-MLV RT domain comprises, relative to the M-MLV (WT) sequence above, a combination of mutations including D200N, L603W, and T330P, and optionally further including T306K and W313F. In some embodiments, an M-MLV RT used herein comprises D200N, L603W, T33OP, T306K and W313F mutations. In some embodiments, a mutant M-MLV RT comprises the following amino acid sequence:

[0307] In some embodiments, a writing domain (e.g., an RT domain) comprises an RNA-binding domain, e.g., that specifically binds to an RNA sequence. In some embodiments, a template

[0308] RNA comprises an RNA sequence that is specifically bound by an RNA-binding domain of a writing domain (e.g., an RT domain).

[0309] In some embodiments, an RT domain only recognizes and reverse transcribes a specific template, e.g., a template RNA of a system of the preset disclosure. In some embodiments, a template RNA comprises a sequence or structure that enables recognition and reverse transcription by a reverse transcription domain. In some embodiments, a template RNA comprises a sequence or structure that enables association with an RNA-binding domain of a gene modifying polypeptide component of a system described herein. In some embodiments, a system of the present disclosure preferably reverse transcribes a template comprising an association sequence over a template lacking an association sequence.

[0310] In some embodiments, a writing domain (e.g., and RT domain) may also comprise DNA- dependent DNA polymerase activity, e.g., comprise enzymatic activity capable of writing DNA into the genome from a template DNA sequence. In some embodiments, DNA-dependent DNA polymerization is employed to complete second-strand synthesis of a target site edit. In some embodiments, DNA-dependent DNA polymerase activity is provided by a DNA polymerase domain in a gene modifying polypeptide. In some embodiments, DNA-dependent DNA polymerase activity is provided by an RT domain that is also capable of DNA-dependent DNA polymerization, e.g., second-strand synthesis. In some embodiments, DNA-dependent DNA polymerase activity is provided by a second polypeptide of a system of the present disclosure. In some embodiments, DNA-dependent DNA polymerase activity is provided by an endogenous host cell polymerase that is optionally recruited to a target site by a component of a system of the present disclosure.

[0311] In some embodiments, an RT domain has a lower probability of premature termination rate (Poff) in vitro relative to a reference RT domain. In some embodiments, a reference RT domain is a viral RT domain, e.g., the RT domain from M-MLV.

[0312] In some embodiments, an RT domain has a lower probability of premature termination rate (Poff) in vitro of less than about 5 x 10-3 / nucleotides, less than about 5 x 10-4 / nucleotides, or less than about 5 x 10-6 / nucleotides, e.g., as measured on a 1094 nucleotide RNA. In some embodiments, an in vitro premature termination rate is determined as described in Bibillo and Eickbush (2002) J Biol Chem 277(38):34836-34845 (incorporated by reference herein its entirety).

[0313] In some embodiments, an RT domain is able to complete at least about 30% or 50% of integrations in cells. The percent of complete integrations can be measured by dividing the number of substantially full-length integration events (e.g., genomic sites that comprise at least 98% of the expected integrated sequence) by the number of total (including substantially full- length and partial) integration events in a population of cells. In some embodiments, the integrations in cells is determined (e.g., across the integration site) using long-read amplicon sequencing, e.g., as described in Karst et al. (2020) bioRxiv doi.org / 10.1101 / 645903 (incorporated by reference herein in its entirety).

[0314] In some embodiments, quantifying integrations in cells comprises counting the fraction of integrations that contain at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of the DNA sequence corresponding to a template RNA (e.g., a template RNA having a length of at least 0.05, at least 0.1, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.5, at least 2, at least 3, at least 4, or at least 5 kb, e.g., a length between 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 1.0-1.2, 1.2-1.4, 1.4- 1.6, 1.6-1.8, 1.8-2.0, 2-3, 3-4, or 4-5 kb).

[0315] In some embodiments, an RT domain is capable of polymerizing dNTPs in vitro. In some embodiments, an RT domain is capable of polymerizing dNTPs in vitro at a rate between 0.1 - 50 nucleotides / sec (e.g., between 0.1-1 , 1- 10, or 10-50 nucleotides / sec). In some embodiments, polymerization of dNTPs by an RT domain is measured by a single-molecule assay, e.g., as described in Schwartz and Quake (2009) PNAS 106(48):20294- 20299 (incorporated by reference in its entirety).

[0316] In some embodiments, an RT domain has an in vitro error rate (e.g., misincorporation of nucleotides) of between 1 x 10’3- l x 10-4or 1 x 10'4- 1 x 10'5substitutions / nucleotide, e.g., as described in Yasukawa et al. (2017) Biochem Biophys Res Commun 492(2): 147- 153 (incorporated herein by reference in its entirety). In some embodiments, an RT domain has an error rate (e.g., misincorporation of nucleotides) in cells (e.g., HEK293T cells, primary liver, or primary lung cells) of between 1 x 10-3- l x 10-4or 1 x 10'4- l 10-5substitutions / nucleotides, e.g., by long-read amplicon sequencing, e.g., as described in Karst et al. (2020) bioRxiv doi.org / 10.1101 / 645903 (incorporated by reference herein in its entirety).

[0317] In some embodiments, an RT domain is capable of performing reverse transcription of a target RNA in vitro. In some embodiments, an RT domain requires a primer of at least 3 nucleotides to initiate reverse transcription of a template. In some embodiments, reverse transcription of a target RNA is determined by detection of cDNA from the target RNA (e.g., when provided with a ssDNA primer, e.g., which anneals to a target with at least 3, 4, 5, 6, 7, 8, 9, or 10 nt at the 3" end), e.g., as described in Bibillo and Eickbush (2002) J Biol Chem 277(38):34836-34845 (incorporated herein by reference in its entirety).

[0318] In some embodiments, an RT domain performs reverse transcription at least 5 or 10 times more efficiently (e.g., by cDNA production), e.g., when converting its RNA template to cDNA, for example, as compared to an RNA template lacking a protein binding motif (e.g., a 3' UTR). In some embodiments, efficiency of reverse transcription is measured as described in Yasukawa et al. (2017) Biochem Biophys Res Commun 492(2): 147- 153 (incorporated by reference herein in its entirety).

[0319] In some embodiments, an RT domain specifically binds a specific RNA template with higher frequency (e.g., about 5 or 10-fold higher frequency) than any endogenous cellular RNA, e.g., when expressed in cells (e.g., HEK293T cells, primary liver cells, or primary lung cells). In some embodiments, frequency of specific binding between an RT domain and a template RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(l l):5490-5501 (incorporated herein by reference in its entirety). Template nucleic acid binding domain

[0320] In some embodiments, a gene modifying polypeptide contains regions capable of associating with a template nucleic acid (e.g., template RNA). In some embodiments, a template nucleic acid binding domain is an RNA binding domain. In some embodiments, an RNA binding domain is a modular domain that can associate with RNA molecules containing specific signatures, e.g., structural motifs. In some embodiments, a template nucleic acid binding domain (e.g., RNA binding domain) is contained within an RT domain, e.g., the reverse transcriptase- derived component has a known signature for RNA preference.

[0321] In some embodiments, a template nucleic acid binding domain (e.g., RNA binding domain) is contained within a target DNA binding domain. For example, in some embodiments, a DNA binding domain is a CRIS PR-associated protein that recognizes the structure of a template nucleic acid (e.g., a template RNA) comprising a gRNA. In some embodiments, a gene modifying polypeptide comprises a DNA-binding domain comprising a CRISPR-associated protein that associates with a gRNA scaffold that allows the DNA-binding domain to bind a target genomic DNA sequence. In some embodiments, a gRNA scaffold and a gRNA spacer is comprised within a template nucleic acid (e.g., template RNA), thus, in some embodiments, a DNA-binding domain is also a template nucleic acid binding domain. In some embodiments, a gene modifying polypeptide possesses RNA binding function in multiple domains, e.g., can bind a gRNA structure in a CRISPR-associated DNA binding domain and an additional sequence or structure in an RT domain.

[0322] In some embodiments, an RNA binding domain is capable of binding to a template RNA with greater affinity than a reference RNA binding domain. In some embodiments, a reference RNA binding domain is an RNA binding domain from Cas9 of S. pyogenes. In some embodiments, an RNA binding domain is capable of binding to a template RNA with an affinity between 100 pM - 10 nM (e.g., between 100 pM-I nM or I nM - 10 nM). In some embodiments, the affinity of an RNA binding domain for a template RNA is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2018) (incorporated by reference herein in its entirety). In some embodiments, the affinity of an RNA binding domain for a template RNA is measured in cells (e.g., by FRET or CLIP-Seq).

[0323] In some embodiments, an RNA binding domain is associated with a template RNA in vitro at a frequency at least about 5-fold higher or at least about 10-fold higher than with a scrambled RNA. In some embodiments, the frequency of association between an RNA binding domain and a template RNA or scrambled RNA is measured by CLIP-scq, c.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(11 ) :5490-5501 (incorporated by reference herein in its entirety). In some embodiments, an RNA binding domain is associated with a template RNA in cells (e.g., in HEK293T cells, primary liver cells, or primary lung cells) at a frequency at least about 5-fold or at least about 10-fold higher than with a scrambled RNA. In some embodiments, the frequency of association between an RNA binding domain and a template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019), supra.

[0324] In some embodiments, an RT domain (e.g., as listed in Table 1) comprises one or more mutations as listed in Table 2A below. In some embodiment, an RT domain as listed in Table 1 comprises one, two, three, four, five, or six of the mutations listed in the corresponding row of Table 2 below.

[0325] Table 2: Exemplary RT domain mutations (relative to corresponding wild-type sequences as listed in the corresponding row of Table 1)

[0326]

[0327]

[0328] Endonuclease domains and DNA binding domains In some embodiments, a gene modifying polypeptide possesses the function of DNA target site cleavage via an endonuclease domain. In some embodiments, a gene modifying polypeptide comprises a DNA binding domain, e.g., for binding to a target nucleic acid. In some embodiments, a domain (e.g., a Cas domain) of a gene modifying polypeptide comprises two or more smaller domains, e.g., a DNA binding domain and an endonuclease domain. It is understood that when a DNA binding domain (e.g., a Cas domain) is said to bind to a target nucleic acid sequence, in some embodiments, the binding is mediated by a gRNA.

[0329] In some embodiments, a domain has two or more functions. For example, in some embodiments, the endonuclease domain is also a DNA-binding domain. In some embodiments, an endonuclease domain is also a template nucleic acid (e.g., a template RNA) binding domain. For example, in some embodiments, a gene modifying polypeptide comprises a CRISPR- associated endonuclease domain that binds a template RNA comprising a gRNA, binds a target DNA sequence (e.g., with complementarity to a portion of the gRNA), and cuts the target DNA sequence. In some embodiments, an endonuclease domain or endonuclease / DNA-binding domain from a heterologous source can be used or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) in a gene modifying system described herein.

[0330] In some embodiments, a nucleic acid encoding an endonuclease domain or endonuclease / DNA binding domain is altered from its natural sequence to have altered codon usage, e.g. improved for human cells. In some embodiments, the endonuclease element is a heterologous endonuclease element, such as a Cas endonuclease (e.g., Cas9), a type-II restriction endonuclease (e.g., Fokl), a meganuclease (e.g., I-Scel), or other endonuclease domain.

[0331] In some embodiments, a DNA-binding domain of a gene modifying polypeptide described herein is selected, designed, or constructed for binding to a desired host DNA target sequence. In some embodiments, a DNA-binding domain of a gene modifying polypeptide is a heterologous DNA-binding element. In some embodiments a heterologous DNA binding element is a zinc-finger element or a TAL effector element, e.g., a zinc-finger or TAL polypeptide or functional fragment thereof. In some embodiments a heterologous DNA binding element is a sequence-guided DNA binding element, such as Cas9, Cpfl, or other CRISPR- related protein that has been altered to have no endonuclease activity. In some embodiments, a heterologous DNA binding element retains endonuclease activity. In some embodiments, a heterologous DNA binding element retains partial endonuclease activity to cleave ssDNA, e.g., possesses nickase activity. In some embodiments, a heterologous DNA-binding domain comprises a Cas9 domain, a TAL domain, a ZF domain, a Myb domain, a combination thereof, or multiples thereof.

[0332] In some embodiments, a DNA-binding domain is modified, for example by site-specific mutation, increasing or decreasing DNA-binding elements (for example, number and / or specificity of zinc fingers), etc., to alter DNA-binding specificity and affinity. In some embodiments a nucleic acid sequence encoding a DNA binding domain is altered from its natural sequence to have altered codon usage, e.g., improved for human cell expression. In some embodiments, a DNA binding domain comprises one or more modifications relative to a wildtype DNA binding domain, e.g., a modification via directed evolution, e.g., phage-assisted continuous evolution (PACE).

[0333] In some embodiments, a DNA binding domain comprises a meganuclease domain (e.g., as described herein), or a functional fragment thereof. In some embodiments, a meganuclease domain possesses endonuclease activity, e.g., double-strand cleavage and / or nickase activity. In some embodiments, a meganuclease domain has reduced activity, e.g., lacks endonuclease activity, e.g., the meganuclease is catalytically inactive. In some embodiments, a catalytically inactive meganuclease is used as a DNA binding domain, e.g., as described in Fonfara et al. Nucleic Acids Res 40(2):847-860 (2012), incorporated herein by reference in its entirety.

[0334] In some embodiments, a gene modifying polypeptide comprises a modification to a DNA-binding domain, e.g., relative to the wild-type polypeptide. In some embodiments, a DNA-binding domain comprises an addition, deletion, replacement, or modification to the amino acid sequence of the original DNA-binding domain. In some embodiments, a DNA-binding domain is modified to include a heterologous functional domain that binds specifically to a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, a functional domain replaces at least a portion (e.g., the entirety of) the prior DNA-binding domain of the gene modifying polypeptide. In some embodiments, a functional domain comprises a zinc finger (e.g., a zinc finger that specifically binds to the target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, a functional domain comprises a Cas domain (e.g., a Cas domain that specifically binds to a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, a Cas domain comprises a Cas9 or a mutant or a variant thereof (e.g., as described herein). In some embodiments, a Cas domain is associated with a guide RNA (gRNA), e.g., as described herein. In some embodiments, a Cas domain is directed to a target nucleic acid (e.g., DNA) sequence of interest by a gRNA. In embodiments, a Cas domain is encoded by the same nucleic acid (e.g., RNA) molecule as a gRNA. In some embodiments, a Cas domain is encoded by a different nucleic acid (e.g., RNA) molecule from the gRNA.

[0335] In some embodiments, a DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with greater affinity than a reference DNA binding domain. In some embodiments, a reference DNA binding domain is a DNA binding domain from Cas9 of S. pyogenes. In some embodiments, a DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with an affinity between 100 pM - 10 nM (e.g., between 100 pM-1 nM or 1 nM - 10 nM).

[0336] In some embodiments, the affinity of a DNA binding domain for a target sequence (e.g., dsDNA target sequence) is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2018) (incorporated by reference herein in its entirety).

[0337] In some embodiments, a DNA binding domain is capable of binding to a target sequence (e.g., dsDNA target sequence), e.g, with an affinity between 100 pM - 10 nM (e.g., between 100 pM-1 nM or 1 nM - 10 nM) in the presence of a molar excess of scrambled sequence competitor dsDNA, e.g., of about 100-fold molar excess.

[0338] In some embodiments, a DNA binding domain is found associated with a target sequence (e.g., a dsDNA target sequence) more frequently than any other sequence in the genome of a target cell, e.g., human target cell, e.g., as measured by ChlP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21 (incorporated herein by reference in its entirety). In some embodiments, a DNA binding domain is found associated with a target sequence (e.g., a dsDNA target sequence) at least about 5-fold or at least about 10-fold more frequently than any other sequence in the genome of a target cell, e.g., as measured by ChlP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010), supra.

[0339] In some embodiments, an endonuclease domain has nickase activity and cleaves one strand of a target DNA. In some embodiments, nickase activity reduces the formation of doublestranded breaks at a target site. In some embodiments, an endonuclease domain creates a staggered nick structure in the first and second strands of a target DNA. In some embodiments, a staggered nick structure generates free 3’ overhangs at a target site. In some embodiments, free 3’ overhangs at a target site improve editing efficiency, e.g., by enhancing access and annealing of a 3’ homology region of a template nucleic acid. In some embodiments, a staggered nick structure reduces the formation of double-stranded breaks at a target site.

[0340] In some embodiments, an endonuclease domain cleaves both strands of a target DNA, e.g., results in blunt-end cleavage of a target with no ssDNA overhangs on either side of the cutsite. The amino acid sequence of an endonuclease domain of a gene modifying system described herein may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of an endonuclease domain described herein, e.g., an endonuclease domain described in Table 4.

[0341] In some embodiments, a heterologous endonuclease is Fokl or a functional fragment thereof. In some embodiments, a heterologous endonuclease is a Holliday junction resolvase or homolog thereof, such as the Holliday junction resolving enzyme from Sulfolobus solfataricus — Ssol Hje (Govindaraju et al., Nucleic Acids Research 44:7, 2016). In some embodiments, a heterologous endonuclease is an endonuclease of a large fragment of a spliceosomal protein, such as Prp8 (Mahbub et al., Mobile DNA 8:16, 2017). In some embodiments, a heterologous endonuclease is derived from a CRIS PR-associated protein, e.g., Cas9. In some embodiments, a heterologous endonuclease is engineered to have only ssDNA cleavage activity, e.g., only nickase activity, e.g., be a Cas9 nickase, e.g., SpCas9 with D10A, H840A, or N863A mutations. Table 4 provides exemplary Cas proteins and mutations associated with nickase activity. In some embodiments, an endonuclease domain is modified, for example by site-specific mutation, to alter DNA endonuclease activity. In some embodiments, an endonuclease domain is modified to reduce DNA-sequence specificity, e.g., by truncation to remove domains that confer DNA- sequence specificity or mutation to inactivate regions conferring DNA-sequence specificity.

[0342] In some embodiments, an endonuclease domain has nickase activity and does not form double-stranded breaks. In some embodiments, an endonuclease domain forms single-stranded breaks at a higher frequency than double-stranded breaks, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the breaks are single-stranded breaks, or less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the breaks are double-stranded breaks. In some embodiments, an endonuclease domain forms substantially no double- stranded breaks. In some embodiments, an endonuclease domain does not form detectable levels of double- stranded breaks. In some embodiments, an endonuclease domain has nickase activity that nicks the first strand of a target site DNA. In some embodiments, an endonuclease domain cuts the genomic DNA of a target site near to the site of alteration on the strand that will be extended by a writing domain (e.g., an RT domain). In some embodiments, an endonuclease domain has nickase activity that nicks the first strand of a target site DNA and does not nick the second strand of the target site DNA. For example, when a gene modifying polypeptide comprises a CRISPR- associated endonuclease domain having nickase activity, in some embodiments, said CRISPR- associated endonuclease domain nicks a target site DNA strand containing a PAM site (e.g., and does not nick the target site DNA strand that does not contain the PAM site). As a further example, when a gene modifying polypeptide comprises a CRISPR-associated endonuclease domain having nickase activity, in some embodiments, said CRISPR-associated endonuclease domain nicks a target site DNA strand that does not containa PAM site (e.g., and does not nick the target site DNA strand that contains the PAM site).

[0343] In some embodiments, an endonuclease domain has nickase activity that nicks the first strand and the second strand of a target site DNA. Without wishing to be bound by any particular theory, after a writing domain (e.g., an RT domain) of a gene modifying polypeptide described herein polymerizes (e.g., reverse transcribes) from a heterologous object sequence of a template nucleic acid (e.g., a template RNA), the cellular DNA repair machinery must repair the nick on the first DNA strand. The target site DNA now contains two different sequences for the first DNA strand: one corresponding to the original genomic DNA (e.g., having a free 5' end) and a second corresponding to that polymerized from the heterologous object sequence (e.g., having a free 3' end). It is thought that the two different sequences equilibrate with one another, first one hybridizing the second strand, then the other, and which sequence the cellular DNA repair apparatus incorporates into its repaired target site may be a stochastic process. Without wishing to be bound by any particular theory, it is thought that introducing an additional nick to the second-strand may bias the cellular DNA repair machinery to adopt the heterologous object sequence-based sequence more frequently than the original genomic sequence (Anzalone et al. Nature 576:149-157 (2019)). In some embodiments, an additional nick is positioned at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, or at least 150 nucleotides 5' or 3' of a target site modification (e.g., an insertion, deletion, or substitution) or to a nick on the first strand.

[0344] Alternatively or additionally, without wishing to be bound by theory, it is thought that an additional nick to the second strand may promote second- strand synthesis. In some embodiments, where a system of the present disclosure has inserted or substituted a portion of a first strand, synthesis of a new sequence corresponding to the insertion / substitution in the second strand is necessary.

[0345] In some embodiments, a gene modifying polypeptide comprises a single domain having endonuclease activity (e.g., a single endonuclease domain) and said domain nicks both the first strand and the second strand. For example, in some embodiments an endonuclease domain may be a CRISPR-associated endonuclease domain, and a template nucleic acid (e.g., template RNA) comprises a gRNA spacer that directs nicking of the first strand and an additional gRNA spacer that directs nicking of the second strand. In some embodiments, a gene modifying polypeptide comprises a plurality of domains having endonuclease activity, and a first endonuclease domain nicks the first strand and a second endonuclease domain nicks the second strand (optionally, the first endonuclease domain does not (e.g., cannot) nick the second strand and the second endonuclease domain does not (e.g., cannot) nick the first strand).

[0346] In some embodiments, an endonuclease domain is capable of nicking a first strand and a second strand. In some embodiments, first and second strand nicks occur at the same position in a target site but on opposite strands. In some embodiments, a second strand nick occurs in a staggered location, e.g., upstream or downstream, from a first nick. In some embodiments, an endonuclease domain generates a target site deletion if a second strand nick is upstream of a first strand nick. In some embodiments, an endonuclease domain generates a target site duplication if a second strand nick is downstream of a first strand nick. In some embodiments, an endonuclease domain generates no duplication and / or deletion if a first and second strand nicks occur in the same position of a target site. In some embodiments, an endonuclease domain has altered activity depending on protein conformation or RNA-binding status, e.g., which promotes the nicking of the first or second strand (e.g., as described in Christensen et al. PNAS 2006; incorporated by reference herein in its entirety).

[0347] In some embodiments, an endonuclease domain comprises a meganuclease, or a functional fragment thereof. In some embodiments, an endonuclease domain comprises a homing endonuclease, or a functional fragment thereof. In some embodiments, an endonuclease domain comprises a mcganuclcasc from the LAGLID ADG, GIY-YIG, HNH, His-Cys Box, or PD-(D / E) XK families, or a functional fragment or variant thereof, e.g., which possess conserved amino acid motifs, e.g., as indicated in the family names. In some embodiments, an endonuclease domain comprises a meganuclease, or fragment thereof, chosen from, e.g., I- SmaMI (Uniprot F7WD42), I-Scel (Uniprot P03882), I- Anil (Uniprot PO388O), I-Dmol (Uniprot P21505), I-Crel (Uniprot P05725), I-TevI (Uniprot P13299), LOnuI (Uniprot Q4VWW5), or I- Bmol (Uniprot Q9ANR6). In some embodiments, a meganuclease is naturally monomeric, e.g., I-Scel, I-TevI, or dimeric, e.g., I-Crel, in its functional form. For example, LAGLID ADG meganucleases with a single copy of the LAGLID ADG motif generally form homodimers, whereas members with two copies of the LAGLID ADG motif arc generally found as monomers. In some embodiments, a meganuclease that normally forms as a dimer is expressed as a fusion, e.g., two subunits are expressed as a single open reading frame (ORF) and, optionally, connected by a linker, e.g., an I-Crel dimer fusion (Rodriguez-Fomes et al. Gene Therapy 2020; incorporated by reference herein in its entirety). In some embodiments, a meganuclease, or a functional fragment thereof, is altered to favor nickase activity for one strand of a doublestranded DNA molecule, e.g., I-Scel (K122I and / or K223I) (Niu et al. J Mol Biol 2008), I-Anil (K227M) (McConnell Smith et al. PNAS 2009), I-Dmol (Q42A and / or K120M) (Molina et al. J Biol Chem 2015). In some embodiments, a meganuclease or functional fragment thereof possessing this preference for single-strand cleavage is used as an endonuclease domain, e.g., with nickase activity. In some embodiments, an endonuclease domain comprises a meganuclease, or a functional fragment thereof, which naturally targets or is engineered to target a safe harbor site, e.g., an I-Crel targeting SH6 site (Rodriguez-Fomes et al., supra). In some embodiments, an endonuclease domain comprises a meganuclease, or a functional fragment thereof, with a sequence-tolerant catalytic domain, e.g., I-TevI recognizing the minimal motif CNNNG (Kleinstiver et al. PNAS 2012). In some embodiments, a target sequence-tolerant catalytic domain is fused to a DNA binding domain, e.g., to direct activity, e.g., by fusing I-TevI to: (i) zinc fingers to create Tev-ZFEs (Kleinstiver et al. PNAS 2012), (ii) other meganucleases to create MegaTevs (Wolfs et al. Nucleic Acids Res 2014), and / or (iii) Cas9 to create TevCas9 (Wolfs et al. PNAS 2016). In some embodiments, an endonuclease domain comprises a restriction enzyme, e.g., a Type IIS or Type IIP restriction enzyme. In some embodiments, an endonuclease domain comprises a Type IIS restriction enzyme, e.g., FokI, or a fragment or variant thereof. In some embodiments, an endonuclease domain comprises a Type IIP restriction enzyme, e.g., PvuII, or a fragment or variant thereof. In some embodiments, a dimeric restriction enzyme is expressed as a fusion such that it functions as a single chain, e.g., a FokI dimer fusion (Minczuk et al. Nucleic Acids Res 36(12):3926-3938 (2008)).

[0348] The use of additional endonuclease domains is described, for example, in Guha and Edgell Int J Mol Sci 18(22):2565 (2017), which is incorporated herein by reference in its entirety.

[0349] In some embodiments, a gene modifying polypeptide comprises a modification to an endonuclease domain, e.g., relative to a wild-type Cas protein. In some embodiments, an endonuclease domain comprises an addition, deletion, replacement, or modification to the amino acid sequence of a wild-type Cas protein. In some embodiments, an endonuclease domain is modified to include a heterologous functional domain that binds specifically to and / or induces endonuclease cleavage of a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, an endonuclease domain comprises a zinc finger. In embodiments, an endonuclease domain comprising a Cas domain is associated with a guide RNA (gRNA), e.g., as described herein. In some embodiments, an endonuclease domain is modified to include a functional domain that does not target a specific target nucleic acid (e.g., DNA) sequence. In some embodiments, an endonuclease domain comprises a FokI domain.

[0350] In some embodiments, an endonuclease domain is associated with a target dsDNA in vitro at a frequency at least about 5-fold or at least about 10-fold higher than with a scrambled dsDNA. In some embodiments, an endonuclease domain is associated with a target dsDNA in vitro at a frequency at least about 5-fold or at least about 10-fold higher than with a scrambled dsDNA, e.g., in a cell (e.g., a HEK293T cell, a primary liver cell, or a primary lung cell). In some embodiments, the frequency of association between an endonuclease domain and a target DNA or scrambled DNA is measured by ChlP-seq, e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21 (incorporated by reference herein in its entirety).

[0351] In some embodiments, an endonuclease domain can catalyze the formation of a nick at a target sequence, e.g., to an increase of at least about 5-fold or at least about 10-fold relative to a non-target sequence (e.g., relative to any other genomic sequence in the genome of the target cell). In some embodiments, the level of nick formation is determined using NickScq, e.g., as described in Elacqua et al. (2019) bioRxiv doi.org / 10.1101 / 867937 (incorporated herein by reference in its entirety).

[0352] In some embodiments, an endonuclease domain is capable of nicking DNA in vitro. In some embodiments, a nick results in an exposed base. In embodiments, an exposed base can be detected using a nuclease sensitivity assay, e.g., as described in Chaudhry and Weinfeld (1995) Nucleic Acids Res 23(19):3805-3809 (incorporated by reference herein in its entirety). In some embodiments, the level of exposed bases (e.g., detected by the nuclease sensitivity assay) is increased by at least about 10%, at least about 50%, or more relative to a reference endonuclease domain. In some embodiments, a reference endonuclease domain is an endonuclease domain from Cas9 of S. pyogenes.

[0353] In some embodiments, an endonuclease domain is capable of nicking DNA in a cell. In some embodiments, an endonuclease domain is capable of nicking DNA in a HEK293T cell. In some embodiments, an unrepaired nick that undergoes replication in the absence of Rad51 results in increased NHEJ rates at the site of the nick, which can be detected, e.g., by using a Rad51 inhibition assay, e.g., as described in Bothmer et al. (2017) Nat Commun 8:13905 (incorporated by reference herein in its entirety). In some embodiments, NHEJ rates are increased above 0-5%. In some embodiments, NHEJ rates are increased to 20-70% (e.g., between 30%-60% or 40-50%), e.g., upon Rad51 inhibition.

[0354] In some embodiments, an endonuclease domain releases a target after cleavage. In some embodiments, release of a target is indicated indirectly by assessing for multiple turnovers by an enzyme, e.g., as described in Yourik at al. RNA 25( 1 ):35-44 (2019) (incorporated herein by reference in its entirety) and shown in FIG. 2 therein. In some embodiments, the keXp of an endonuclease domain is 1 x 10’3- 1 x 10’5min’1as measured by such methods.

[0355] In some embodiments, an endonuclease domain has a catalytic efficiency (fcCat / m) greater than about 1 x 108s’1M’1in vitro. In some embodiments, an endonuclease domain has a catalytic efficiency greater than about 1 x 105, greater than about 1 x 106, greater than about 1 x 107, or greater than about 1 x 108, s’1M’1in vitro. In some embodiments, catalytic efficiency is determined as described in Chen et al. (2018) Science 360(6387):436-439 (incorporated herein by reference in its entirety). In some embodiments, an endonuclease domain has a catalytic efficiency (fcat / ^m) greater than about 1 x 108s"1M'1in cells. In some embodiments, an endonuclease domain has a catalytic efficiency greater than about 1 x 105, greater than about 1 x 106, greater than about 1 x 107, or greater than about 1 x 108s1M1in cells.

[0356] Gene modifying polypeptides comprising Cas domains

[0357] In some embodiments, a gene modifying polypeptide described herein comprises a Cas domain. In some embodiments, a Cas domain can direct a gene modifying polypeptide to a target site specified by a gRNA spacer, thereby modifying a target nucleic acid sequence in “cis”. In some embodiments, a gene modifying polypeptide is fused to a Cas domain. In some embodiments, a gene modifying polypeptide comprises a CRISPR / Cas domain (also referred to herein as a CRISPR-associated protein). In some embodiments, a CRISPR / Cas domain comprises a protein involved in the clustered regulatory interspaced short palindromic repeat (CRISPR) system, e.g., a Cas protein, and optionally binds a guide RNA, e.g., single guide RNA (sgRNA).

[0358] CRISPR systems are adaptive defense systems originally discovered in bacteria and archaea. CRISPR systems use RNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases (e. g., Cas9 or Cpfl) to cleave foreign DNA. For example, in a typical CRISPR- Cas system, an endonuclease is directed to a target nucleotide sequence (e. g., a site in the genome that is to be sequence-edited) by sequence-specific, non-coding “guide RNAs” that target single- or double-stranded DNA sequences. Three classes (I-III) of CRISPR systems have been identified. The class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”). The crRNA contains a “spacer” sequence, a typically about 20-nucleotide RNA sequence that corresponds to a target DNA sequence (“protospacer”). In the wild-type system, and in some engineered systems, crRNA also contains a region that binds to the tracrRNA to form a partially doublestranded structure that is cleaved by RNase III, resulting in a crRNA / tracrRNA hybrid molecule. A crRNA / tracrRNA hybrid then directs the Cas endonuclease to recognize and cleave a target DNA sequence. A target DNA sequence is generally adjacent to a “protospacer adjacent motif’ (“PAM”) that is specific for a given Cas endonuclease and required for cleavage activity at a target site matching the spacer of the crRNA. CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements, e.g., as listed for exemplary Cas enzymes in Table 3; examples of PAM sequences include 5'-NGG (Streptococcus pyogenes; SEQ ID NO: 11,019), 5'-NNAGAA (Streptococcus thermophilus CRISPR1; SEQ ID NO: 11,020), 5 '-NGGNG (Streptococcus thermophilus CRISPR3; SEQ ID NO: 11,021), and 5'- NNNGATT (Neisseria meningiditis; SEQ ID NO: 11,022). Some endonucleases, e.g., Cas9 endonucleases, are associated with G-rich PAM sites, e. g., 5'-NGG (SEQ ID NO: 11,023), and perform blunt-end cleaving of the target DNA at a location 3 nucleotides upstream from (5' from) the PAM site. Another class II CRISPR system includes the type V endonuclease Cpfl, which is smaller than Cas9; examples include AsCpfl (from Acidaminococcus sp.) and LbCpfl (from Lachnospiraceae sp.). Cpfl -associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words, a Cpfl system, in some embodiments, comprises only Cpfl nuclease and a crRNA to cleave a target DNA sequence. Cpfl endonucleases, are typically associated with T-rich PAM sites, e. g., 5'-TTN. Cpfl can also recognize a 5'-CTA PAM motif. Cpfl typically cleaves a target DNA by introducing an offset or staggered double-strand break with a 4- or 5-nucleotide 5' overhang, for example, cleaving a target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream from (3' from) a PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA. See, e.g., Zetsche et al. (2015) Cell, 163:759 - 771.

[0359] A variety of CRISPR associated (Cas) genes or proteins can be used in the technologies provided by the present disclosure and the choice of Cas protein will depend upon the particular conditions of the method. Specific examples of Cas proteins include class II systems including Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Cpfl, C2C1, or C2C3. In some embodiments, a Cas protein, e.g., a Cas9 protein, may be from any of a variety of prokaryotic species. In some embodiments a Cas protein, e.g., a Cas9 protein, is selected to recognize a particular protospacer-adjacent motif (PAM) sequence. In some embodiments, a DNA-binding domain or endonuclease domain includes a sequence targeting polypeptide, such as a Cas protein, e.g., Cas9. In some embodiments a Cas protein, e.g., a Cas9 protein, may be obtained from a bacteria or archaea or synthesized using known methods. In some embodiments, a Cas protein may be from a gram-positive bacteria or a gram-negative bacteria. In some embodiments, a Cas protein may be from a Streptococcus (e.g., a S. pyogenes, or a S. thermophilus), a Francisella (e.g., an F. novicida), a Staphylococcus (e.g., an S. aureus), an Acidaminococcus (e.g., an Acidaminococcus sp. BV3L6), a Neisseria (e.g., an N. meningitidis), a Cryptococcus, a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillonella, or a Marinobacter.

[0360] In some embodiments, a gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 4000, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the amino acid sequence of SEQ ID NO: 4000, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, is positioned at the N- terminal end of a gene modifying polypeptide. In some embodiments, the amino acid sequence of SEQ ID NO: 4000, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity thereto, is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the N-terminal end of a gene modifying polypeptide.

[0361] In some embodiments, a gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 4001, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the amino acid sequence of SEQ ID NO: 4001, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, is positioned at the C- terminal end of a gene modifying polypeptide. In some embodiments, an amino acid sequence of SEQ ID NO: 4001 below, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the C -terminal end of a gene modifying polypeptide.

[0362] Exemplary benchmarking sequence

[0363] In some embodiments, a gene modifying polypeptide may comprise a Cas domain as listed in Table 3 or Table 4, or a functional fragment thereof, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0364] Table 3: CRISPR / Cas Proteins, Species, and Mutations

[0365] Table 4: Amino Acid Sequences of StlCas9 Proteins, Species, and Mutations

[0366] In some embodiments, a Cas protein requires a protospacer adjacent motif (PAM) to be present in or adjacent to a target DNA sequence for the Cas protein to bind and / or function. In some embodiments, a PAM is or comprises, from 5' to 3', NGG (SEQ ID NO: 11,024), YG (SEQ ID NO: 11,025), NNGRRT (SEQ ID NO: 11,026), NNNRRT (SEQ ID NO: 11,027), NGA (SEQ ID NO: 11,029), TYCV (SEQ ID NO: 11,030), TATV (SEQ ID NO: 11,031), NTTN (SEQ ID NO: 11,032), or NNNGATT (SEQ ID NO: 11,033), where N stands for any nucleotide, Y stands for C or T, R stands for A or G, and V stands for A or C or G. In some embodiments, a Cas protein is a protein listed in Table 3 or Table 4. In some embodiments, a Cas protein comprises one or more mutations altering its PAM. In some embodiments, a Cas protein comprises E1369R, E1449H, and R1556A mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises E782K, N968K, and R1015H mutations or analogous substitutions to the amino acids corresponding to said positions. Tn some embodiments, a Cas protein comprises DI 135V, R1335Q, and T1337R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R and K607R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R, K548V, and N552R mutations or analogous substitutions to the amino acids corresponding to said positions. Exemplary advances in the engineering of Cas enzymes to recognize altered PAM sequences are reviewed in Collias et al Nature Communications 12:555 (2021), incorporated herein by reference in its entirety.

[0367] In some embodiments, a Cas protein is catalytically active and cuts one or both strands of a target DNA site. In some embodiments, cutting a target DNA site is followed by formation of an alteration, e.g., an insertion or deletion, e.g., by the cellular repair machinery.

[0368] In some embodiments, a Cas protein is modified to deactivate or partially deactivate its nuclease, e.g., a nuclease-deficient Cas9. Whereas wild-type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by a gRNA, a number of CRISPR endonucleases having modified functionalities are available, for example: a “nickase” version of Cas9 that has been partially deactivated generates only a single-strand break; a catalytically inactive Cas9 (“dCas9”) does not cut target DNA. In some embodiments, dCas9 binding to a DNA sequence may interfere with transcription at that site by steric hindrance. In some embodiments, dCas9 binding to an anchor sequence may interfere with (e.g., decrease or prevent) genomic complex (e.g., ASMC) formation and / or maintenance. In some embodiments, a DNA-binding domain comprises a catalytically inactive Cas9, e.g., dCas9. Many catalytically inactive Cas9 proteins arc known in the art. In some embodiments, dCas9 comprises mutations in each endonuclease domain of the Cas protein, e.g., D10A and H840A or N863A mutations. In some embodiments, a catalytically inactive or partially inactive CRISPR / Cas domain comprises a Cas protein comprising one or more mutations, e.g., one or more of the mutations listed in Table 3. In some embodiments, a Cas protein described on a given row of Table 3 comprises one, two, three, or all of the mutations listed in the same row of Table 3. In some embodiments, a Cas protein, e.g., not described in Table 3, comprises one, two, three, or all of the mutations listed in a row of Table 3 or a corresponding mutation at a corresponding site in that Cas protein.

[0369] In some embodiments, a catalytically inactive, e.g., dCas9, or partially deactivated Cas9 protein comprises a Dl l mutation (e.g., Dl l A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, c.g., dCas9, or partially deactivated Cas9 protein comprises a H969 mutation (c.g., H969A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N995 mutation (e.g., N995A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises mutations at one, two, or three of positions Dl l, H969, and N995 (e.g., D11A, H969A, and N995A mutations) or analogous substitutions to the amino acids corresponding to said positions.

[0370] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a DIO mutation (e.g., a D10A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H557 mutation (e.g., a H557A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a DIO mutation (e.g., a D10A mutation) and a H557 mutation (e.g., a H557A mutation) or analogous substitutions to the amino acids corresponding to said positions.

[0371] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D839 mutation (e.g., a D839A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H840 mutation (e.g., a H840A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N863 mutation (e.g., a N863A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a DIO mutation (e.g., D10A), a D839 mutation (e.g., D839A), a H840 mutation (e.g., H840A), and a N863 mutation (e.g., N863A) or analogous substitutions to the amino acids corresponding to said positions.

[0372] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a E993 mutation (e.g., a E993A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D917 mutation (e.g., a D917A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a a E1006 mutation (e.g., a E1006A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D1255 mutation (e.g., a D1255A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D917 mutation (e.g., D917A), a E1006 mutation (e.g., E1006A), and a D1255 mutation (e.g., D1255A) or analogous substitutions to the amino acids corresponding to said positions.

[0373] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D16 mutation (e.g., a D16A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D587 mutation (e.g., a D587A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a partially deactivated Cas domain has nickase activity. In some embodiments, a partially deactivated Cas9 domain is a Cas9 nickase domain. In some embodiments, the catalytically inactive Cas domain or dead Cas domain produces no detectable double strand break formation. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H588 mutation (e.g., a H588A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N611 mutation (e.g., a N611A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D16 mutation (e.g., D16A), a D587 mutation (e.g., D587A), a H588 mutation (e.g., H588A), and a N611 mutation (e.g., N611A) or analogous substitutions to the amino acids corresponding to said positions.

[0374] In some embodiments, a DNA-binding domain or endonuclease domain may comprise a Cas molecule comprising or linked (e.g., covalently) to a gRNA (e.g., a template nucleic acid, e.g., template RNA, comprising a gRNA). In some embodiments, an endonuclease domain or DNA binding domain comprises a Streptococcus pyogenes Cas9 (SpCas9) or a functional fragment or variant thereof. In some embodiments, an endonuclease domain or DNA binding domain comprises a modified SpCas9. In some embodiments, a modified SpCas9 comprises a modification that alters protospacer- adjacent motif (PAM) specificity. In some embodiments, a PAM has specificity for the nucleic acid sequence 5'-NGT-3'. In some embodiments, a modified SpCas9 comprises one or more amino acid substitutions, e.g., at one or more of positions LI 111, DI 135, G1218, E1219, A1322, or R1335, e.g., selected from Li l HR, DI 135V, G1218R, E1219F, A1322R, or R1335V. In some embodiments, a modified SpCas9 comprises the amino acid substitution T1337R and one or more additional amino acid substitutions, e.g., selected from Li l HR, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereto. In some embodiments, a modified SpCas9 comprises: (i) one or more amino acid substitutions selected from D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, and T1337; and (ii) one or more amino acid substitutions selected from Li l HR, G1218R, E1219F, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereto.

[0375] In some embodiments, an endonuclease domain or DNA binding domain comprises a Cas domain, e.g., a Cas9 domain. In some embodiments, an endonuclease domain or DNA binding domain comprises a nuclease-active Cas domain, a Cas nickase (nCas) domain, or a nucleaseinactive Cas (dCas) domain. In some embodiments, an endonuclease domain or DNA binding domain comprises a nuclease-active Cas9 domain, a Cas9 nickase (nCas9) domain, or a nuclease-inactive Cas9 (dCas9) domain. In some embodiments, an endonuclease domain or DNA binding domain comprises a Cas9 domain of Cas9 (e.g., dCas9 and nCas9), Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, or Casl2i. In some embodiments, an endonuclease domain or DNA binding domain comprises a Cas9 (e.g., dCas9 and nCas9), Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, or Casl2i. In some embodiments, an endonuclease domain or DNA binding domain comprises an S. pyogenes or an S. thermophilus Cas9, or a functional fragment thereof. In some embodiments, an endonuclease domain or DNA binding domain comprises a Cas9 sequence, e.g., as described in Chylinski, Rhun, and Charpentier (2013) RNA Biology 10:5, 726-737; incorporated herein by reference. In some embodiments, an endonuclease domain or DNA binding domain comprises the HNH nuclease subdomain and / or the RuvCl subdomain of a Cas, e.g., Cas9, e.g., as described herein, or a variant thereof. In some embodiments, an endonuclease domain or DNA binding domain comprises Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, or Casl2i. In some embodiments, an endonuclease domain or DNA binding domain comprises a Cas polypeptide (e.g., enzyme), or a functional fragment thereof. In some embodiments, a Cas polypeptide (e.g., enzyme) is selected from Cast, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csxl2), CaslO, CaslOd, Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, Casl2i, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csxl l, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2b / C2cl, Casl2c / C2c3, SpCas9(K855A), eSpCas9(l.l), SpCas9-HFl, hyper accurate Cas9 variant (HypaCas9), homologues thereof, modified or engineered versions thereof, and / or functional fragments thereof. In some embodiments, a Cas9 comprises one or more substitutions, e.g., selected from H840A, D10A, P475A, W476A, N477A, DI 125 A, W1126A, and D1127A. In some embodiments, a Cas9 comprises one or more mutations at positions selected from: DIO, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987, e.g., one or more substitutions selected from D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A. In some embodiments, an endonuclease domain or DNA binding domain comprises a Cas (e.g., Cas9) sequence from Corynebacterium ulcerans, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquis, Streptococcus thermophilus, Listeria innocua, Campylobacter jejuni, Neisseria meningitidis, Streptococcus pyogenes, or Staphylococcus aureus, or a fragment or variant thereof.

[0376] In some embodiments, an endonuclease domain or DNA binding domain comprises a Cpfl domain, e.g., comprising one or more substitutions, e.g., at position D917, E1006A, D1255 or any combination thereof, e.g., selected from D917A, E1006A, D1255A, D917A / E1006A,

[0377] D917A / D1255A, E1006A / D1255A, and D917A / E1006A / D1255A.

[0378] In some embodiments, an endonuclease domain or DNA binding domain comprises spCas9, spCas9-VRQR (SEQ ID NO: 5019), spCas9- VRER (SEQ ID NO: 5020), xCas9 (sp), saCas9, saCas9-KKH, spCas9-MQKSER (SEQ ID NO: 5021), spCas9-LRKIQK (SEQ ID NO: 5022), or spCas9- LRVSQL (SEQ ID NO: 5023).

[0379] In some embodiments, a gene modifying polypeptide has an endonuclease domain comprising a Cas9 nickase, e.g., Cas9 H840A. In some embodiments, the Cas9 H840A has the following amino acid sequence:

[0380] In some embodiments, a gene modifying polypeptide comprises a dCas9 sequence comprising a D10A and / or H840A mutation, e.g., the following sequence:

[0381] TAL Effectors and Zinc Finger Nucleases

[0382] In some embodiments, an endonuclease domain or DNA-binding domain comprises a TAL effector molecule. A TAL effector molecule, e.g., a TAL effector molecule that specifically binds a DNA sequence, typically comprises a plurality of TAL effector domains or fragments thereof, and optionally one or more additional portions of naturally occurring TAL effectors (e.g., N- and / or C-terminal of the plurality of TAL effector domains). Many TAL effectors are known to those of skill in the art and are commercially available, e.g., from Thermo Fisher Scientific.

[0383] Naturally occurring TALEs are natural effector proteins secreted by numerous species of bacterial pathogens including the plant pathogen Xanlhomonas which modulates gene expression in host plants and facilitates bacterial colonization and survival. The specific binding of TAL effectors is based on a central repeat domain of tandemly arranged nearly identical repeats of typically 33 or 34 amino acids (the repeat-variable di-residues, RVD domain). Members of the TAL effectors family differ mainly in the number and order of their repeats. The number of repeats typically ranges from 1.5 to 33.5 repeats and the C-tcrminal repeat is usually shorter in length (e.g., about 20 amino acids) and is generally referred to as a “half-repeat.” Each repeat of the TAL effector generally features a one-repeat-to-one-base-pair correlation with different repeat types exhibiting different base-pair specificity (one repeat recognizes one base-pair on the target gene sequence). Generally, the smaller the number of repeats, the weaker the protein-DNA interactions. A number of 6.5 repeats has been shown to be sufficient to activate transcription of a reporter gene (Scholze et al., 2010).

[0384] Repeat to repeat variations occur predominantly at amino acid positions 12 and 13, which have therefore been termed “hypervariable” and which are responsible for the specificity of the interaction with the target DNA promoter sequence, as shown in Table 5 listing exemplary repeat variable diresidues (RVD) and their correspondence to nucleic acid base targets.

[0385] Table 5: RVDs and Nucleic Acid Base Specificity

[0386] Accordingly, it is possible to modify the repeats of a TAL effector to target specific DNA sequences. Further studies have shown that the RVD NK can target G. Target sites of TAL effectors also tend to include a T flanking the 5' base targeted by the first repeat, but the exact mechanism of this recognition is not known. More than 113 TAL effector sequences are known to date. Non-limiting examples of TAL effectors from Xanthomonas include, Hax2, Hax3, Hax4, AvrXa7, AvrXalO and AvrBs3.

[0387] Accordingly, a TAL effector domain of a TAL effector molecule described herein may be derived from a TAL effector from any bacterial species (e.g., Xanthomonas species such as the African strain of Xanthomonas oryzae pv. Oryzae (Yu et al. 2011), Xanthomonas campestris pv. raphani strain 756C and Xanthomonas oryzae pv. Oryzicola strain BLS256 (Bogdanove et al. 2011). In some embodiments, a TAL effector domain comprises an RVD domain as well as flanking sequence(s) (sequences on the N-terminal and / or C-terminal side of the RVD domain) also from the naturally occurring TAL effector. It may comprise more or fewer repeats than the RVD of the naturally occurring TAL effector. A TAL effector molecule can be designed to target a given DNA sequence based on the above code and others known in the art. The number of TAL effector domains (e.g., repeats (monomers or modules)) and their specific sequence can be selected based on the desired DNA target sequence. For example, TAL effector domains, e.g., repeats, may be removed or added in order to suit a specific target sequence. In someembodiments, a TAL effector molecule comprises between 6.5 and 33.5 TAL effector domains, e.g., repeats. In some embodiments, a TAL effector molecule of the present invention comprises between 8 and 33.5 TAL effector domains, e.g., repeats, e.g., between 10 and 25 TAL effector domains, e.g., repeats, e.g., between 10 and 14 TAL effector domains, e.g., repeats.

[0388] In some embodiments, a TAL effector molecule comprises TAL effector domains that correspond to a perfect match to a DNA target sequence. In some embodiments, a mismatch between a repeat and a target base-pair on a DNA target sequence is permitted as along as it allows for the function of the polypeptide comprising the TAL effector molecule. Wihtout wishing to be bound by any particular theory, TALE binding is inversely correlated with the number of mismatches. In some embodiments, a TAL effector molecule of a gene modfiying polypeptide comprises no more than 7 mismatches, no more than 6 mismatches, no more than 5 mismatches, no more than 4 mismatches, no more than 3 mismatches, no more than 2 mismatches, or no more than 1 mismatch, and optionally no mismatch, with a target DNA sequence. Without wishing to be bound by any particular theory, the smaller the number of TAL effector domains in a TAL effector molecule, the smaller the number of mismatches will be tolerated and still allow for the function of a gene modifying polypeptide comprising the TAL effector molecule. Binding affinity is thought to depend on the sum of matching repeat-DNA combinations. For example, TAL effector molecules having 25 TAL effector domains or more may be able to tolerate up to 7 mismatches.

[0389] In addition to TAL effector domains, a TAL effector molecule may comprise additional sequences derived from a naturally occurring TAL effector. The length of C-terminal and / or N- terminal sequence(s) included on each side of a TAL effector domain portion of a TAL effector molecule can vary and be selected by one skilled in the art, for example based on the studies of Zhang et al. (2011). Zhang et al., have characterized a number of C-terminal and N-terminal truncation mutants in Hax3 derived TAL-effector based proteins and have identified key elements, which contribute to optimal binding to the target sequence and thus activation of transcription. Generally, it was found that transcriptional activity is inversely correlated with the length of N-terminus. Regarding the C-terminus, an important element for DNA binding residues within the first 68 amino acids of the Hax 3 sequence was identified. Accordingly, in some embodiments, the first 68 amino acids on the C-terminal side of a TAL effector domains of the naturally occurring TAL effector is included in a TAL effector molecule. Accordingly, in some embodiments, a TAL effector molecule comprises 1) one or more TAL effector domains derived from a naturally occurring TAL effector; 2) at least 70, at least 80, at least 90, at least 100, at least 110, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280 or more amino acids from the naturally occurring TAL effector on the N-terminal side of the TAL effector domains; and / or 3) at least 68, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260 or more amino acids from the naturally occurring TAL effector on the C-terminal side of the TAL effector domains.

[0390] In some embodiments, an endonuclease domain or DNA-binding domain is or comprises a Zn finger molecule. A Zn finger molecule comprises a Zn finger protein, e.g., a naturally occurring Zn finger protein or engineered Zn finger protein, or fragment thereof. Many Zn finger proteins are known to those of skill in the art and are commercially available, e.g., from Sigma-Aldrich.

[0391] In some embodiments, a Zn finger molecule comprises a non-naturally occurring Zn finger protein that is engineered to bind to a target DNA sequence of choice. See, for example, Beerli, et al. (2002) Nature Biotechnol. 20:135-141; Pabo, et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan, et al. (2001) Nature Biotechnol. 19:656-660; Segal, et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo, et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005 / 0064474; 2007 / 0218528; 2005 / 0267061, all incorporated herein by reference in their entireties.

[0392] An engineered Zn finger protein may have a novel binding specificity, compared to a naturally-occurring Zn finger protein. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual Zn finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by reference herein in their entireties.

[0393] Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98 / 37186; WO 98 / 53057; WO 00 / 27878; WO 01 / 88197; and GB 2,338,237. In addition, enhancement of binding specificity for zinc finger proteins has been described, for example, in International Patent Publication No. WO 02 / 077227.

[0394] In addition, as disclosed in these and other references, zinc finger domains and / or multifingered zinc finger proteins may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02 / 077227.

[0395] Zn finger proteins and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and described in detail in U.S. Pat. Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; International Patent Publication Nos. WO 95 / 19431; WO 96 / 06166; WO 98 / 53057; WO 98 / 54311; WO 00 / 27878; WO 01 / 60970; WO 01 / 88197; WO 02 / 099084; WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536; and WO 03 / 016496.

[0396] In addition, as disclosed in these and other references, Zn finger proteins and / or multifingered Zn finger proteins may be linked together, e.g., as a fusion protein, using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. The Zn finger molecules described herein may include any combination of suitable linkers between the individual zinc finger proteins and / or multi-fingered Zn finger proteins of the Zn finger molecule.

[0397] In some embodiments, a DNA-binding domain or endonuclease domain comprises a Zn finger molecule comprising an engineered zinc finger protein that binds (in a sequence-specific manner) to a target DNA sequence. In some embodiments, a Zn finger molecule comprises one Zn finger protein or fragment thereof. In some embodiments, a Zn finger molecule comprises a plurality of Zn finger proteins (or fragments thereof), e.g., 2, 3, 4, 5, 6 or more Zn finger proteins (and optionally no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 Zn finger proteins). In some embodiments, a Zn finger molecule comprises at least three Zn finger proteins. In some embodiments, a Zn finger molecule comprises four, five or six Zn finger proteins. In some embodiments, a Zn finger molecule comprises 8, 9, 10, 11 or 12 finger proteins. In some embodiments, a Zn finger molecule comprising three Zn finger proteins recognizes a target DNA sequence comprising 9 or 10 nucleotides. In some embodiments, a Zn finger molecule comprising four Zn finger proteins recognizes a target DNA sequence comprising 12 to 14 nucleotides. In some embodiments, a Zn finger molecule comprising six Zn finger proteins recognizes a target DNA sequence comprising 18 to 21 nucleotides.

[0398] In some embodiments, a Zn finger molecule comprises a two-handed Zn finger protein. Two handed zinc finger proteins are those proteins in which two clusters of zinc finger proteins are separated by intervening amino acids so that the two zinc finger domains bind to two discontinuous target DNA sequences. An example of a two-handed type of zinc finger binding protein is SIP1, where a cluster of four zinc finger proteins is located at the amino terminus of the protein and a cluster of three Zn finger proteins is located at the carboxyl terminus (see Remade, et al. (1999) EMBO Journal 18(18):5073-5084). Each cluster of zinc fingers in these proteins is able to bind to a unique target sequence and the spacing between the two target sequences can comprise many nucleotides.

[0399] Linkers

[0400] In some embodiments, a gene modifying polypeptide may comprise a linker, e.g., a peptide linker, e.g., a linker as described in Table 6. In some embodiments, a gene modifying polypeptide comprises, in an N-terminal to C-terminal direction, a Cas domain (e.g., a Cas domain of Table 4), a linker of Table 6 (or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), and an RT domain (e.g., an RT domain of Table 1). In some embodiments, a gene modifying polypeptide comprises a flexible linker between an endonuclease and ab RT domain, c.g., a linker comprising the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 11,002). In some embodiments, an RT domain of a gene modifying polypeptide may be located C-terminal to an endonuclease domain. In some embodiments, an RT domain of a gene modifying polypeptide may be located N-terminal to the endonuclease domain.

[0401] Table 6: Exemplary linker sequences

[0402] In some embodiments, a linker of a gene modifying polypeptide comprises a motif chosen from: (SGGS)n(SEQ ID NO: 5025), (GGGS)„ (SEQ ID NO: 5026), (GGGGS)n (SEQ ID NO: 5027), (G)n, (EAAAK)U(SEQ ID NO: 5028), (GGS)U, or (XP)U. Gene modifying polypeptide selection by pooled screening

[0403] Candidate gene modifying polypeptides may be screened to evaluate a candidate’s gene editing ability. For example, an RNA gene modifying system designed for the targeted editing of a coding sequence in the human genome may be used. In some embodiments, such a gene modifying system may be used in conjunction with a pooled screening approach. For example, a library of gene modifying polypeptide candidates and a template guide

[0404] RNA (tgRNA) may be introduced into mammalian cells to test the candidates’ gene editing abilities by a pooled screening approach. In some embodiments, a library of gene modifying polypeptide candidates is introduced into mammalian cells followed by introduction of tgRNA into the cells. Representative, non-limiting examples of mammalian cells that may be used in screening include HEK293T cells, U2OS cells, HeLa cells, HepG2 cells, Huh7 cells, K562 cells, or iPS cells. A gene modifying polypeptide candidate may comprise 1 ) a Cas-nuclease, for example a wild-type Cas nuclease, c.g., a wild-type Cas9 nuclease, a mutant Cas nuclease, c.g., a Cas nickase, for example, a Cas9 nickase such as a Cas9 N863A nickase, or a Cas nuclease selected from Table 3 or Table 4, 2) a peptide linker, e.g., a sequence from Table 6, Table 7, or Table 8, that may exhibit varying degrees of length, flexibility, hydrophobicity, and / or secondary structure; and 3) a reverse transcriptase (RT), e.g. an RT domain from Table 7, Table 8, or Table 1. A gene modifying polypeptide candidate library comprises: a plurality of different gene modifying polypeptide candidates that differ from each other with respect to one, two or all three of a Cas nuclease, a peptide linker or an RT domain component, or a plurality of nucleic acid expression vectors that encode such gene modifying polypeptide candidates.

[0405] For screening of gene modifying polypeptide candidates, a two-component system may be used that comprises a gene modifying polypeptide component and a tgRNA component. A gene modifying component may comprise, for example, an expression vector, e.g., an expression plasmid or lentiviral vector, that encodes a gene modifying polypeptide candidate, for example, comprises a human codon-optimized nucleic acid that encodes a gene modifying polypeptide candidate, e.g., a Cas-linker-RT fusion as described above. In some embodiments, a lentiviral cassette is utilized that comprises: (i) a promoter for expression in mammalian cells, e.g., a CMV promoter; (ii) a gene modifying library candidate, e.g. a Cas-linker-RT fusion comprising a Cas nuclease of Table 3 or Table 4, a peptide linker of Table 6, and an RT of Table 1, for example a Cas-linker-RT fusion as in Table 7 or Table 8; (iii) a self-cleaving polypeptide, e.g., a T2A peptide; (iv) a marker enabling selection in mammalian cells, e.g., a puromycin resistance gene; and (v) a termination signal, e.g., a poly A tail.

[0406] A tgRNA component may comprise a tgRNA or expression vector, e.g., an expression plasmid, that produces the tgRNA, for example, utilizes a U6 promoter to drive expression of the tgRNA, wherein the tgRNA is a non-coding RNA sequence that is recognized by Cas and localizes it to the genomic locus of interest, and that also templates reverse transcription of a desired edit into the genome by an RT domain.

[0407] To prepare a pool of cells expressing gene modifying polypeptide library candidates, mammalian cells, e.g., HEK293T or U2OS cells, may be transduced with pooled gene modifying polypeptide candidate expression vector preparations, e.g., lentiviral preparations, of the gene modifying candidate polypeptide library. In some embodiments, lentiviral plasmids are utilized, and HEK293 Lenti-X cells are seeded in 15 cm plates (~12xl06cells) prior to lentiviral plasmid transfection. In some embodiments, lentiviral plasmid transfection may be performed using the Lentiviral Packaging Mix (Biosettia) and transfection of the plasmid DNA for the gene modifying candidate library is performed the following day using Lipofectamine 2000 and Opti-MEM media according to the manufacturer’s protocol. In some embodiments, extracellular DNA may be removed by a full media change the next day and virus-containing media may be harvested 48 hours after. Lentiviral media may be concentrated using Lenti-X Concentrator (TaKaRa Biosciences) and 5 mL lentiviral aliquots may be made and stored at -80°C. Lentiviral titering is performed by enumerating colony forming units post-selection, e.g., post Puromycin selection.

[0408] For monitoring gene editing of a target DNA, mammalian cells, e.g., HEK293T or U2OS cells, carrying a target DNA may be utilized. In some embodiments for monitoring gene editing of a target DNA, mammalian cells, e.g., HEK293T or U2OS cells, carrying a target DNA genomic landing pad may be utilized. In some embodiments, a target DNA genomic landing pad may comprise a gene to be edited for treatment of a disease or disorder of interest. In some embodiments, a target DNA is a gene sequence that expresses a protein that exhibits detectable characteristics that may be monitored to determine whether gene editing has occurred. For example, in some embodiments, a blue fluorescence protein (BFP)- or green fluorescence protein (GFP)-expressing genomic landing pad is utilized. In some embodiments, mammalian cells, e.g., HEK293T or U2OS cells, comprising a target DNA, e.g., a target DNA genomic landing pad, are seeded in culture plates at 500x - 3000x cells per gene modifying library candidate and transduced at a 0.2 - 0.3 multiplicity of infection (MOI) to minimize multiple infections per cell. Puromycin (2.5 ug / mL) may be added 48 hours post infection to allow for selection of infected cells. In such an embodiment, cells may be kept under puromycin selection for at least 7 days and then scaled up for tgRNA introduction, e.g., tgRNA electroporation.

[0409] To ascertain whether gene editing occurs, mammalian cells containing a target DNA to be edited may be infected with gene modifying polypeptide library candidates then transfected with tgRNA designed for use in editing of the target DNA. Subsequently, the cells may be analyzed to determine whether editing of the target locus has occurred according to the designed outcome, or whether no editing or imperfect editing has occurred, e.g., by using cell sorting and sequence analysis. In some embodiments, to ascertain whether genome editing occurs, BFP- or GFP- cxprcssing mammalian cells, e.g., HEK293T or U2OS cells, may be infected with gene modifying library candidates and then transfected or electroporated with tgRNA plasmid or RNA, e.g., by electroporation of 250,000 cells / well with 200 ng of a tgRNA plasmid designed to convert BFP- to-GFP or GFP-to-BFP, at a cell count ensuring >250x - lOOOx coverage per library candidate. In such an embodiment, the genome-editing capacity of the various constructs in this assay may be assessed by sorting the cells by Fluorescence-Activated Cell Sorting (FACS) for expression of the color-converted fluorescent protein (FP) at 4-10 days post-electroporation. Cells are sorted and harvested as distinct populations of unedited cells (exhibiting original florescence protein signal), edited cells (exhibiting converted fluorescence protein signal), and imperfect edit (exhibiting no florescence protein signal) cells. A sample of unsorted cells may also be harvested as the input population to determine candidate enrichment during analysis.

[0410] To determine which gene modifying library candidates exhibit genome-editing capacity in an assay, genomic DNA (gDNA) is harvested from the sorted cell populations, and analyzed by sequencing the gene modifying library candidates in each population. Briefly, gene modifying candidates may be amplified from the genome using primers specific to the gene modifying polypeptide expression vector, e.g., the lentiviral cassette, amplified in a second round of PCR to dilute genomic DNA, and then sequenced, for example, sequenced by a next-generation sequencing platform. After quality control of sequencing reads, reads of at least about 1500 nucleotides and generally no more than about 3200 nucleotides are mapped to the gene modifying polypeptide library sequences and those containing a minimum of about an 80% match to a library sequence are considered to be successfully aligned to a given candidate for purposes of this pooled screen. In order to identify candidates capable of performing gene editing in the assay, e.g., the BFP-to-GFP or GFP-to-BFP edit, the read count of each library candidate in the edited population is compared to its read count in the initial, unsorted population.

[0411] For purposes of pooled screening, gene modifying candidates with genome-editing capacity are identified based on enrichment in the edited (converted FP) population relative to unsorted (input) cells. In some embodiments, an enrichment of at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100-fold over the input indicates potentially useful gene editing activity, e.g., at least 2-fold enrichment. In some embodiments, the enrichment is converted to a log-value by taking the log base 2 of the enrichment ratio. In some embodiments, a log2 enrichment score of at least 0, at least 1, at least 2, at least 3, at least 4, at least 5, at least 5.5, at least 6.0, at least 6.1, at least 6.2, at least 6.3, at least 6.4, at least 6.5, or at least 6.6 indicates potentially useful gene editing activity, e.g., a log2 enrichment score of at least 1.0. In particular embodiments, enrichment values observed for gene modifying candidates may be compared to enrichment values observed under similar conditions utilizing a reference, e.g., Element ID No: 17380.

[0412] In some embodiments, multiple tgRNAs may be used to screen a gene modifying candidate library. In some embodiments, a plurality of tgRNAs may be utilized to optimize template / Cas- linker-RT fusion pairs, e.g., for gene editing of particular target genes, for example, gene targets for the treatment of disease. In some embodiments, a pooled approach to screening gene modifying candidates may be performed using a multiplicity of different tgRNAs in an arrayed format.

[0413] In some embodiments, multiple types of edits, e.g., insertions, substitutions, and / or deletions of different lengths, may be used to screen the gene modifying candidate library.

[0414] In some embodiments, multiple target sequences, e.g., different fluorescent proteins, may be used to screen the gene modifying candidate library. In some embodiments, multiple target sequences, e.g., different fluorescent proteins, may be used to screen the gene modifying candidate library. In some embodiments, multiple cell types, e.g., HEK293T or U2OS, may be used to screen a gene modifying candidate library. A person of ordinary skill in the art will appreciate that a given candidate may exhibit altered editing capacity or even the gain or loss of any observable or useful activity across different conditions, including tgRNA sequence (e.g., nucleotide modifications, PBS length, RT template length), target sequence, target location, type of edit, location of mutation relative to the first-strand nick of the gene modifying polypeptide, or cell type. Thus, in some embodiments, gene modifying library candidates are screened across multiple parameters, e.g., with at least two distinct tgRNAs in at least two cell types, and gene editing activity is identified by enrichment in any single condition. In some embodiments, a candidate with more robust activity across different tgRNA and cell types is identified by enrichment in at least two conditions, e.g., in all conditions screened. For clarity, candidates found to exhibit little to no enrichment under any given condition are not assumed to be inactive across all conditions and may be screened with different parameters or reconfigured at the polypeptide level, e.g., by swapping, shuffling, or evolving domains (e.g., RT domain), linkers, or other signals (e.g., NLS).

[0415] Sequences of exemplary Cas9-linker-RT fusions

[0416] In some embodiments, a gene modifying polypeptide comprises a linker sequence and an RT sequence. In some embodiments, a gene modifying polypeptide comprises a linker sequence as listed in Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain as listed in Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a linker sequence as listed in Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; and the amino acid sequence of an RT domain as listed in Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker sequence as listed in a row of Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; and (ii) the amino acid sequence of an RT domain as listed in the same row of Table 7 or Table 8, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0417] Exemplary Gene Modifying Polypeptides

[0418] In some embodiments, a gene modifying polypeptide (e.g., a gene modifying polypeptide that is part of a system described herein) comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743 of the sequence listing, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1 -7743, or an amino acid sequence having at least 80% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1 -7743, or an amino acid sequence having at least 90% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 95% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide described herein comprises an RT and linker sequence from any of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto and a StlCas9 domain described herein. In some embodiments, a gene modifying polypeptide described herein comprises an RT and linker sequence from any of SEQ ID NOs: 1-7743, and a StlCas9 domain described herein.

[0419] In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in Table 12, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0420] In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a linker comprising a linker sequence as listed in Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0421] Table 7: Selection of exemplary gene modifying polypeptides

[0422] In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a linker comprising a linker sequence as listed in Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0423] Table 8: Selection of exemplary gene modifying polypeptides

[0424] Subsequences of Exemplary Gene Modifying Polypeptides

[0425] In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C- terminal order, one or more (e.g., 1, 2, 3, 4, 5, or all 6) of an N-terminal methionine residue, a first nuclear localization signal (NLS), a DNA binding domain, a linker, an RT domain, and / or a second NLS. In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a NLS (e.g., a first NLS), a DNA binding domain, a linker, and an RT domain, wherein the linker and RT domain are the linker and RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker and RT domain. In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a DNA binding domain, a linker, an RT domain, and an NLS (e.g., a second NLS) wherein the linker and RT domain are the linker and RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker and RT domain. In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a first NLS, a DNA binding domain, a linker, an RT domain, and a second NLS, wherein the linker and RT domain are the linker and RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker and RT domain. In some embodimetns, the gene modifying polypeptide further comprises an N-terminal methionine residue.

[0426] In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C- terminal order, one or more (e.g., 1, 2, 3, 4, 5, or all 6) of an N-terminal methionine residue, a first nuclear localization signal (NLS) (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and / or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), a DNA binding domain (e.g., a Cas domain, e.g., a SpyCas9 domain, e.g., as listed in Table 4, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; or a DNA binding domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and / or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), a linker (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and / or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), an RT domain (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and / or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), and a second NLS (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1- 7743 and / or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto). In some embodiments, the gene modifying polypeptide further comprises (e.g., C-terminal to the second NLS) a T2A sequence and / or a puromycin sequence (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and / or as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto). In some embodiments, a nucleic acid encoding a gene modifying polypeptide (e.g., as described herein) encodes a T2A sequence, e.g., wherein the T2A sequence is situated between a region encoding the gene modifying polypeptide and a second region, wherein the second region optionally encodes a selectable marker, e.g., puromycin.

[0427] In some embodiments, the first NLS comprises a first NLS sequence of a gene modifying polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In certain embodiments, the first NLS comprises a first NLS sequence of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%idcntity thereto. In some embodiments, a first NLS sequence comprises a C-myc NLS. In some embodiments, a first NLS comprises the amino acid sequence PAAKRVKLD (SEQ ID NO: 11,095), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0428] In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between a first NLS and a DNA binding domain. In some embodiments, a spacer sequence between a first NLS and a DNA binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between a first NLS and a DNA binding domain comprises the amino acid sequence GG.

[0429] In some embodiments, a DNA binding domain comprises a DNA binding domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a DNA binding domain comprises a DNA binding domain of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a DNA binding domain comprises a Cas domain (e.g., as listed in Table 4). In some embodiments, a DNA binding domain comprises the amino acid sequence of a SpyCas9 polypeptide (e.g., as listed in Table 4, e.g., a Cas9 N863A polypeptide), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a DNA binding domain comprises the amino acid sequence:

[0430] DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDK KHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRG HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLT LLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEE LLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILT FRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDN EENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDS LHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKG QKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKN YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILD SRMNTKYDENDKL1REVKV1TLKSKLVSDFRKDFQFYKVRE1NNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK RMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 11,096) or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0431] In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between a DNA binding domain and a linker. In some embodiments, a spacer sequence between a DNA binding domain and a linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between a DNA binding domain and a linker comprises the amino acid sequence GG.

[0432] In some embodiments, a linker comprises a linker sequence of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In certain embodiments, the linker comprises a linker sequence of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the linker comprises an amino acid sequence as listed in Table 6, Table 7 or Table 8 , or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0433] In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between a linker and an RT domain. In some embodiments, a spacer sequence between a linker and an RT domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between a linker and an RT domain comprises the amino acid sequence GG. In some embodiments, an RT domain comprises an RT domain sequence of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises a RT domain sequence of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises an amino acid sequence as listed in Table 7, Table 8, or Table 1, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain has a length of about 400-500, about 500-600, about 600-700, about 700-800, about 800-900, or about 900-1000 amino acids.

[0434] In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between an RT domain and a second NLS. In some embodiments, a spacer sequence between an RT domain and a second NLS comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between an RT domain and a second NLS comprises the amino acid sequence AG.

[0435] In some embodiments, a second NLS comprises a second NLS sequence of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743. In some embodiments, a second NLS comprises a second NLS sequence of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8. In some embodiments, a second NLS sequence comprises a plurality of partial NLS sequences. In some embodiments, an NLS sequence, e.g., a second NLS sequence, comprises a first partial NLS sequence, e.g., comprising the amino acid sequence KRTADGSEFE (SEQ ID NO: 11,097), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In embodiments, an NLS sequence, e.g., a second NLS sequence, comprises a second partial NLS sequence. In some embodiments, an NLS sequence, e.g., a second NLS sequence, comprises an SV40A5 NLS, e.g., a bipartite SV40A5 NLS, e.g., comprising the amino acid sequence KRTADGSEFESPKKKAKVE (SEQ ID NO: 11,098), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an NLS sequence, e.g., a second NLS sequence, comprises the amino acid sequence KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 11,099), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0436] In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between a second NLS and a T2A sequence and / or puromycin sequence. In some embodiments, a spacer sequence between a second NLS and a T2A sequence and / or puromycin sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between a second NLS and a T2A sequence and / or puromycin sequence comprises the amino acid sequence GSG.

[0437] Linkers and RT domains

[0438] In some embodiments, a gene modifying polypeptide comprises a linker (e.g., as described herein) and an RT domain (e.g., as described herein). In some embodiments, a gene modifying polypeptide comprises, in N-tcrminal to C-tcrminal order, a linker (e.g., as described herein) and an RT domain (e.g., as described herein).

[0439] In some embodiments, a linker comprises a linker sequence as listed in Table 6, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In certain embodiments, the linker comprises a linker sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a linker comprises a linker sequence of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a linker comprises a linker sequence present in any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a linker comprises a linker sequence of an exemplary gene modifying polypeptide listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises an RT domain sequence as listed in Table 1, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises an RT domain sequence of an exemplary gene modifying polypeptide listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0440] In some embodiments, a gene modifying polypeptide comprises a portion of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.

[0441] In some embodiments, a gene modifying polypeptide comprises a linker of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker. In some embodiments, a gene modifying polypeptide comprises a linker of a gene modifying polypeptide of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker. In some embodiments, a gene modifying polypeptide comprises a linker of a gene modifying polypeptide of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said linker. In some embodiments, a gene modifying polypeptide comprises a linker of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or a linker comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0442] In some embodiments, a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said RT domain. In some embodiments, a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity said RT domain. In some embodiments, a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity said RT domain. In some embodiments, a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8, or an RT domain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0443] In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) of a gene modifying polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 80% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 90% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 95% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 99% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) of a gene modifying polypeptide having the amino acid sequence of any one of SEQ ID NOs: 6001-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) of a gene modifying polypeptide having the amino acid sequence of any one of SEQ ID NOs: 4501-4541. In some embodiments, a linker and am RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) from a single row of any of Table 12, Table 7, or Table 8 (e.g., from a single exemplary gene modifying polypeptide as listed in any of Table 12, Table 7, or Table 8). Insome embodiments, a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) from two different amino acid sequences selected from SEQ ID NOs: 1-7743. In some embodiments, a linker and an RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto) from different rows of any of Table 12, Table 7, or Table 8.

[0444] In some embodiments, a gene modifying polypeptide further comprises a first NLS (e.g., a 5’ NLS), e.g., as described herein. In some embodiments, a gene modifying polypeptide further comprises a second NLS (e.g., a 3’ NLS), e.g., as described herein. In some embodiments, a gene modifying polypeptide further comprises an N-terminal methionine residue.

[0445] RT Families and Mutants

[0446] In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, ML VMS, PERV, SFV1, SFV3L, WMSV, XMRV6, BLVAU, BLVJ, HTL1A, HTL1C, HTL1L, HTL32, HTL3P, HTLV2, JSRV, MLVF5, MLVRD, MMTVB, MPMV, SFVCP, SMRVH, SRV1, SRV2, and WDSV. In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, MLVMS, PERV, SFV1, SFV3L, WMSV, and XMRV6.

[0447] In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from an MLVMS RT domain. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 1 of Table 9, or a point mutation corresponding thereto. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 3 of Table 9 (Genl MLVMS), or a point mutation corresponding thereto. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations at an amino acid position of the RT domain as listed in columns 1 and 2 of Table 10, or an amino acid position corresponding thereto.

[0448] In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from an AVIRE RT domain. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 2 of Table 9, or a point mutation corresponding thereto. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 4 of Table 9 (Gen2 AVIRE), or a point mutation corresponding thereto. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations at an amino acid position of the RT domain as listed in columns 3 and 4 of Table 10, or an amino acid position corresponding thereto. In some embodiments, an RT domain comprises an IENSSP (e.g., at the C-terminus).

[0449] Table 9: Exemplary point mutations in ML VMS and AVIRE RT domains

[0450] Table 10: Positions that can be mutated in exemplary ML VMS and AVIRE RT domains

[0451] In some embodiments, a gene modifying polypeptide comprises a gamma retrovirus derived RT domain. In some embodiments, a gamma retrovirus-derived RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, ML VMS, PERV, SFV1 , SFV3L, WMSV, and XMRV6. In some embodiments, a gamma retrovirus-derived RT domain of a gene modifying polypeptide is not derived from PERV. In some embodiments, an RT domain includes one, two, three, four, five, six or more mutations shown in Table 2 and corresponding to mutations D200N, L603W, T330P, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L67 IP, E69K, or D653N in the RT domain of murine leukemia virus reverse transcriptase. In some embodiments, a gene modifying polypeptide further comprises a linker having at least 99% identity to a linker domains of any one of SEQ ID NOs: 1-7743. In some embodiments, the gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO: 11,041.

[0452] In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of an AVIRE RT (e.g., an AVIRE_P03360 sequence, e.g., SEQ ID NO: 8001), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of an AVIRE RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, G33OP, L605W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an AVIRE RT further comprising one, two, or three mutations selected from the group consisting of D200N, G33OP, and L605W, or a corresponding position in a homologous RT domain.

[0453] In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a BAEVM RT (e.g., an BAEVM_P10272 sequence, e.g., SEQ ID NO: 8004), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a BAEVM RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L602W, T304K, and W311F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a BAEVM RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L602W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of an FFV RT (c.g., an FFV_O93209 sequence, c.g., SEQ ID NO: 8012), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, three, or four mutations selected from the group consisting of D21N, T293N, T419P, and L393K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, or three mutations selected from the group consisting of D21N, T293N, and T419P, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising the mutation D21N. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, or three mutations selected from the group consisting of T207N, T333P, and L307K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising one or two mutations selected from the group consisting of T207N and T333P, or a corresponding position in a homologous RT domain.

[0454] In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of an FLV RT (e.g., an FLV_P10273 sequence, e.g., SEQ ID NO: 8019), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of an FLV RT further comprising one, two, three, or four mutations selected from the group consisting of D199N, L602W, T305K, and W312F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an FLV RT further comprising one or two mutations selected from the group consisting of D199N and L602W, or a corresponding position in a homologous RT domain.

[0455] In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a FOAMV RT (e.g., a FOAMV_P14350 sequence, e.g., SEQ ID NO: 8021), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, S420P, and L396K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and S420P, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV RT further comprising the mutation D24N, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one, two, or three mutations selected from the group consisting of T207N, S331P, and L307K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one or two mutations selected from the group consisting of T207N and S331P, or a corresponding position in a homologous RT domain.

[0456] In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a GALV RT (e.g., an GALV_P21414 sequence, e.g., SEQ ID NO: 8027), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L600W, T304K, and W31 IF, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L600W, or a corresponding position in a homologous RT domain.

[0457] In embodiments, an RT domain comprises the amino acid sequence of an RT domain of a KORV RT (e.g., an KORV_Q9TTC1 sequence, e.g., SEQ ID NO: 8047), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, four, five, or six mutations selected from the group consisting of D32N, D322N, E452P, L274W, T428K, and W435F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, or four mutations selected from the group consisting of D32N, D322N, E452P, and L274W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising the mutation D32N. Tn some embodiments, an RT domain comprises the amino acid sequence of a KORV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D231N, E361P, L633W, T337K, and W344F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a KORV RT further comprising one, two, or three mutations selected from the group consisting of D231N, E361P, and L633W, or a corresponding position in a homologous RT domain.

[0458] In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVAV RT (e.g., an MLVAV_P03356 sequence, e.g., SEQ ID NO: 8053), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a MLVAV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T33OP, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVAV RT further comprising one, two, or three mutations selected from the group consisting of D200N, T33OP, and L603W, or a corresponding position in a homologous RT domain.

[0459] In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVBM RT (e.g., an MLVBM_Q7SVK7 sequence, e.g., SEQ ID NO: 8056), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a MLVBM RT further comprising one, two, three, four, or five mutations selected from the group consisting of D199N, T329P, L602W, T305K, and W312F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVBM RT further comprising one, two, and three mutations selected from the group consisting of D200N, T33OP, and L603W, or a corresponding position in a homologous RT domain.

[0460] In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVCB RT (e.g., an MLVCB_P08361 sequence, e.g., SEQ ID NO: 8062), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a MLVCB RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T33OP, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVCB RT further comprising one, two, and three mutations selected from the group consisting of D200N, T33OP, and L603W, or a corresponding position in a homologous RT domain.

[0461] In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVFF RT, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a MLVFF RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVFF RT further comprising one, two, and three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain.

[0462] In embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVMS RT (e.g., an MLVMS_reference sequence, e.g., SEQ ID NO: 8137; or an MLVMS_P03355 sequence, e.g., SEQ ID NO: 8070), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, three, four, five, or six mutations selected from the group consisting of D200N, T330P, L603W, T306K, W313F, and H8Y, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, or three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain.

[0463] In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a PERV RT (e.g., an PERV_Q4VFZ2 sequence, e.g., SEQ ID NO: 8099), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a PERV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D196N, E326P, L599W, T302K, and W309F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a PERV RT further comprising one, two, or three mutations selected from the group consisting of D196N, E326P, and L599W, or a corresponding position in a homologous RT domain.

[0464] In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a SFV1 RT (e.g., an SFV1_P23O74 sequence, e.g., SEQ ID NO: 8105), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a SFV 1 RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, N420P, and L396K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV 1 RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and N420P, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV1 RT further comprising the D24N, or a corresponding position in a homologous RT domain.

[0465] In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a SFV3L RT (e.g., an SFV3L_P27401 sequence, e.g., SEQ ID NO: 8111), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, N422P, and L396K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and N422P, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising the mutation D24N, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, or three mutations selected from the group consisting of T307N, N333P, and L307K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one or two mutations selected from the group consisting of T307N and N333P, or a corresponding position in a homologous RT domain.

[0466] In embodiments, an RT domain comprises the amino acid sequence of an RT domain of a WMSV RT (e.g., an WMSV_P03359 sequence, e.g., SEQ ID NO: 8131), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a WMSV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L600W, T304K, and W31 IF, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a WMSV RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L600W, or a corresponding position in a homologous RT domain.

[0467] In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a XMRV6 RT (e.g., an XMRV6_A1Z651 sequence, e.g., SEQ ID NO: 8134), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a XMRV6 RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T33OP, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a XMRV6 RT further comprising one, two, or three mutations selected from the group consisting of D200N, T33OP, and L603W, or a corresponding position in a homologous RT domain.

[0468] In some embodiments, an RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain of an AVIRE RT, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In embodiments, an RT domain comprises the amino acid sequence of an RT domain comprised in a sequence listed in column 1 of Table 11, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO:11,041.

[0469] In some embodiments, an RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain of an MLVMS RT, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain comprised in a sequence listed in any of columns 2-6 of Table 11, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO:11,041.

[0470] Table 11: Exemplary gene modifying polypeptides comprising an AVIRE RT domain or an MLVMS RT domain.

[0471]

[0472]

[0473]

[0474]

[0475]

[0476] Systems

[0477] In some embodiments, the present disclosure provides a system comprising a nucleic acid molecule encoding a gene modifying polypeptide (e.g., as described herein) and a template nucleic acid (e.g., a template RNA, e.g., as described herein). In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises one or more silent mutations in the coding region (e.g., in the sequence encoding an RT domain) relative to a nucleic acid molecule as described herein. In some embodiments, a system further comprises a gRNA (e.g., a gRNA that binds to a polypeptide that induces a nick, e.g., in the opposite strand of a target DNA bound by a gene modifying polypeptide).

[0478] In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide encodes a polypeptide as listed in any of Tables 12, 7, or 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0479] In some embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 1-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion. In some embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 6001-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion. In some embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 4501-4541, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion. In some embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of a polypeptide listed in any of Table 12, Table 7, or Table 8, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.

[0480] In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding a linker of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding a linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding a linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding a linker of a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0481] In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding an RT domain of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding an RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001- 7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding an RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541 , or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises a sequence encoding an RT domain of a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0482] In some embodiments, the present disclosure provides a system comprising a gene modifying polypeptide (e.g., as described herein) and a template nucleic acid (e.g., a template RNA, e.g., as described herein).

[0483] In some embodiments, a gene modifying polypeptide comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0484] In some embodiments, a gene modifying polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 1-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion. In some embodiments, a gene modifying polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 6001-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion. In some embodiments, a gene modifying polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 4501-4541 , wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion. In some embodiments, a gene modifying polypeptide comprises a portion of a polypeptide listed in any of Table 12, Table 7, or Table 8, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to said portion.

[0485] In some embodiments, a gene modifying polypeptide comprises a linker of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a sequence encoding the linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a sequence encoding the linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises the linker of a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0486] In some embodiments, a gene modifying polypeptide comprises an RT domain of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a sequence encoding an RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a sequence encodingan RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an RT domain of a polypeptide as listed in any of Table 12, Table 7, or Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. Table 12: Exemplary amino acid sequences for gene modifying polypeptides

[0487] In some embodiments, a gene modifying polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 26002, 26004, 10001, 10011, 10118, 10119, 10120, 31453, 31454, 31455, 31458, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. Localization sequences for gene modifying systems

[0488] In some embodiments, a gene modifying system RNA further comprises an intracellular localization sequence, c.g., a nuclear localization sequence (NLS). In some embodiments, a gene modifying polypeptide comprises an NLS as comprised in SEQ ID NO: 4000 and / or SEQ ID NO: 4001, or an NLS having an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0489] A nuclear localization sequence may be an RNA sequence that promotes the import of an RNA into the nucleus. In some embodiments, a nuclear localization signal is located on a template RNA. In some embodiments, a gene modifying polypeptide is encoded on a first RNA, and a template RNA is a second, separate, RNA, and a nuclear localization signal is located on the template RNA and not on an RNA encoding the gene modifying polypeptide. While not wishing to be bound by theory, in some embodiments, an RNA encoding a gene modifying polypeptide is targeted primarily to the cytoplasm to promote its translation, while a template RNA is targeted primarily to the nucleus to promote insertion into the genome. In some embodiments a nuclear localization signal is at the 3' end, 5' end, or in an internal region of a template RNA. In some embodiments, a nuclear localization signal is 3' of a heterologous sequence (e.g., is directly 3' of the heterologous sequence) or is 5' of the heterologous sequence (e.g., is directly 5' of the heterologous sequence). In some embodiments, a nuclear localization signal is placed outside of a 5' UTR or outside of a 3' UTR of a template RNA. In some embodiments, a nuclear localization signal is placed between a 5' UTR and a 3' UTR, wherein optionally the nuclear localization signal is not transcribed with a transgene (e.g., the nuclear localization signal is in an anti-sense orientation or is downstream of a transcriptional termination signal or polyadenylation signal). In some embodiments, a nuclear localization sequence is situated inside of an intron. In some embodiments, a plurality of the same or different nuclear localization signals are in an RNA, e.g., in a template RNA. In some embodiments, a nuclear localization signal is less than 5, less than 10, less than 25, less than 50, less than 75, less than 100, less than 150, less than 200, less than 250, less than 300, less than 350, less than 400, less than 450, less than 500, less than 600, less than 700, less than 800, less than 900 or less than 1000 bp in length. Various RNA nuclear localization sequences can be used. For example, Lubelsky and Ulitsky, Nature 555 (107-111), 2018 describe RNA sequences which drive RNA localization into the nucleus. In some embodiments, a nuclear localization signal is a SINE-derived nuclear RNA localization (SIRLOIN) signal. In some embodiments, a nuclear localization signal binds a nuclear-enriched protein. In some embodiments, a nuclear localization signal binds the HNRNPK protein. In some embodiments, a nuclear localization signal is rich in pyrimidines, e.g., is a C / T rich, C / U rich, C rich, T rich, or U rich region. In some embodiments, a nuclear localization signal is derived from a long non-coding RNA. In some embodiments, a nuclear localization signal is derived from MALAT1 long non-coding RNA or is the 600 nucleotide M region of MALAT 1 (described in Miyagawa et al., RNA 18, (738-751), 2012). In some embodiments a nuclear localization signal is derived from BORG long non-coding RNA or is a AGCCC motif (described in Zhang et al., Molecular and Cellular Biology 34, 2318-2329 (2014). In some embodiments, a nuclear localization sequence is described in Shukla et al., The EMBO Journal c98452 (2018). In some embodiments, a nuclear localization signal is derived from a retrovirus.

[0490] In some embodiments, a gene modifying polypeptide described herein comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example a nuclear localization sequence (NLS). In some embodiments, an NLS is a bipartite NLS. In some embodiments, an NLS facilitates the import of a protein comprising an NLS into the cell nucleus. In some embodiments, an NLS is fused to the N-terminus of a gene modifying polypeptide as described herein. In some embodiments, an NLS is fused to the C-terminus of a gene modifying polypeptide. In some embodiments, an NLS is fused to the N-terminus or the C-terminus of a Cas domain. In some embodiments, a linker sequence is disposed between an NLS and a neighboring domain of a gene modifying polypeptide.

[0491] In some embodiments, an NLS comprises the amino acid sequence MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 5009), PKKRKVEGADKRTADGSEFESPKKKRKV(SEQ ID NO: 5010), RKSGKIAAIWKRPRKPKKKRKV (SEQ ID NO: 5011) KRTADGSEFESPKKKRKV(SEQ ID NO: 5012), KKTELQTTNAENKTKKL (SEQ ID NO: 5013), or KRGINDRNFWRGENGRKTR (SEQ ID NO: 5014), KRPAATKKAGQAKKKK (SEQ ID NO: 5015), PAAKRVKLD (SEQ ID NO:4644), KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 4649), KRTADGSEFE (SEQ ID NO: 4650), KRTADGSEFESPKKKAKVE (SEQ ID NO: 4651), AGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 4001), or a functional fragment or variant thereof. Exemplary NLS sequences are also described in PCT / EP2000 / 011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, an NLS comprises an amino acid sequence as disclosed in Table 13. An NLS of Table 13 may be utilized with one or more copies in a polypeptide in one or more locations in a polypeptide, e.g., 1, 2, 3 or more copies of an NLS in an N-terminal domain, between peptide domains, in a C- terminal domain, or in a combination of locations, in order to improve subcellular localization to the nucleus. Multiple unique sequences may be used within a single polypeptide. Sequences may be naturally monopartite or bipartite, e.g., having one or two stretches of basic amino acids, or may be used as chimeric bipartite sequences. Sequence references correspond to UniProt accession numbers, except where indicated as SeqNLS for sequences mined using a subcellular localization prediction algorithm (Lin et al BMC Bioinformat 13:157 (2012), incorporated herein by reference in its entirety).

[0492] Table 13: Exemplary nuclear localization signals for use in gene modifying systems

[0493] In some embodiments, an NLS is a bipartite NLS. A bipartite NLS typically comprises two basic amino acid clusters separated by a spacer sequence (which may be, e.g., about 10 amino acids in length). A monopartite NLS typically lacks a spacer. An example of a bipartite NLS is the nucleoplasmin NLS, having the sequence KR[PAATKKAGQA]KKKK (SEQ ID NO: 5015), wherein the spacer is indicated in square brackets. Another exemplary bipartite NLS has the sequence PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 5016). Exemplary NLSs are described in International Application W02020051561, which is herein incorporated by reference in its entirety, including for its disclosures regarding nuclear localization sequences.

[0494] In some embodiments, a gene editor system polypeptide (e.g., a gene modifying polypeptide as described herein) further comprises an intracellular localization sequence, e.g., a nuclear localization sequence and / or a nucleolar localization sequence. A nuclear localization sequence and / or nucleolar localization sequence may be an amino acid sequences that promotes the import of the protein into the nucleus and / or nucleolus, where it can promote integration of heterologous sequence into the genome. In some embodiments, a gene editor system polypeptide (e.g., (e.g., a gene modifying polypeptide as described herein) further comprises a nucleolar localization sequence. In some embodiments, a gene modifying polypeptide is encoded on a first RNA, and a template RNA is a second, separate, RNA, and a nucleolar localization signal is encoded on the RNA encoding the gene modifying polypeptide and not on the template RNA. In some embodiments, a nucleolar localization signal is located at the N-terminus, C- terminus, or in an internal region of a gene modifying polypeptide. In some embodiments, a plurality of the same or different nucleolar localization signals are used. In some embodiments, a nuclear localization signal is less than 5, less than 10, less than 25, less than 50, less than 75, or less than 100 amino acids in length. Various polypeptide nucleolar localization signals can be used. For example, Yang et al., Journal of Biomedical Science 22, 33 (2015), describe a nuclear localization signal that also functions as a nucleolar localization signal. In some embodiments, a nucleolar localization signal may also be a nuclear localization signal. In some embodiments, a nucleolar localization signal may overlap with a nuclear localization signal. In some embodiments, a nucleolar localization signal may comprise a stretch of basic residues. In some embodiments, a nucleolar localization signal may be rich in arginine and lysine residues. In some embodiments, a nucleolar localization signal may be derived from a protein that is enriched in the nucleolus. In some embodiments, a nucleolar localization signal may be derived from a protein enriched at ribosomal RNA loci. In some embodiments, a nucleolar localization signal may be derived from a protein that binds rRNA. In some embodiments, a nucleolar localization signal may be derived from MSP58. In some embodiments, a nucleolar localization signal may be a monopartite motif. In some embodiments, a nucleolar localization signal may be a bipartite motif. In some embodiments, a nucleolar localization signal may comprise multiple monopartite or bipartite motifs. In some embodiments, a nucleolar localization signal may comprise a mix of monopartite and bipartite motifs. In some embodiments, a nucleolar localization signal may be a dual bipartite motif. In some embodiments, a nucleolar localization motif may be a KRASSQALGTIPKRRSSSRFIKRKK (SEQ ID NO: 5017). In some embodiments, a nucleolar localization signal may be derived from nuclear factor-KB-inducing kinase. In some embodiments, a nucleolar localization signal may be an RKKRKKK motif (SEQ ID NO: 5018) (described in Birbach et al., Journal of Cell Science, 117 (3615-3624), 2004).

[0495] Evolved Variants of Gene Modifying Polypeptides and Systems

[0496] In some embodiments, the present disclosure provides evolved variants of gene modifying polypeptides as described herein. Evolved variants can, in some embodiments, be produced by mutagenizing a reference gene modifying polypeptide, or one of the fragments or domains comprised therein. In some embodiments, one or more domains (e.g., a reverse transcriptase domain) is evolved. One or more of such evolved variant domains can, in some embodiments, be evolved alone or together with other domains. An evolved variant domain or domains may, in some embodiments, be combined with unevolved cognate component(s) or evolved variants of the cognate component(s), e.g., which may have been evolved in either a parallel or serial manner.

[0497] In some embodiments, a process of mutagenizing a reference gene modifying polypeptide, or fragment or domain thereof, comprises mutagenizing a reference gene modifying polypeptide or fragment or domain thereof. In some embodiments, mutagenesis comprises a continuous evolution method (e.g., PACE) or non-continuous evolution method (e.g., PANCE), e.g., as described herein. In some embodiments, an evolved gene modifying polypeptide, or a fragment or domain thereof, comprises one or more amino acid variations introduced into its amino acid sequence relative to the amino acid sequence of a reference gene modifying polypeptide, or fragment or domain thereof. In some embodiments, amino acid sequence variations may include one or more mutated residues (e.g., conservative substitutions, nonconservative substitutions, or a combination thereof) within the amino acid sequence of a reference gene modifying polypeptide, e.g., as a result of a change in the nucleotide sequence encoding the gene modifying polypeptide that results in, e.g., a change in the codon at any particular position in the coding sequence, the deletion of one or more amino acids (e.g., a truncated protein), the insertion of one or more amino acids, or any combination of the foregoing. An evolved variant gene modifying polypeptide may include variants in one or more components or domains of the gene modifying polypeptide (e.g., variants introduced into a reverse transcriptase domain).

[0498] In some embodiments, the present disclosure provides gene modifying polypeptides, systems, kits, and methods using or comprising an evolved variant of a gene modifying polypeptide, e.g., employs an evolved variant of a gene modifying polypeptide or a gene modifying polypeptide produced or producible by PACE or PANCE. In some embodiments, an unevolved reference gene modifying polypeptide is a gene modifying polypeptide as disclosed herein.

[0499] The term “phage-assisted continuous evolution (PACE),”as used herein, generally refers to continuous evolution that employs phage as viral vectors. Examples of PACE technology have been described, for example, in International PCT Application No. PCT / US 2009 / 056194, filed September 8, 2009, published as WO 2010 / 028347 on March 11, 2010; International PCT Application, PCT / US2011 / 066747, filed December 22, 2011, published as WO 2012 / 088381 on June 28, 2012; U.S. Patent No. 9,023,594, issued May 5, 2015; U.S. Patent No. 9,771,574, issued September 26, 2017; U.S. Patent No. 9,394,537, issued July 19, 2016; International PCT Application, PCT / US2015 / 012022, filed January 20, 2015, published as WO 2015 / 134121 on September 11, 2015; U.S. Patent No. 10,179,911, issued January 15, 2019; and International PCT Application, PCT / US2016 / 027795, filed April 15, 2016, published as WO 2016 / 168631 on October 20, 2016, the entire contents of each of which are incorporated herein by reference.

[0500] The term “phage-assisted non-continuous evolution (PANCE),” as used herein, generally refers to non-continuous evolution that employs phage as viral vectors. Examples of PANCE technology have been described, for example, in Suzuki T. et al, Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase, Nat Chem Biol. 13(12): 1261-1266 (2017), incorporated herein by reference in its entirety. Briefly, PANCE is a technique for rapid in vivo directed evolution using serial flask transfers of evolving selection phage (SP), which contain a gene of interest to be evolved, across fresh host cells (e.g., E. coli cells). Genes inside the host cell may be held constant while genes contained in the SP continuously evolve. Following phage growth, an aliquot of infected cells may be used to transfect a subsequent flask containing host E. coli. This process can be repeated and / or continued until the desired phenotype is evolved, e.g., for as many transfers as desired.

[0501] Methods of applying PACE and PANCE to gene modifying polypeptides may be readily appreciated by askilled artisan by reference to, inter alia, the foregoing references. Additional exemplary methods for directing continuous evolution of genome-modifying proteins or systems, e.g., in a population of host cells, e.g., using phage particles, can be applied to generate evolved variants of gene modifying polypeptides, or fragments or subdomains thereof. Non-limiting examples of such methods are described in International PCT Application, PCT / US2009 / 056194, filed September 8, 2009, published as WO 2010 / 028347 on March 11, 2010; International PCT Application, PCT / US2011 / 066747, filed December 22, 2011, published as WO 2012 / 088381 on June 28, 2012; U.S. Patent No. 9,023,594, issued May 5, 2015; U.S. Patent No. 9,771,574, issued September 26, 2017; U.S. Patent No. 9,394,537, issued July 19, 2016; International PCT Application, PCT / US2015 / 012022, filed January 20, 2015, published as WO 2015 / 134121 on September 11, 2015; U.S. Patent No. 10,179,911, issued January 15, 2019; International Application No. PCT / US2019 / 37216, filed June 14, 2019, International Patent Publication WO 2019 / 023680, published January 31, 2019, Interna...

Claims

CLAIMS1. A nucleic acid molecule comprising: an StlCas9 scaffold; wherein the StlCas9 scaffold comprises a chemically modified nucleotide at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or all of) positions 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 relative to SEQ ID NO: 25999.

2. A nucleic acid molecule comprising: an StlCas9 scaffold comprising: a) a Repeat: anti-repeat (RAR) region, wherein optionally the RAR region comprises a RAR lower stem, a RAR upper stem, and an RAR loop (e.g., a tetraloop); b) a stem-loop 1 (SL1) region that is optionally 3’ of the RAR region, and c) optionally, a stem loop 2 (SL2) region that is optionally 3’ of the SL1 region; wherein the StlCas9 scaffold comprises a chemically modified nucleotide in one or both of the RAR region or the SL1 region.

3. The nucleic acid of claim 1 or 2, wherein at least 15-20%, 20-30%, 30-40%, 40-50%, 50- 60%, 60-70, or 70-75% of nucleotides in the StlCas9 scaffold are chemically modified.

4. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a RAR region, wherein the RAR region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 chemically modified nucleotides (e.g., wherein the chemically modified nucleotides have the same chemical modification).

5. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a SL1 region, wherein the SL1 region comprises 1, 2, 3, 4, 5, 6, 7, or 8 chemicallymodified nucleotides (e.g., wherein the chemically modified nucleotides have the same chemical modification).

6. The nucleic acid of any of the preceding claims, wherein positions 1, 2, and 3 (if present) do not comprise a 2’-O-methyl chemically modified nucleotide.

7. The nucleic acid of any of the preceding claims, wherein positions 43 through 54 (if present) do not comprise a 2’-O-methyl chemically modified nucleotide.

8. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 4 through 6 (if present).

9. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 13 through 15 (if present).

10. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 16 through 18 (if present).

11. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 19 through 21 (if present).

12. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 22 through 24 (if present).

13. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 34 through 36 (if present).

14. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 37 through 39 (if present).

15. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 40 through 42 (if present).

16. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 13 through 24 (if present).

17. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold comprises a chemically modified nucleotide at each of positions 34 through 42 (if present).

18. The nucleic acid of any of the preceding claims, wherein the chemically modified nucleotide is a modification to a sugar group, e.g., a modification to the 2’-0 of ribose, e.g., a 2’-O-Methyl chemically modified nucleotide.

19. The nucleic acid of any of the preceding claims, which further comprises a second chemically modified nucleotide.

20. The nucleic acid of any of the preceding claims, which comprises a sequence according to SEQ ID NO: 26000, or a sequence having at least 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity thereto.

21. The nucleic acid of any of the preceding claims, wherein the StlCas9 scaffold binds to an StlCas9 protein having a sequence of SEQ ID NO: 23818.

22. A nucleic acid comprising a Repeat: anti-repeat (RAR) region which comprises a chemically modified nucleotide.

23. A nucleic acid comprising a stem-loop 1 (SL1) region which comprises a chemically modified nucleotide.

24. A nucleic acid molecule comprising: an StlCas9 scaffold;wherein the StlCas9 scaffold comprises a chemically modified nucleotide.

25. A template RNA comprising (tgRNA) comprising, from 5’ to 3’:(1) a gRNA spacer;(2) a chemically modified StlCas9 scaffold comprising a nucleic acid of any of claims 1-24;(3) a heterologous object sequence; and(4) a primer binding site (PBS) sequence.

26. A system comprising: a nucleic acid of any of claims 1-24 or a template RNA of claim 25; and a polypeptide comprising a StlCas9 domain, or a nucleic acid encoding the polypeptide.

27. A gene modifying system comprising: a template RNA of claim 25 ; and a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, the gene modifying polypeptide comprising:(1) a StlCas9 domain;(2) a linker; and(3) a reverse transcriptase (RT) domain.

28. A pharmaceutical composition, comprising the nucleic acid or template RNA of any one of claims 1-25 or the system of claim 27, and a pharmaceutically acceptable excipient or carrier.

29. A host cell (e.g., a mammalian cell, e.g., a human cell) comprising the gene modifying system, template RNA, or nucleic acid of any one of the preceding claims.

30. A method of making the nucleic acid or template RNA of any one of claims 1-25, the method comprising synthesizing the template RNA in vitro (e.g., by in vitro transcription or solid state synthesis).

31. A method for modifying a target site (c.g., a target site in the human SERPINA1 gene) in a cell, the method comprising contacting the cell with the gene modifying system of claim 27, or DNA encoding the same, or the pharmaceutical composition of claim 28, thereby modifying the target site.

32. A method for treating a subject having a disease or condition associated with a mutation in a gene (e.g., the human SERPINA1 gene), the method comprising administering to the subject the gene modifying system of claim 27, or DNA encoding the same, or the pharmaceutical composition of claim 28, thereby treating the subject having a disease or condition.

33. A method for treating a subject having AATD, the method comprising administering to the subject the gene modifying system of claim 27, or DNA encoding the same, or the pharmaceutical composition of claim 28, thereby treating the subject having AATD.

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