PAH Modulating Compositions and Methods

Abstract

The present disclosure provides compositions, systems and methods for targeting, editing, modifying or manipulating the genome of a host cell, for example, at one or more locations in the DNA sequence of a cell, tissue or subject. A gene modification system for treating phenylketonuria (PKU) is described.

Description

[Technical Field]

[0001] Sequence Listing This application has been submitted electronically in XML format conforming to WIPO Standard ST.26 and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The XML copy, created on September 8, 2022, is named V2065-7025WO_SL.xml and is 15,726,933 bytes in size.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 241,897, filed September 8, 2021, and U.S. Provisional Patent Application No. 63 / 367,025, filed June 24, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Integration of a nucleic acid of interest into a genome occurs at low frequency in the absence of specialized proteins to facilitate the insertion event and has little site specificity. Some existing methods, such as CRISPR / Cas9, are more suitable for small edits that rely on host repair pathways and are less effective at integrating long sequences. Other existing methods, such as Cre / loxP, require a first step of inserting a loxP site into the genome, followed by 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, modifying, or deleting a sequence of interest in a genome.

[0004] PKU is a genetic disorder involving an autosomal recessive inborn error of metabolism caused by a deficiency of the hepatic enzyme PAH. PAH catalyzes the hydroxylation of phenylalanine to tyrosine, the rate-limiting step in phenylalanine metabolism. This reaction depends on the cofactors tetrahydrobiopterin (BH4), molecular oxygen, and iron. Loss-of-function mutations in one or both copies of the PAH gene lead to a nonfunctional or inefficient enzyme. This ultimately results in a severe form of PKU, where phenylalanine accumulates in the blood to toxic levels, potentially resulting in decreased plasma tyrosine levels. In addition, this deficiency prevents the normal synthesis of downstream products, including dopamine, norepinephrine, and melanin.

[0005] The PAH genomic sequence and its flanking regions span approximately 171 kb and contain 13 exons. Studies of pathogenic allelic variants have identified over 500 different disease-causing mutations in the PAH gene (Mitchell, et al. Genet Med. 2011;13:697-707). Approximately 62% of these mutations have been characterized as missense, 13% as deletions, 11% as splices, 6% as silent, 5% as nonsense, 2% as insertions, and less than 1% as exon deletions or duplications. The effects of several PAH mutations on enzyme activity have been documented using enzyme kinetics and crystallography studies. Mutations affecting catalytic binding modes, including Y138F, S23A, and Y377F, have been observed to reduce the propensity for tetramer formation (Flydal, et al. PNAS. 2019;116(23):11229-34). Other residues that interact with BH4 in the precatalytic conformation (amino acids 245-255, 286, 322, and 325) also interact with BH4 in the catalytic conformation, and these sites are indeed involved in significant destabilization of PAH.

[0006] The distribution pattern of naturally occurring N-terminal PAH mutations has been determined to be nonrandom and clusters within residues 46–48 (the GAL motif) and 65–69 (the IESRP motif), both motifs highly conserved in pyruvate dehydrogenase (PDH) (Gjetting, et al. Am. / J. Hum. Genet. 2001;68:1353–60). Structure-function studies have demonstrated that mutations in these regions dramatically reduce phenylalanine binding. Many missense mutations identified to date in PKU result in phenotypic outcomes related to PAH enzyme misfolding, increased protein turnover, and loss of enzyme function. Residues in exons 7–9 and in the interdomain region within this subunit appear to play important structural roles and constitute hotspots for destabilization. Additionally, using recombinant forms of hPAH, mutations in the BH4-reactive domain, including R408W and Y414C, showed residual activity but disrupted allostery, suggesting altered protein conformation (Gersting, et al. Hum. Genet. 2008;83:5-17). Mutational and structure-function analyses have identified robust genotype-phenotype correspondences regarding the role of PAH in PKU. However, no successful cure has been observed beyond lifelong symptom management strategies.

[0007] Dietary phenylalanine (Phe) therapy has remained the mainstay of treatment for PKU since its adoption in 1953. In the 1970s, combination therapy with tetrahydrobiopterin (BH4) and neurotransmitter precursors (L-dopa / carbidopa and 5-hydroxytryptophan) showed promise in modulating PKU. Since its establishment, synthetic drugs such as sapropterin have been formulated as small molecule isomers of BH4. However, this form of therapy is generally only useful in a subset of patients with mild PAH-deficient PKU. Treatment response is thought to be related to mutations in the PAH gene that result in some residual enzyme activity. When blood levels of Phe are high, they compete with other large neutral amino acids (LNAAs) for transport across the blood-brain barrier. LNAA supplementation has been shown to reduce brain Phe concentrations, despite the observed increase in plasma Phe levels. Similarly, dietary supplementation with glycomacropeptide (GMP) has been observed to significantly reduce urea production and improve protein retention and Phe utilization, but these strategies rarely address elevated blood Phe levels or genotypic factors. Modern non-dietary approaches include the development of PAH-based fusion proteins and enzyme replacement therapy. Enzyme replacement therapy can involve administering phenylalanine ammonia lyase (PAL) to patients. PAL is an enzyme that catalyzes the conversion of Phe to trans-cinnamic acid and trace amounts of ammonia. Initial studies using PAL administered to PKU patients in enteric-coated gelatin capsules demonstrated a reduction in Phe levels, but repeated in vivo administration resulted in the development of an immune response. However, these approaches are clinically impractical because the limited half-life of the circulating enzyme may require multiple intravenous injections. Gene therapy, for example, using viral vectors, has shown some promise in rescuing PAH function. However, the effectiveness of this strategy is hampered by extremely low gene transfer rates and transient transgene expression. Therefore, new, more effective treatments for targeting PAH in PKU are needed. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Mitchell,et al.Genet Med.2011;13:697-707 [Non-patent document 2] Flydal,et al.PNAS.2019;116(23):11229-34 [Non-patent document 3] Gjetting,et al.Am. / J.Hum.Genet.2001;68:1353-60 [Non-patent document 4] Gersting,et al.Hum.Genet.2008;83:5-17 Summary of the Invention [Means for solving the problem]

[0009] The present disclosure relates to novel compositions, systems, and methods for modifying the genome of one or more locations in a host cell, tissue, or subject, either in vivo or in vitro. The present disclosure provides genetic modification systems capable of modulating phenylalanine hydroxylase (PAH) activity (e.g., inserting, modifying, or deleting sequences of interest), and methods for treating phenylketonuria (PKU) by modifying genomic sequences, such as correcting mutations, within the PAH gene on human chromosome 12q23.2, which is implicated as a driver gene in PKU, by administering one or more such systems.

[0010] In one aspect, the present disclosure relates to a system for modifying DNA to correct a human PAH gene mutation that causes PKU, the system comprising: (a) a nucleic acid encoding a genetically modified polypeptide capable of target-primed reverse transcription, the polypeptide comprising (i) a reverse transcriptase domain and (ii) a Cas9 nickase that binds to DNA and has endonuclease activity; and (b) a template RNA comprising: (i) a gRNA spacer complementary to a first portion of the human PAH gene; (ii) a gRNA scaffold that binds to the polypeptide; (iii) a heterologous target sequence comprising a mutation region for correcting the mutation; and (iv) a primer binding site (PBS) sequence comprising at least 3, 4, 5, 6, 7, or 8 bases of 100% homology to the target DNA strand at the 3' end of the template RNA. In some embodiments, the PAH gene may comprise a R408W mutation. In some embodiments, the PAH gene may comprise a R261Q mutation. In some embodiments, the PAH gene may comprise a R243Q mutation. In some embodiments, the PAH gene can include an IVS10-11G>A mutation. The template RNA sequence can include a sequence set forth herein, for example, in Table 1A, Table IB, Table 1C, Table ID, Table 3A, Table 3B, Table 3C, Table 3D, Table 4A, Table 4B, Table 4C, Table 4D, Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A.

[0011] The gRNA spacer may comprise at least 15 bases at the 5' end of the template RNA that are 100% homologous to the target DNA. The template RNA may further comprise a PBS sequence that comprises at least 5 bases that are at least 80% homologous to the target DNA strand. The template RNA may comprise one or more chemical modifications.

[0012] The domains of the genetically modified polypeptide can be connected by a peptide linker. The polypeptide can include one or more peptide linkers. The genetically modified polypeptide can further include a nuclear localization signal. The polypeptide can include two or more nuclear localization signals, for example, multiple adjacent nuclear localization signals or one or more nuclear localization signals in different regions of the polypeptide, for example, one or more nuclear localization signals at the N-terminus of the polypeptide and one or more nuclear localization signals at the C-terminus of the polypeptide. The nucleic acid encoding the genetically modified polypeptide can encode one or more intein domains.

[0013] Introduction of the system into a target cell can result in the 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 the system into a target cell can result in a deletion, where the deletion is less than 2, 3, 4, 5, 10, 50, or 100 base pairs of genomic DNA upstream or downstream of the insertion. Introduction of the system into a target cell can result in a substitution, for example, of 1, 2, or 3 nucleotides, for example, consecutive nucleotides.

[0014] The heterologous sequence of interest can be at least 5, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, or 700 base pairs.

[0015] In one aspect, the present disclosure relates to a pharmaceutical composition comprising the above-described system 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 one aspect, the present disclosure relates to a pharmaceutical composition comprising the above-described system and a plurality of 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, for example, wherein the above-described system is delivered by two different excipients or carriers, for example, two lipid nanoparticles, two viral vectors, or one lipid nanoparticle and one viral vector. The viral vector may be an adeno-associated virus (AAV).

[0016] In one aspect, the present disclosure relates to a host cell (e.g., a mammalian cell, e.g., a human cell) comprising the above-described system.

[0017] In one aspect, the present disclosure relates to a method for correcting a mutation in a human PAH gene in a cell, tissue, or subject, the method comprising administering the system described above to the cell, tissue, or subject, wherein optionally, the correction of the mutant PAH gene comprises an amino acid substitution of W408R, Q261R, and / or Q243R (reverting the pathogenic substitutions R408W, R261Q, or R243Q). In another aspect, the correction of the mutant PAH gene comprises a nucleic acid substitution of IVS10-11A>G (reverting the pathogenic substitution IVS10-11G>A). The system can be introduced in vivo, in vitro, ex vivo, or in situ. The nucleic acid of (a) can be integrated into the genome of a host cell. In some embodiments, the nucleic acid of (a) is not integrated into the genome of a host cell. In some embodiments, the heterologous sequence of interest is inserted at only one target site within the host cell genome. The heterologous sequence of interest can be inserted at two or more target sites within the host cell genome, for example, at the same corresponding sites on two homologous chromosomes or at two different sites on the same or different chromosomes. The heterologous sequence of interest can encode a mammalian polypeptide or a fragment or variant thereof. The components of the system can be delivered on one, two, three, four, or more different nucleic acid molecules. The system can be introduced into the 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.

[0018] Features of the compositions or methods may include one or more of the following enumerated embodiments.

[0019] Enumerated Embodiments 1. A template RNA, e.g., 5' to 3': (i) a gRNA spacer complementary to a first portion of a human PAH gene, the gRNA spacer having a sequence including the core nucleotide of a gRNA spacer sequence of Table 1A, Table IB, Table 1C, or Table 1D, or a sequence having one, two, or three substitutions therewith, and optionally including one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the gRNA spacer (e.g., including one or more flanking nucleotides adjacent to the core nucleotide), or having the sequence of a spacer selected from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A; (ii) a gRNA scaffold that binds to a genetically modified polypeptide (e.g., binds to a Cas domain of the genetically modified polypeptide); (iii) a heterologous sequence of interest comprising a mutation region for introducing a mutation into a second portion of the human PAH gene (e.g., for correcting a mutation therein), wherein optionally, the heterologous sequence of interest comprises, from 5' to 3', a post-edited homology region, a mutation region, and a pre-edited homology region; and (iv) a primer binding site (PBS) sequence containing at least 3, 4, 5, 6, 7, or 8 bases of 100% identity to the third portion of the human PAH gene; A template RNA containing

[0020] 2. The template RNA of embodiment 1, wherein the heterologous target sequence comprises the core nucleotide of an RT template sequence from Table 3A, Table 3B, Table 3C, or Table 3D, or a sequence having one, two, or three substitutions thereto, and optionally one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the RT template sequence; or wherein the heterologous target sequence comprises the sequence of an RT template sequence from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A.

[0021] 3. The template RNA of embodiment 1, wherein the heterologous target sequence comprises the core nucleotide of the RT template sequence of Table 3A, Table 3B, Table 3C, or Table 3D corresponding to the gRNA spacer sequence or a sequence having one, two, or three substitutions thereto, and optionally comprises one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the RT template sequence (e.g., comprising one or more flanking nucleotides adjacent to the core nucleotide), or wherein the heterologous target sequence comprises the sequence of an RT template sequence from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A.

[0022] 4. The template RNA of any one of embodiments 1 to 3, wherein the PBS sequence has a sequence that includes the core nucleotide of a PBS sequence from the same row of Table 3A, Table 3B, Table 3C, or Table 3D as the RT template sequence, or a sequence with one, two, or three substitutions thereto, and optionally includes one or more contiguous nucleotides starting from the 5' end of a flanking nucleotide of the PBS sequence (e.g., includes one or more flanking nucleotides adjacent to the core nucleotide).

[0023] 5. The template RNA of any one of embodiments 1-3, wherein the PBS sequence has a sequence that includes the core nucleotide of a PBS sequence of Table 3A, Table 3B, Table 3C or Table 3D corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence with one, two or three substitutions thereto, and optionally includes one or more contiguous nucleotides starting from the 5' end of a flanking nucleotide of the PBS sequence; or the PBS sequence comprises a PBS sequence from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence with one, two or three substitutions thereto.

[0024] 6. The template RNA of any one of embodiments 1-5, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold in Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0025] 7. The template RNA of any one of embodiments 1-5, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold in Table 12 corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0026] 8. A template RNA, for example, having from 5' to 3': (i) a gRNA spacer complementary to the first portion of the human PAH gene; (ii) a gRNA scaffold that binds to a genetically modified polypeptide (e.g., binds to a Cas domain of the genetically modified polypeptide); (iii) a heterologous target sequence comprising a mutation region for introducing a mutation into a second portion of the human PAH gene, the heterologous target sequence comprising the core nucleotide of an RT template sequence of Table 3A, Table 3B, Table 3C or Table 3D or a sequence having one, two or three substitutions thereto, and optionally comprising one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the RT template sequence, or a heterologous target sequence comprising an RT template sequence of Table 5A-5F, Table 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A; and (iv) a PBS sequence containing at least 3, 4, 5, 6, 7, or 8 bases that are 100% identical to the third portion of the human PAH gene. A template RNA containing

[0027] 9. The template RNA of embodiment 8, wherein the gRNA spacer comprises the core nucleotide of a gRNA spacer sequence of Table 1A, Table IB, Table 1C, or Table 1D, or a sequence having one, two, or three substitutions thereto, and optionally one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the gRNA spacer, or the gRNA spacer comprises a gRNA spacer sequence of Table 5A-5F, Table 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A.

[0028] 10. The template RNA of any one of embodiments 1 to 9, wherein the gRNA spacer comprises ACCTCAATCCTTTTGGGTGTA (SEQ ID NO: 16355) or a sequence having one, two, or three substitutions therein.

[0029] 11. The template RNA of any one of embodiments 1 to 9, wherein the gRNA spacer comprises CCTCAATCCTTTGGGTGTAT (SEQ ID NO: 16332) or a sequence having one, two, or three substitutions therein.

[0030] 12. The template RNA of any one of embodiments 1 to 9, wherein the gRNA spacer comprises TGGGTCGTAGCGAACTGAGA (SEQ ID NO: 16102) or a sequence having one, two, or three substitutions therein.

[0031] 13. The template RNA of any one of embodiments 1 to 9, wherein the gRNA spacer comprises GGGTCGTAGCGAACTGAGAA (SEQ ID NO: 16084) or a sequence having one, two, or three substitutions therein.

[0032] 14. The template RNA of any one of embodiments 1 to 9, wherein the gRNA spacer comprises TAGCGAACTGAGAAGGGCCA (SEQ ID NO: 16011) or a sequence having one, two, or three substitutions therein.

[0033] 15. The template RNA of any one of embodiments 1 to 9, wherein the gRNA spacer comprises ACTTGCTGCCACAATACCT (SEQ ID NO: 16032) or a sequence having one, two, or three substitutions therein.

[0034] 16. The template RNA of embodiment 8, wherein the heterologous sequence of interest comprises the core nucleotides of the gRNA spacer sequence of Table 1A, Table IB, Table 1C or Table 1D corresponding to the RT template sequence, or a sequence having one, two or three substitutions thereto, and optionally one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the gRNA spacer, or the heterologous sequence of interest comprises the nucleotides of the gRNA spacer sequence of Table 5A-5F, Table 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A corresponding to the RT template sequence, or a sequence having one, two or three substitutions thereto.

[0035] 17. The template RNA of any one of embodiments 8-16, wherein the PBS sequence has a sequence that includes the core nucleotide of a PBS sequence from the same row as the RT template sequence of Table 3A, Table 3B, Table 3C, or Table 3D, or a sequence with one, two, or three substitutions thereto, and optionally includes one or more contiguous nucleotides starting from the 5' end of a flanking nucleotide of the PBS sequence.

[0036] 18. The template RNA of any one of embodiments 8-16, wherein the PBS sequence has a sequence that includes the core nucleotide of a PBS sequence of Table 3A, Table 3B, Table 3C or Table 3D corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence with one, two or three substitutions thereto, and optionally includes one or more contiguous nucleotides starting from the 5' end of a flanking nucleotide of the PBS sequence; or the PBS sequence has a sequence that includes a PBS sequence of Table 5A-5F, Table 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A corresponding to the RT template sequence, the gRNA spacer sequence, or both.

[0037] 19. The template RNA of any one of embodiments 8-18, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold in Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0038] 20. The template RNA of any one of embodiments 8-18, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold in Table 12 corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0039] 21. A gene modification system for modifying DNA, comprising: (a) a first RNA comprising, from 5' to 3', (i) a guide RNA sequence complementary to a first portion of a human PAH gene, the guide RNA sequence comprising a core nucleotide of a spacer sequence in Table 1A, Table IB, Table 1C, or Table ID, or a sequence having one, two, or three substitutions therewith, and optionally comprising one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the guide RNA sequence, or a spacer sequence selected from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A; and (ii) a sequence (e.g., a scaffold region) that binds to a genetically modified polypeptide (e.g., binds to a Cas domain of a genetically modified polypeptide), and (b)(iii) a heterologous sequence of interest comprising a nucleotide substitution to introduce a mutation into a second portion of the human PAH gene (wherein, optionally, the heterologous sequence of interest comprises, from 5' to 3', a post-edited homology region, a mutation region, and a pre-edited homology region); (iv) a primer region comprising at least 5, 6, 7, or 8 bases of 100% identity to the third portion of the human PAH gene; and (v) a second RNA comprising an RRS (RNA-binding protein recognition sequence) that binds to the genetic modification protein. A genetic modification system comprising:

[0040] 22. The genetic modification system of embodiment 21, wherein the heterologous sequence of interest comprises the core nucleotide of an RT template sequence from Table 3A, Table 3B, Table 3C, or Table 3D, or a sequence having one, two, or three substitutions thereto, and optionally one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the RT template sequence, or wherein the heterologous sequence of interest comprises the sequence of an RT template sequence from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A, or a sequence having one, two, or three substitutions thereto.

[0041] 23. The genetic modification system of embodiment 21, wherein the heterologous sequence of interest comprises the core nucleotide of the RT template sequence of Table 3A, Table 3B, Table 3C or Table 3D corresponding to the gRNA spacer sequence, or a sequence having one, two or three substitutions thereto, and optionally one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the RT template sequence, or the heterologous sequence of interest comprises the sequence of the RT template sequence from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A corresponding to the gRNA spacer sequence, or a sequence having one, two or three substitutions thereto.

[0042] 24. The genetic modification system of any one of embodiments 21-23, wherein the PBS sequence has a sequence that includes the core nucleotide of a PBS sequence from the same row as the RT template sequence of Table 3A, Table 3B, Table 3C, or Table 3D, or a sequence with 1, 2, or 3 substitutions thereto, and optionally includes one or more contiguous nucleotides starting from the 5' end of a flanking nucleotide of the PBS sequence, or the PBS sequence has the sequence of a PBS sequence from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A, or a sequence with 1, 2, or 3 substitutions thereto.

[0043] 25. The genetic modification system of any one of embodiments 21-23, wherein the PBS sequence has a sequence that includes the core nucleotide of a PBS sequence of Table 3A, Table 3B, Table 3C or Table 3D corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence with one, two or three substitutions thereto, and optionally includes one or more contiguous nucleotides starting from the 5' end of a flanking nucleotide of the PBS sequence; or the PBS sequence has the sequence of a PBS sequence from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A corresponding to the RT template sequence, or a sequence with one, two or three substitutions thereto.

[0044] 26. The genetic modification system of any one of embodiments 21-25, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold in Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0045] 27. The genetic modification system of any one of embodiments 21-25, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold in Table 12 corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0046] 28. A gene modification system for modifying DNA, comprising: (a) a first RNA comprising, from 5' to 3', (i) a guide RNA sequence complementary to a first portion of a human PAH gene, and (ii) a sequence (e.g., a scaffold region) that binds to a genetically modified polypeptide (e.g., binds to a Cas domain of the genetically modified polypeptide); and (b)(iii) a heterologous target sequence comprising nucleotide substitutions to introduce mutations into a second portion of the human PAH gene, the heterologous target sequence comprising the core nucleotide of the RT template sequence of Table 3A, Table 3B, Table 3C or Table 3D or a sequence having one, two or three substitutions thereto, and optionally comprising one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the RT template sequence, or an RT sequence from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A or a sequence having one, two or three substitutions thereto; (iv) a primer region comprising at least 5, 6, 7 or 8 bases of 100% homology to the third portion of the human PAH gene; and (v) an RRS (RNA binding protein recognition sequence) that binds to the genetic modification protein. A genetic modification system comprising:

[0047] 29. The genetic modification system of embodiment 28, wherein the gRNA spacer comprises the core nucleotide of a gRNA spacer sequence of Table 1A, Table IB, Table 1C or Table 1D, or a sequence having one, two or three substitutions thereto, and optionally one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the gRNA spacer, or wherein the gRNA spacer comprises a gRNA spacer sequence from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A, or a sequence having one, two or three substitutions thereto.

[0048] 30. The genetic modification system of embodiment 28, wherein the heterologous sequence of interest comprises the core nucleotide of a gRNA spacer sequence of Table 1A, Table IB, Table 1C or Table 1D corresponding to the RT template sequence, or a sequence having one, two or three substitutions thereto, and optionally one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the gRNA spacer, or wherein the heterologous sequence of interest comprises a gRNA spacer sequence from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A corresponding to the RT template sequence, or a sequence having one, two or three substitutions thereto.

[0049] 31. The genetic modification system of any one of embodiments 28-30, wherein the PBS sequence has a sequence that includes the core nucleotide of a PBS sequence from the same row of Table 3A, Table 3B, Table 3C, or Table 3D as the RT template sequence, or a sequence with 1, 2, or 3 substitutions thereto, and optionally includes one or more contiguous nucleotides starting from the 5' end of a flanking nucleotide of the PBS sequence, or the PBS sequence has a sequence that includes a PBS sequence from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A, or a sequence with 1, 2, or 3 substitutions thereto.

[0050] 32. The genetic modification system of any one of embodiments 28-30, wherein the PBS sequence has a sequence that includes the core nucleotide of a PBS sequence of Table 3A, Table 3B, Table 3C or Table 3D corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence with one, two or three substitutions thereto, and optionally includes one or more contiguous nucleotides starting from the 5' end of a flanking nucleotide of the PBS sequence; or the PBS sequence has a sequence that includes a PBS sequence from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence with one, two or three substitutions thereto.

[0051] 33. The genetic modification system of any one of embodiments 28-32, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold in Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0052] 34. The genetic modification system of any one of embodiments 28-32, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold in Table 12 corresponding to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0053] 35. A gRNA comprising: (i) a gRNA spacer sequence complementary to a first portion of a human PAH gene, wherein the gRNA spacer has a sequence that includes the core nucleotides of a gRNA spacer sequence of Table 1A, Table IB, Table 1C or Table 1D, Table 2A, Table 2B, Table 2C or Table 2D, or Table 4A, Table 4B, Table 4C or Table 4D, or a sequence having one, two or three substitutions therein, and optionally includes one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the gRNA spacer, or a gRNA spacer sequence from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A, or a sequence having one, two or three substitutions therein; and (ii) a gRNA scaffold.

[0054] 36. The gRNA of embodiment 35, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold in Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0055] 37. The gRNA of embodiment 35, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold in Table 12 corresponding to the gRNA spacer sequence, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0056] 38. (iii) a template RNA comprising: (iii) a heterologous target sequence comprising a mutation region for introducing a mutation into a second portion of the human PAH gene, the heterologous target sequence comprising the core nucleotide of an RT template sequence of Table 3A, Table 3B, Table 3C or Table 3D or a sequence having one, two or three substitutions thereto, and optionally comprising one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the RT template sequence, or comprising an RT template sequence from Tables 5A to 5F, Tables 8A to 8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A or a sequence having one, two or three substitutions thereto; and (iv) a PBS sequence comprising at least 5, 6, 7 or 8 bases that are 100% homologous to the third portion of the human PAH gene.

[0057] 39. The template RNA of embodiment 38, wherein the PBS sequence has a sequence that includes the core nucleotide of a PBS sequence from the same row of Table 3A, Table 3B, Table 3C, or Table 3D as the RT template sequence, or a sequence with one, two, or three substitutions thereto, and optionally includes one or more contiguous nucleotides starting from the 5' end of a flanking nucleotide of the PBS sequence.

[0058] 40. The template RNA of embodiment 38, wherein the PBS sequence has a sequence that includes the core nucleotide of the PBS sequence of Table 3A, Table 3B, Table 3C or Table 3D that corresponds to the RT template sequence, or a sequence with one, two or three substitutions thereto, and optionally includes one or more contiguous nucleotides starting from the 5' end of a flanking nucleotide of the PBS sequence, or the PBS sequence comprises a PBS sequence from Tables 5A-5F, 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A that corresponds to the RT template sequence, or a sequence with one, two or three substitutions thereto.

[0059] 41. The template RNA of any one of embodiments 1 to 20 or 38 to 40, the genetic modification system of any one of embodiments 21 to 35, or the gRNA of any one of embodiments 35 to 37, wherein the mutation introduced by the system is a W408R, Q261R, Q243R and / or IVS10-11A>G mutation in the PAH gene (e.g., to correct a pathogenic R408W, R261Q, R243Q and / or IVS10-11G>A mutation).

[0060] 42. The template RNA of any one of embodiments 1 to 20 or 38 to 41 or the genetic modification system of any one of embodiments 35 to 37 or 41, wherein the pre-editing sequence comprises from about 1 nucleotide to about 35 nucleotides in length (e.g., from about 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, or 30 to 35 nucleotides in length).

[0061] 43. The template RNA of any one of embodiments 1 to 20 or 38 to 42 or the genetic modification system of any one of embodiments 35 to 37, 41, or 42, wherein the mutated region comprises a single nucleotide.

[0062] 44. The template RNA of any one of embodiments 1 to 20 or 38 to 42 or the genetic modification system of any one of embodiments 35 to 37, 41, or 42, wherein the mutation region is at least 2 nucleotides in length.

[0063] 45. The template RNA of any one of embodiments 1 to 20, 38 to 42, or 44, or the genetic modification system of any one of embodiments 35 to 37, 41, 42, or 44, wherein the mutation region is up to 32 (e.g., up to 5, 10, 15, 20, 25, 30, or 32) nucleotides in length and comprises 1, 2, or 3 sequence differences compared to the second portion of the human PAH gene.

[0064] 46. ​​The template RNA of any one of embodiments 1 to 20, 38 to 42, 44, or 45, or the genetic modification system of any one of embodiments 35 to 37, 41, 42, 44, or 45, wherein the mutated region comprises two sequence differences compared to the second part of the human PAH gene.

[0065] 47. The template RNA of any one of embodiments 1-20, 38-42, or 44-46, or the genetic modification system of any one of embodiments 35-37, 41, 42, or 44-46, wherein the mutation region comprises a first region (e.g., a first nucleotide) designed to correct a pathogenic mutation in the PAH gene and a second region (e.g., a second nucleotide) designed to inactivate the PAM sequence (e.g., a "PAM-kill" mutation as described herein).

[0066] 48. The template RNA of any one of embodiments 1 to 20 or 38 to 46 or the genetic modification system of any one of embodiments 35 to 37 or 41 to 46, wherein the mutated region comprises less than 80%, 70%, 60%, 50%, 40%, or 30% identity to the corresponding portion of the human PAH gene.

[0067] 49. The template RNA of any one of embodiments 1-48, wherein the template RNA comprises one or more silent mutations (e.g., silent substitutions), e.g., as exemplified in Tables 7A-7C, 8A-8D, E6, or E6A.

[0068] 50. The template RNA of any one of embodiments 1 to 49, wherein the mutation region comprises a first region designed to correct a pathogenic mutation in the PAH gene and a second region designed to introduce a silent substitution.

[0069] 51. The template RNA of any one of embodiments 1 to 50, comprising one or more chemically modified nucleotides.

[0070] 52. A genetic modification system comprising: The template RNA according to any one of embodiments 1 to 20, 38 to 46, or the system according to any one of embodiments 35 to 37, or 41 to 46, and Genetically modified polypeptides or nucleic acids (e.g., RNA) encoding genetically modified polypeptides A genetic modification system comprising:

[0071] 53. A genetically modified polypeptide, A reverse transcriptase (RT) domain (e.g., an RT domain from a retrovirus or a polypeptide domain having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto); and a Cas domain (e.g., a Cas9 domain) that binds to a target DNA molecule and is heterologous to the RT domain; and Optionally, a linker placed between the RT domain and the Cas domain 53. The genetic modification system of embodiment 52, comprising:

[0072] 54.RT domain is (a) an RT domain of Table 6; or (b) RT domains derived from murine leukemia virus (MMLV), porcine endogenous retrovirus (PERV); avian reticuloendotheliosis virus (AVIRE), feline leukemia virus (FLV), simian foamy virus (SFV) (e.g., SFV3L), bovine leukemia virus (BLV), Mason-Pfizer simian virus (MPMV), human foamy virus (HFV), or bovine foamy / syncytial virus (BFV / BSV). 54. The genetic modification system of embodiment 53, comprising:

[0073] 55. The genetic modification system of embodiment 53 or 54, wherein the Cas domain comprises a Cas domain of Table 7 or Table 8.

[0074] 56.Cas domain, (a) Cas9 domain; (b) an SpCas9 domain, a BlatCas9 domain, an Nme2Cas9 domain, a PnpCas9 domain, a SauCas9 domain, a SauCas9-KKH domain, a SauriCas9 domain, a SauriCas9-KKH domain, a ScaCas9-Sc++ domain, a SpyCas9 domain, a SpyCas9-NG domain, a SpyCas9-SpRY domain, or a St1Cas9 domain; and / or (c) The genetic modification system of any one of embodiments 53 to 55, wherein the Cas9 domain comprises an N670A mutation, an N611A mutation, an N605A mutation, an N580A mutation, an N588A mutation, an N872A mutation, an N863 mutation, an N622A mutation, or an H840A mutation.

[0075] 57. The genetic modification system of embodiment 56, wherein the Cas9 domain binds to a PAM sequence listed in Table 7 or Table 12.

[0076] 58. The gene modification system of embodiment 57, wherein the second portion of the human PAH gene overlaps with the PAM recognized by the Cas domain, e.g., the second portion of the human PAH gene is within the PAM or the PAM is within the second portion of the human PAH gene.

[0077] 59. The genetic modification system of any one of embodiments 52 to 58, wherein the gRNA spacer is a gRNA spacer according to Table 1A, Table IB, Table 1C or Table 1D, and the Cas domain comprises a Cas domain listed in the same row of Table 1A, Table IB, Table 1C or Table 1D, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0078] 60. The genetic modification system of any one of embodiments 52-58, wherein the template RNA comprises a template RNA sequence of Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0079] 61. (a) the template RNA comprises the sequence of a template RNA sequence of Table 3A to Table 3D, Table 5A to Table 5F, Table 8A to Table 8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A; (b) the Cas domain comprises a Cas domain of Table 7 or Table 8; (c) the linker comprises a linker sequence in Table 10 (e.g., any of SEQ ID NOs: 5217, 5106, 5190, and 5218); and (d) The genetic modification system of any one of embodiments 52 to 60, wherein the genetically modified polypeptide comprises one or two NLS sequences from Table 11 (e.g., any of SEQ ID NOs: 5245, 5290, 5323, 5330, 5349, 5350, 5351, and 4001).

[0080] 62. A genetic modification system described in any one of embodiments 52 to 61, which creates a first nick in the first strand of the human PAH gene.

[0081] 63. The gene modification system of embodiment 62, further comprising a second strand-targeting gRNA that directs a second nick in the second strand of the human PAH gene.

[0082] 64. The second strand targeting gRNA is (i) a sequence comprising the core nucleotides of a left gRNA spacer sequence or a right gRNA spacer sequence from Table 2A, Table 2B, Table 2C, or Table 2D, and optionally comprising one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the left gRNA spacer sequence or the right gRNA spacer sequence; or (ii) a second strand targeting gRNA comprising a spacer sequence of Table 6A or a spacer sequence having one, two, or three substitutions thereto; 64. The genetic modification system of embodiment 63, comprising:

[0083] 65. The genetic modification system of embodiment 63, wherein the second strand targeting gRNA comprises a sequence comprising the core nucleotide of a left gRNA spacer sequence or a right gRNA spacer sequence from Table 2A, Table 2B, Table 2C, or Table 2D corresponding to the gRNA spacer sequence of (i), and optionally one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the left gRNA spacer sequence or the right gRNA spacer sequence.

[0084] 66. The second strand targeting gRNA is (i) a sequence comprising the core nucleotides of a second nicked gRNA sequence from Table 4A, Table 4B, Table 4C, Table 4D, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and optionally one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the second nicked gRNA sequence; or (ii) the genetic modification system of embodiment 63, comprising a second strand targeting gRNA comprising a spacer sequence from Table 6A or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0085] 67. The genetic modification system of embodiment 63, wherein the second strand targeting gRNA comprises a sequence comprising the core nucleotides of the second nicked gRNA sequence from Table 4A, Table 4B, Table 4C, or Table 4D corresponding to the gRNA spacer sequence of (i), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and optionally one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the second nicked gRNA sequence.

[0086] 68. The genetic modification system of any one of embodiments 52-67, wherein the second strand targeting gRNA has a "PAM-in orientation" with the template RNA of the genetic modification system, e.g., as illustrated in Tables 2A-2D, 4A-4D, or 6A.

[0087] 69. A genetic modification system described in any one of embodiments 52 to 68, wherein the second strand targeting gRNA targets a sequence that overlaps with the target mutation in the template RNA.

[0088] 70. The second strand targeting gRNA is (i) a sequence complementary to the PAH mutation (e.g., a spacer sequence); (ii) a sequence complementary to the wild-type sequence at the target locus (e.g., a spacer sequence); (iii) a sequence (e.g., a spacer sequence) complementary to a SNP proximal to the target locus, e.g., a SNP contained in the genomic DNA of a subject (e.g., a patient); (iv) a sequence (e.g., a spacer sequence) that is complementary to or includes one or more silent substitutions proximal to the target locus. 70. The genetic modification system of embodiment 69, comprising:

[0089] 71. The template RNA, genetic modification system, or gRNA of any one of embodiments 1 to 70, wherein the gRNA spacer comprises about 1, 2, 3 or more flanking nucleotides of the gRNA spacer.

[0090] 72. The template RNA or gene modification system according to any one of embodiments 1 to 71, wherein the heterologous sequence of interest comprises about 2, 3, 4, 5, 10, 20, 30, 40 or more flanking nucleotides of the RT template sequence.

[0091] 73. The template RNA or gene modification system according to any one of embodiments 1 to 72, wherein the heterologous sequence of interest comprises about 8 to 30, 9 to 25, 10 to 20, 11 to 16, or 12 to 15 (e.g., about 11 to 16) nucleotides.

[0092] 74. The template RNA or gene modification system according to any one of embodiments 1 to 73, wherein the mutation region comprises 1, 2, or 3 nucleotide positions that differ in sequence compared to the corresponding portion of the human PAH gene.

[0093] 75. The template RNA or gene modification system according to any one of embodiments 1 to 74, wherein the mutation region comprises at least two nucleotide positions that differ in sequence compared to the corresponding portion of the human PAH gene.

[0094] 76. A template RNA or gene modification system described in any one of embodiments 1 to 75, wherein the post-edited homology region and / or the pre-edited homology region comprises 100% identity to the PAH gene.

[0095] 77. The template RNA or gene modification system according to any one of embodiments 1 to 76, wherein the PBS sequence additionally comprises about 1, 2, 3, 4, 5, 6, 7 or more flanking nucleotides.

[0096] 78. The template RNA or gene modification system according to any one of embodiments 1 to 77, wherein the PBS sequence comprises about 5 to 20, 8 to 16, 8 to 14, 8 to 13, 9 to 13, 9 to 12, or 10 to 12 (e.g., about 9 to 12) nucleotides.

[0097] 79. The template RNA or gene modification system according to any one of embodiments 1 to 78, wherein the PBS sequence is bound within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the nick site in the PAH gene.

[0098] 80. A genetic modification system according to any one of embodiments 1 to 79, wherein the domains of the genetically modified polypeptide are joined by peptide linkers.

[0099] 81. The genetic modification system of embodiment 80, wherein the linker comprises the sequence of a linker in Table 10 (e.g., any of SEQ ID NOs: 5217, 5106, 5190, and 5218).

[0100] 82. The genetic modification system of any one of embodiments 1 to 81, wherein the genetically modified polypeptide further comprises one or more nuclear localization sequences (NLS).

[0101] 83. The genetic modification system of embodiment 82, wherein the genetically modified polypeptide comprises a first NLS and a second NLS.

[0102] 84. The genetic modification system of embodiment 82 or 83, wherein the NLS comprises the sequence of an NLS in Table 11 (e.g., any of SEQ ID NOs: 5245, 5290, 5323, 5330, 5349, 5350, 5351, and 4001).

[0103] 85. A template RNA comprising a template RNA sequence of Table 4A-4D, Table 5A-5F, Table 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0104] 86. A template RNA comprising the sequence of a template RNA of Table 4A to Table 4D, Table 5A to Table 5F, Table 8A to Table 8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A.

[0105] 87. A genetic modification system comprising: (i) a template RNA comprising a template RNA sequence of Table 4A, Table 4B, Table 4C, or Table 4D, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and (ii) a second nicked gRNA sequence from the same row as (i) of Table 4A, Table 4B, Table 4C, or Table 4D, to which it has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity. A genetic modification system comprising:

[0106] 88. A gene modification system comprising: (i) a template RNA comprising the sequence of a template RNA of Table 4A, Table 4B, Table 4C, or Table 4D; and (ii) a second nicked gRNA sequence from the same row as (i) of Table 4A, Table 4B, Table 4C, or Table 4D; A genetic modification system comprising:

[0107] 89. A DNA encoding the template RNA of any one of embodiments 1 to 20, 38 to 48, 71 to 79, 85 or 86, or the gRNA of any one of embodiments 34 to 37.

[0108] 90. A pharmaceutical composition comprising a system according to any one of embodiments 49 to 84, 87 or 88 or one or more nucleic acids encoding same, and a pharmaceutically acceptable excipient or carrier.

[0109] 91. The pharmaceutical composition according to embodiment 90, 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.

[0110] 92. The pharmaceutical composition of embodiment 91, wherein the viral vector is an adeno-associated virus.

[0111] 93. A host cell (e.g., a mammalian cell, e.g., a human cell) comprising a template RNA or a gene modification system according to any one of embodiments 1 to 92.

[0112] 94. A method for producing a template RNA according to any one of embodiments 1-20, 38-48, 71-79, 85, or 86, comprising synthesizing the template RNA by in vitro transcription (e.g., solid-state synthesis) or by introducing DNA encoding the template RNA into a host cell under conditions that allow for the production of the template RNA.

[0113] 95. A method for modifying a target site in a human PAH gene in a cell, comprising contacting the cell with a gene modification system described in any one of embodiments 49 to 84, 87 or 88 or DNA encoding same, thereby modifying the target site in the human PAH gene in the cell.

[0114] 96. A method for modifying a target site in a human PAH gene in a cell, comprising contacting the cell with (i) a template RNA or a DNA encoding the same described in any one of embodiments 49 to 84, 87, or 88; and (ii) a genetically modified polypeptide or a nucleic acid encoding the genetically modified polypeptide, thereby modifying the target site in the human PAH gene in the cell.

[0115] 97. A method for treating a subject having a disease or condition associated with a mutation in the human PAH gene, comprising administering to the subject a genetic modification system described in any one of embodiments 49 to 84, 87, or 88, or a DNA encoding the same, thereby treating the subject having a disease or condition associated with a mutation in the human PAH gene.

[0116] 98. A method for treating a subject having a disease or condition associated with a mutation in the human PAH gene, comprising administering to the subject (ii) a template RNA or a DNA encoding the same described in any one of embodiments 49 to 84, 87, or 88; and (ii) a genetically modified polypeptide or a nucleic acid encoding the genetically modified polypeptide, thereby treating the subject having a disease or condition associated with a mutation in the human PAH gene.

[0117] 99. The method of embodiment 97 or 98, wherein the disease or condition is phenylketonuria (PKU).

[0118] 100. The method of any one of embodiments 97-99, wherein the subject has the R408W, R261Q, R243Q and / or IVS10-11G>A mutation.

[0119] 101. A method for treating a subject with PKU, comprising administering to the subject a genetic modification system described in any one of embodiments 49 to 84, 87 or 88 or DNA encoding the same, thereby treating the subject with PKU.

[0120] 102. A method for treating a subject with PKU, comprising administering to the subject (i) a template RNA or a DNA encoding the same described in any one of embodiments 49 to 84, 87 or 88, and (ii) a genetically modified polypeptide or a nucleic acid encoding the genetically modified polypeptide, thereby treating the subject with PKU.

[0121] 103. A genetic modification system or method according to any one of embodiments 1 to 102, wherein introduction of the system into target cells results in correction of a pathogenic mutation in the PAH gene.

[0122] 104. The genetic modification system or method of any one of embodiments 1 to 103, wherein the pathogenic mutation is a W408R, Q261R, Q243R and / or IVS10-11A>G mutation, and the correction comprises an amino acid substitution of R408W, R261Q, or R243Q or a nucleic acid substitution of IVS10-11G>A.

[0123] 105. The genetic modification system or method of any one of embodiments 1 to 104, wherein the mutation correction occurs in at least 30% (e.g., 30%, 40%, 50%, 60%, 70% or more) of the target nucleic acids.

[0124] 106. A genetic modification system or method described in any one of embodiments 1 to 105, wherein correction of the mutation occurs in at least 30% (e.g., 30%, 40%, 50%, 60%, 70% or more) of the target cells.

[0125] 107. The genetic modification system or method of any one of embodiments 1 to 106, wherein the genetic modification system comprises a second strand-targeting gRNA and wherein mutation correction in the target cell population is increased compared to a target cell population treated with a genetic modification system comprising a template RNA without the second strand-targeting gRNA.

[0126] 108. The genetic modification system or method of any one of embodiments 1-107, wherein the template RNA comprises one or more silent substitutions (e.g., as exemplified in Table 7A, Table X4, and Table X4A), and mutation correction in the target cell population is increased compared to a target cell population treated with a genetic modification system comprising template RNA that does not comprise the one or more silent substitutions.

[0127] 109. The method of any one of embodiments 1-108, wherein the cell is a mammalian cell, such as a human cell.

[0128] 110. The method of any one of embodiments 1-109, wherein the subject is a human.

[0129] 111. The method of any one of embodiments 1-110, wherein the contacting is performed ex vivo, for example, the DNA of the cell or the subject is modified ex vivo.

[0130] 112. The method of any one of embodiments 1-111, wherein the contacting is performed in vivo, for example, the DNA of the cell or the subject is modified in vivo.

[0131] 113. The method of any one of embodiments 1 to 112, wherein contacting the cell or the subject with the system comprises contacting the cell or a cell in the subject with a nucleic acid (e.g., DNA or RNA) encoding the genetically modified polypeptide under conditions that allow production of the genetically modified polypeptide.

[0132] Additional Enumerated Embodiments A1.5' to 3', (i) a gRNA spacer complementary to a first portion of a human PAH gene, the gRNA spacer having a nucleotide sequence comprising ACCTCAATCCTTTTGGGTGTA (SEQ ID NO: 16355), or a nucleotide sequence having one, two, or three substitutions therein; (ii) a gRNA scaffold that binds to the Cas domain of a gene-modifying polypeptide; (iii) a heterologous sequence of interest comprising a mutation region for correcting a mutation in a second portion of the human PAH gene; and (iv) a primer binding site (PBS) sequence containing at least 5 bases of 100% identity to the third portion of the human PAH gene; A template RNA containing

[0133] A2. The template RNA of embodiment A1, wherein the gRNA spacer has a nucleotide sequence comprising ACCTCAATCCTTTTGGGTGTA (sequence number 16355).

[0134] A3. The template RNA of embodiment A1 or A2, wherein the heterologous sequence of interest comprises a sequence of at least 30 nucleotides from the 3' end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccatac (SEQ ID NO: 24984), or a sequence having 1, 2, 3, or 4 substitutions thereto.

[0135] A4. The template RNA of any one of embodiments A1 to A3, wherein the heterologous sequence of interest comprises the sequence tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccatac (SEQ ID NO: 24984), or a sequence having at least 40, 50, 60, or 70 nucleotides from the 3' end of said sequence or 1, 2, 3, or 4 substitutions thereto.

[0136] A5. The template RNA of any one of embodiments A1 to A4, wherein the heterologous sequence of interest comprises a sequence of at least 30 nucleotides from the 3' end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccatac (SEQ ID NO: 24984).

[0137] A6. The template RNA of any one of embodiments A1 to A5, wherein the heterologous sequence of interest comprises the sequence tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccatac (SEQ ID NO: 24984), or at least 40, 50, 60, or 70 nucleotides from the 3' end of said sequence.

[0138] A7. The template RNA of any one of embodiments A1 to A6, wherein the PBS comprises a sequence of at least 5, 6, or 8 nucleotides from the 5' end of a sequence according to acccaaagg (SEQ ID NO: 37628), or a sequence having one substitution therein.

[0139] A8. The template RNA of any one of embodiments A1 to A7, wherein the PBS comprises the sequence acccaaagg (SEQ ID NO: 37628).

[0140] A9.5' to 3', (i) a gRNA spacer complementary to a first portion of a human PAH gene, the gRNA spacer having a nucleotide sequence comprising CCTCAATCCTTTGGGTGTAT (SEQ ID NO: 16332), or a nucleotide sequence having one, two, or three substitutions therein; (ii) a gRNA scaffold that binds to the Cas domain of the gene-modifying polypeptide; (iii) a heterologous sequence of interest comprising a mutation region for correcting a mutation in a second portion of the human PAH gene; and (iv) a primer binding site (PBS) sequence containing at least 5 bases of 100% identity to the third portion of the human PAH gene; A template RNA containing

[0141] A10. The template RNA of embodiment A9, wherein the gRNA spacer has a nucleotide sequence comprising CCTCAATCCTTTGGGTGTAT (sequence number 16332).

[0142] A11. The template RNA of any one of embodiments A1 to A10, wherein the heterologous sequence of interest comprises a sequence of at least 50 nucleotides from the 3' end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccata (SEQ ID NO: 24975), or a sequence having 1, 2, 3, or 4 substitutions thereto.

[0143] A12. The template RNA of any one of embodiments A1 to A11, wherein the heterologous sequence of interest comprises the sequence tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccata (SEQ ID NO: 24975), or a sequence having at least 60 or 70 nucleotides from the 3' end of said sequence or 1, 2, 3, or 4 substitutions thereto.

[0144] A13. The template RNA of any one of embodiments A1 to A12, wherein the heterologous sequence of interest comprises a sequence of at least 50 nucleotides from the 3' end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccata (SEQ ID NO: 24975).

[0145] A14. The template RNA of any one of embodiments A1 to A13, wherein the heterologous sequence of interest comprises the sequence tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccata (SEQ ID NO: 24975), or at least 60 or 70 nucleotides from the 3' end of said sequence.

[0146] A15. The template RNA of any one of embodiments A1 to A14, wherein the PBS comprises a sequence of at least 5, 6, 7, or 8 nucleotides from the 5' end of a sequence according to cacccaaag (SEQ ID NO: 37629) or a sequence having one substitution therein.

[0147] A16. The template RNA of any one of embodiments A1 to A15, wherein the PBS comprises the sequence cacccaaag (SEQ ID NO: 37629).

[0148] A17.5' to 3' (i) a gRNA spacer complementary to a first portion of a human PAH gene, the gRNA spacer having a nucleotide sequence comprising TGGGTCGTAGCGAACTGAGA (SEQ ID NO: 16102), or a nucleotide sequence having one, two, or three substitutions therein; (ii) a gRNA scaffold that binds to the Cas domain of the gene-modifying polypeptide; (iii) a heterologous sequence of interest comprising a mutation region for correcting a mutation in a second portion of the human PAH gene; and (iv) a primer binding site (PBS) sequence containing at least 5 bases of 100% identity to the third portion of the human PAH gene; A template RNA containing

[0149] A18. The template RNA of embodiment A17, wherein the gRNA spacer has a nucleotide sequence comprising TGGGTCGTAGCGAACTGAGA (SEQ ID NO: 16102).

[0150] A19. The template RNA of any one of embodiments A1 to A18, wherein the heterologous sequence of interest comprises a sequence of at least 10 nucleotides from the 3' end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttct (SEQ ID NO: 24863), or a sequence having 1, 2, 3, or 4 substitutions thereto.

[0151] A20. The template RNA of any one of embodiments A1 to A19, wherein the heterologous sequence of interest comprises the sequence tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttct (SEQ ID NO: 24863), or a sequence having at least 20, 30, 40, or 50 nucleotides from the 3' end of said sequence or 1, 2, 3, or 4 substitutions thereto.

[0152] A21. The template RNA of any one of embodiments A1 to A20, wherein the heterologous sequence of interest comprises a sequence of at least 10 nucleotides from the 3' end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttct (SEQ ID NO: 24863).

[0153] A22. The template RNA of any one of embodiments A1 to A21, wherein the heterologous sequence of interest comprises the sequence tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttct (SEQ ID NO: 24863), or at least 20, 30, 40, or 50 nucleotides from the 3' end of said sequence.

[0154] A23. The template RNA of any one of embodiments A1 to A22, wherein the PBS comprises a sequence of at least 5, 6, 7, or 8 nucleotides from the 5' end of a sequence according to cagttcgct (SEQ ID NO: 37630), or a sequence having one substitution therein.

[0155] A24. The template RNA of any one of embodiments A1 to A23, wherein the PBS comprises the sequence cagttcgct (SEQ ID NO: 37630).

[0156] A25.5' to 3', (i) a gRNA spacer complementary to a first portion of a human PAH gene, the gRNA spacer having a nucleotide sequence comprising GGGTCGTAGCGAACTGAGAA (SEQ ID NO: 16084), or a nucleotide sequence having one, two, or three substitutions therein; (ii) a gRNA scaffold that binds to the Cas domain of the gene-modifying polypeptide; (iii) a heterologous sequence of interest comprising a mutation region for correcting a mutation in a second portion of the human PAH gene; and (iv) a primer binding site (PBS) sequence containing at least 5 bases of 100% identity to the third portion of the human PAH gene; A template RNA containing

[0157] A26. The template RNA of embodiment A25, wherein the gRNA spacer has a nucleotide sequence comprising GGGTCGTAGCGAACTGAGAA (SEQ ID NO: 16084).

[0158] A27. The template RNA of any one of embodiments A1 to A26, wherein the heterologous sequence of interest comprises a sequence of at least 9 nucleotides from the 3' end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttc (SEQ ID NO: 24856), or a sequence having 1, 2, 3, or 4 substitutions thereto.

[0159] A28. The template RNA of any one of embodiments A1 to A27, wherein the heterologous sequence of interest comprises the sequence tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttc (SEQ ID NO: 24856), or a sequence having at least 10, 20, 30, 40, or 50 nucleotides from the 3' end of said sequence or 1, 2, 3, or 4 substitutions therein.

[0160] A29. The template RNA of any one of embodiments A1 to A28, wherein the heterologous sequence of interest comprises a sequence of at least 9 nucleotides from the 3' end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttc (SEQ ID NO: 24856).

[0161] A30. The template RNA of any one of embodiments A1 to A29, wherein the heterologous sequence of interest comprises the sequence tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttc (SEQ ID NO: 24856), or at least 10, 20, 30, 40, or 50 nucleotides from the 3' end of said sequence.

[0162] A31. The template RNA of any one of embodiments A1 to A30, wherein the PBS comprises a sequence of at least 5, 6, 7, or 8 nucleotides from the 5' end of a sequence according to tcagttcgc (SEQ ID NO: 37631), or a sequence having one substitution therein.

[0163] A32. The template RNA of any one of embodiments A1 to A31, wherein the PBS comprises the sequence tcagttcgc (SEQ ID NO: 37631).

[0164] A33.5' to 3', (i) a gRNA spacer complementary to a first portion of a human PAH gene, the gRNA spacer having a nucleotide sequence comprising TAGCGAACTGAGAAGGGCCA (SEQ ID NO: 16011), or a nucleotide sequence having one, two, or three substitutions therein; (ii) a gRNA scaffold that binds to the Cas domain of a gene-modifying polypeptide; (iii) a heterologous sequence of interest comprising a mutation region for correcting a mutation in a second portion of the human PAH gene; and (iv) a primer binding site (PBS) sequence containing at least 5 bases of 100% identity to the third portion of the human PAH gene; A template RNA containing

[0165] A34. The template RNA of embodiment A33, wherein the gRNA spacer has a nucleotide sequence comprising TAGCGAACTGAGAAGGGCCA (SEQ ID NO: 16011).

[0166] A35. The template RNA of any one of embodiments A1 to A34, wherein the heterologous sequence of interest comprises a sequence of at least 3 nucleotides from the 3' end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCgg (SEQ ID NO: 24817), or a sequence having 1, 2, 3, or 4 substitutions thereto.

[0167] A36. The template RNA of any one of embodiments A1 to A35, wherein the heterologous sequence of interest comprises the sequence tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCgg (SEQ ID NO: 24817), or a sequence having at least 5, 10, 20, 30, 40, or 50 nucleotides from the 3' end of said sequence or 1, 2, 3, or 4 substitutions therein.

[0168] A37. The template RNA of any one of embodiments A1 to A36, wherein the heterologous sequence of interest comprises a sequence of at least 3 nucleotides from the 3' end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCgg (SEQ ID NO: 24817).

[0169] A38. The template RNA of any one of embodiments A1 to A37, wherein the heterologous sequence of interest comprises the sequence tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCgg (SEQ ID NO: 24817), or at least 5, 10, 20, 30, 40, or 50 nucleotides from the 3' end of said sequence.

[0170] A39. The template RNA of any one of embodiments A1 to A38, wherein the PBS comprises a sequence of at least 5, 6, 7, or 8 nucleotides from the 5' end of a sequence according to cccttctca (SEQ ID NO: 37632), or a sequence having one substitution therein.

[0171] A40. The template RNA of any one of embodiments A1 to A39, wherein the PBS comprises the sequence cccttctca (SEQ ID NO: 37632).

[0172] A41.5' to 3', (i) a gRNA spacer complementary to a first portion of a human PAH gene, the gRNA spacer having a nucleotide sequence comprising ACTTGCTGCCACAATACCT (SEQ ID NO: 16032), or a nucleotide sequence having one, two, or three substitutions therein; (ii) a gRNA scaffold that binds to the Cas domain of a gene-modifying polypeptide; (iii) a heterologous sequence of interest comprising a mutation region for correcting a mutation in a second portion of the human PAH gene; and (iv) a primer binding site (PBS) sequence containing at least 5 bases of 100% identity to the third portion of the human PAH gene; A template RNA containing

[0173] A42. The template RNA of embodiment A41, wherein the gRNA spacer has a nucleotide sequence comprising ACTTGCTGCCACAATACCT (SEQ ID NO: 16032).

[0174] A43. The template RNA of any one of embodiments A1 to A42, wherein the heterologous sequence of interest comprises a sequence of at least 4 nucleotides from the 3' end of a sequence according to caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggccGagg (SEQ ID NO: 24825), or a sequence having 1, 2, 3, or 4 substitutions thereto.

[0175] A44. The template RNA of any one of embodiments A1 to A43, wherein the heterologous sequence of interest comprises the sequence caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggccGagg (SEQ ID NO: 24825), or a sequence having at least 5, 10, 20, 30, 40, or 50 nucleotides from the 3' end of said sequence or 1, 2, 3, or 4 substitutions thereto.

[0176] A45. The template RNA of any one of embodiments A1 to A44, wherein the heterologous sequence of interest comprises a sequence of at least 4 nucleotides from the 3' end of a sequence according to caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggccGagg (SEQ ID NO: 24825).

[0177] A46. The template RNA of any one of embodiments A1 to A45, wherein the heterologous sequence of interest comprises the sequence caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggccGagg (SEQ ID NO: 24825), or at least 5, 10, 20, 30, 40, or 50 nucleotides from the 3' end of said sequence.

[0178] A47. The template RNA of any one of embodiments A1 to A46, wherein the PBS comprises a sequence of at least 5, 6, 7, 8, 9, 10, or 15 nucleotides from the 5' end of a sequence according to tattgtggcagcaaagt (SEQ ID NO: 37633), or a sequence having one substitution therein.

[0179] A48. The template RNA of any one of embodiments A1 to A47, wherein the PBS comprises the sequence tattgtggcagcaaagt (SEQ ID NO: 37633).

[0180] A49. The template RNA of any one of embodiments A1 to A48, wherein the gRNA scaffold has a sequence according to GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 37627), or a sequence with at least 90% identity thereto.

[0181] A50. The template RNA of any one of embodiments A1 to A49, wherein the gRNA scaffold has a sequence according to GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 37627).

[0182] A51. The template RNA of any one of embodiments A1 to A50, wherein the mutation region comprises a single nucleotide.

[0183] A52. The template RNA of any one of embodiments A1 to A51, wherein the mutation region is at least 2 nucleotides in length.

[0184] A53. The template RNA of any one of embodiments A1 to A52, wherein the mutation region is up to 20 nucleotides in length and comprises 1, 2, or 3 sequence differences compared to the second portion of the human PAH gene.

[0185] A54. The template RNA of any one of embodiments A1 to A53, wherein the mutation region comprises a first region designed to correct a pathogenic mutation in the PAH gene and a second region designed to inactivate the PAM sequence.

[0186] A55. The template RNA of any one of embodiments A1 to A54, wherein the mutation region comprises a first region designed to correct a pathogenic mutation in the PAH gene and a second region designed to introduce a silent substitution.

[0187] A56. The template RNA of any one of embodiments A1 to A55, wherein the template RNA is configured to edit the pathogenic R408W mutation in the human PAH gene.

[0188] A57. The template RNA of embodiment A56, wherein said template RNA is configured to convert the R408W mutation to arginine.

[0189] A58. The template RNA of any one of embodiments A1 to A57, comprising one or more chemically modified nucleotides.

[0190] A59. A genetic modification system, A template RNA according to any one of embodiments A1 to A58, and Genetically modified polypeptides or nucleic acids encoding genetically modified polypeptides A genetic modification system comprising:

[0191] A60. The genetic modification system of embodiment A59, wherein the genetically modified polypeptide comprises an RT domain having a sequence according to SEQ ID NO: 8,003 or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0192] A61. The genetic modification system of embodiment A59, wherein the genetically modified polypeptide comprises an RT domain having a sequence according to SEQ ID NO: 8,020, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0193] A62. The genetic modification system of embodiment 5A9, wherein the genetically modified polypeptide comprises an RT domain having a sequence according to SEQ ID NO: 8,074 or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0194] A63. The genetic modification system of embodiment A59, wherein the genetically modified polypeptide comprises an RT domain having a sequence according to SEQ ID NO: 8,113, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0195] A64. The genetic modification system of embodiment A59, wherein the genetically modified polypeptide comprises a DNA-binding domain having a sequence of a Cas9 nickase comprising an N863A mutation, e.g., a sequence according to SEQ ID NO: 11,096, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0196] A65. The genetic modification system of embodiment A59, which creates a first nick in the first strand of the human PAH gene.

[0197] A66. The genetic modification system of embodiment A65, further comprising a second strand-targeting gRNA that directs a second nick into the second strand of the human PAH gene.

[0198] A67. The genetic modification system of embodiment A66, wherein the first nick and the second nick are separated by 80 to 120 nucleotides.

[0199] A68. The genetic modification system of embodiment A66, wherein the template RNA and second strand targeting gRNA are configured to create an outside nick configuration.

[0200] A69. The gene modification system of embodiment A66, wherein the second strand targeting gRNA comprises a spacer sequence complementary to a human PAH gene harboring a disease mutation or a wild-type sequence.

[0201] A70. A method for modifying a target site in a human PAH gene in a cell, comprising contacting the cell with a genetic modification system described in embodiment 59, thereby modifying the target site in the human PAH gene in the cell.

[0202] A71. The method of embodiment A70, wherein correction of the mutation occurs in at least 30% of the target nucleic acids.

[0203] A72. A method of treating a subject having a disease or condition associated with a mutation in the human PAH gene, wherein the disease or condition is phenylketonuria (PKU) or hyperphenylalaninemia (e.g., mild or severe hyperphenylalaninemia), the method comprising administering to the subject a genetic modification system described in embodiment 59, thereby treating the subject having the disease or condition associated with a mutation in the human PAH gene.

[0204] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0205] [Figure 1] 1 illustrates a genetic modification system as described herein. The diagram on the left shows a genetic modification polypeptide, which includes a Cas nickase domain (e.g., spCas9 N863A) and a reverse transcriptase domain (RT domain) connected by a linker. The diagram on the right shows a template RNA, which includes, from 5' to 3', a gRNA spacer, a gRNA scaffold, a heterologous sequence of interest, and a primer binding site sequence (PBS sequence). The heterologous sequence of interest may include a mutation region containing one or more sequence differences compared to the target site. The heterologous sequence of interest may also include pre-editing and post-editing homology regions flanking the mutation region. Without wishing to be bound by theory, it is believed that the gRNA spacer of the template RNA binds to the second strand of the target site in the genome, and the gRNA scaffold of the template RNA binds to the genetic modification polypeptide, thereby positioning the genetic modification polypeptide to the target site in the genome, for example. The Cas domain of the genetically modified polypeptide is believed to nick the target site (e.g., the first strand of the target site), thereby allowing, for example, the PBS sequence to bind to a sequence on the first strand of the target site adjacent to the site to be modified. The RT domain of the genetically modified polypeptide is believed to polymerize a sequence complementary to the heterologous target sequence, for example, by using the first strand of the target site, which binds to a complementary sequence containing the PBS sequence of the template RNA, as a primer, and the heterologous target sequence of the template RNA as a template. Without wishing to be bound by theory, it is believed that reverse transcription then proceeds through the pre-editing homology region, then through the mutation region, and then through the post-editing homology region, thereby generating a DNA strand containing the mutation specified by the heterologous target sequence. [Figure 2] Graph showing the rewriting rates achieved using RNAV209-013 or RNAV214-040 genetically modified polypeptides with the indicated template RNAs. [Figure 3]1 is a graph showing the relative amount of Fah mRNA compared to the wild type when template RNA is used together with RNAV209-013 or RNAV214-040 genetically modified polypeptides. [Figure 4] 1 is a graph showing the rate of Cas9-positive hepatocytes 6 hours after administration of LNPs carrying various genetically modified polypeptides and template RNA. [Figure 5] 1 is a graph showing rewrite levels in liver samples 6 days after administration of LNPs with various genetically modified polypeptides and template RNA. [Figure 6] 1 is a graph showing restoration of wild-type Fah mRNA in liver samples compared to littermate heterozygous mice after administration of LNPs with various genetically modified polypeptides and template RNA. [Figure 7] 1 is a graph showing Fah protein distribution in liver samples after administration of LNPs with various genetically modified polypeptides and template RNA. [Figure 8] A series of Western blots showing Cas9-RT expression 6 hours after injection of Cas9-RT mRNA + TTR-guided LNP. Each lane represents an individual animal, where 20 μg of tissue homogenate was added per lane. The positive control was from an in vitro cell experiment (described above) in which Cas9-RT was expressed. GAPDH was used in each sample as a loading control. n=4 per vehicle or treatment group. [Figure 9] Figure 1 shows gene editing of the TTR locus after treatment with Cas9-RT mRNA + TTR-guided LNP. Indel levels detected at the TTR locus as measured by TIDE analysis of Sanger sequencing of the protospacer-targeted TTR locus. [Figure 10] Figure 1 shows that TTR serum levels are reduced after treatment with Cas9-RT mRNA + TTR guide LNPs. Measurement of circulating TTR levels 5 days after treatment of mice with LNPs encapsulating Cas9-RT + TTR guide RNA. [Figure 11]Figure 1 shows Cas9-RT expression after injection of Cas9-RT mRNA + TTR-guided LNP. Relative expression was quantified by ProteinSimple Jess capillary electrophoresis Western blot. Numbers indicate the number of animals in each group. Vehicle n=2, Cas9-RT + TTR-guided n=3. [Figure 12] (Figure 1) Gene editing of the TTR locus after injection of Cas9-RT mRNA + TTR-guided LNP. The level of indels detected at the TTR locus was measured by amplicon sequencing of the protospacer-targeted TTR locus. Each animal had eight different biopsies taken from the whole liver, where the percentage of reads showing indels was measured by amplicon sequencing. [Figure 13] 1 is a graph showing rewrite rates in primary mouse hepatocytes nucleofected with various gene modification systems. [Figure 14] Graph showing editing rates in primary mouse hepatocytes nucleofected with various gene modification systems containing a second nicked gRNA. [Figure 15] 1 is a heat map showing the rewriting efficiency of various gene modification systems with or without a second nicked gRNA. [Figure 16] 1 is a graph showing the percentage of mouse hepatocytes expressing Cas9 6 hours after administration of various gene modification systems. [Figure 17] 1 is a pair of Western blots showing Cas9 expression in mouse liver samples 6 hours after administration of various gene modification systems. [Figure 18] 1 is a graph showing phenylalanine (Phe) levels present in plasma samples 7 days after administration of various genetic modification systems. [Figure 19] 19A and 19B are graphs showing the rewrite rate (FIG. 19A) and indel rate (FIG. 19B) in mouse liver 7 days after administration of various gene modification systems. [Figure 20-1]20A-20C are graphs showing rewrite rates in liver samples, plasma Phe levels (FIG. 20B), and indel rates in mouse livers (FIG. 20C) 7 days after administration of various gene modification systems. [Figure 20-2] 20A-20C are graphs showing rewrite rates in liver samples, plasma Phe levels (FIG. 20B), and indel rates in mouse livers (FIG. 20C) 7 days after administration of various gene modification systems. [Figure 21] A pair of graphs showing rewrite and indel rates in liver samples (FIG. 21A) and plasma Phe levels (FIG. 21B) 7 days after administration of various gene modification systems with or without a second nicked gRNA. [Figure 22] 1 is a graph showing plasma phenylalanine (Phe) levels versus rewrite rate in samples obtained from mice treated with various gene modification systems. [Figure 23] 1 is a graph showing the rewriting rates of HEK293T cells harboring the cynomolgus monkey (M. fascicularis) PAH gene for four different mutant forms using template RNAs with four different spacer sequences. [Figure 24] 24A and 24B are graphs showing the rewrite rate (FIG. 24A) and indel rate (FIG. 24B) of mouse hepatocytes, or plasma Phe concentration (FIG. 24C), several days after administration of LNPs containing various gene modification systems. [Figure 25-1] 25A and 25B are heat maps showing the rewriting rates for each combination of template RNA and second strand targeting RNA in primary human hepatocytes (FIG. 25A) and primary mouse hepatocytes (FIG. 25C) after transfection (FIG. 25A and FIG. 25B) or LNP delivery (FIG. 25C) of various gene modification systems. [Figure 25-2] Same as above. [Figure 26] 26A and 26B are graphs showing the rewrite rate (FIG. 26A) and indel rate (FIG. 26B) of liver samples 7 and 28 days after LNP delivery of the gene modification system to mice. [Figure 27] 1 is a graph showing Phe concentrations in plasma samples 7 and 28 days after LNP delivery of the gene modification system to mice. [Figure 28] 1 is a graph showing Phe concentrations in brain samples 7 and 28 days after LNP delivery of the gene modification system to mice. [Figure 29] FIG. 29 is a graph showing brain Phe concentrations versus plasma Phe concentrations from the samples used to generate FIGS. 27 and 28. [Figure 30-1] 1 is a heat map showing the rewriting rate for each combination of template RNA and second strand targeting RNA after mRNA delivery of the gene modification system into primary cynomolgus monkey hepatocytes. [Figure 30-2] 1 is a heat map showing the rewriting rate for each combination of template RNA and second strand targeting RNA after mRNA delivery of the gene modification system into primary cynomolgus monkey hepatocytes. [Figure 31] FIG. 31A is a graph stratified by silent substitution showing the total rewrite rate after mRNA delivery of various gene modification systems into human iPSC-derived hepatoblasts using hPKU3 template RNA containing various silent substitutions (FIG. 31A), and a chart showing the specific silent substitutions used in FIG. 31A (FIG. 31B). [Figure 32] FIG. 32A is a graph stratified by silent substitution showing the total rewrite rate after mRNA delivery of various gene modification systems into human iPSC-derived hepatoblasts using hPKU4 template RNA containing various silent substitutions (FIG. 32A), and a chart showing the specific silent substitutions used in FIG. 32A (FIG. 32B). [Figure 33] Graph (33A) and chart (33B) show the total rewrite rate stratified by silent substitution following mRNA delivery of various gene modification systems into human iPSC-derived hepatoblasts using hPKU5 template RNA containing various silent substitutions. [Figure 34]FIG. 34A is a graph stratified by silent substitution showing the total rewrite rate after mRNA delivery of various gene modification systems into human iPSC-derived hepatoblasts using hPKU6 template RNA containing various silent substitutions (FIG. 34A), and a chart showing the specific silent substitutions used in FIG. 34A (FIG. 34B). [Figure 35] 1 is a graph showing serum Phe levels in mice after treatment with LNPs containing various gene modification systems. [Figure 36] 36A and 36B are graphs showing the rewrite rate (FIG. 36A) and indel rate (FIG. 36B) in mouse livers after treatment with LNPs containing various gene modification systems. DETAILED DESCRIPTION OF THE INVENTION

[0206] definition The term "expression cassette," as used herein, refers to a nucleic acid construct that contains sufficient nucleic acid elements for expression of a nucleic acid molecule of the invention.

[0207] "gRNA spacer," as used herein, refers to a portion of a nucleic acid that has complementarity to a target nucleic acid and, together with the gRNA scaffold, can target a Cas protein to the target nucleic acid.

[0208] "gRNA scaffold," as used herein, refers to a portion of a nucleic acid that can bind to a Cas protein and, together with a gRNA spacer, target the Cas protein to a target nucleic acid. In some embodiments, the gRNA scaffold comprises a crRNA sequence, a tetraloop, and a tracrRNA sequence.

[0209] "Genetically modified 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%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a retroviral reverse transcriptase that 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., within a mammalian host cell, such as a genomic DNA molecule within the host cell). In some embodiments, the genetically modified polypeptide is capable of integrating the sequence substantially independent of the host machinery. In some embodiments, the genetically modified polypeptide integrates the sequence at a random location within the genome; in some embodiments, the genetically modified polypeptide integrates the sequence at a specific target site. In some embodiments, the genetically modified polypeptide comprises one or more domains that collectively: 1) bind to a template nucleic acid; 2) facilitate binding to a target DNA molecule; and 3) facilitate integration of at least a portion of the template nucleic acid into the target DNA. Genetically modified polypeptides include both naturally occurring polypeptides and engineered variants thereof, e.g., having one or more amino acid substitutions relative to the naturally occurring sequence. Genetically modified polypeptides also include heterologous constructs, for example, where one or more of the domains listed above are heterologous to one another, whether by heterologous fusion (or other conjugate) of otherwise wild-type domains as well as fusion of modified domains, for example, by substitution or fusion of heterologous subdomains or other replacement domains. Exemplary genetically modified polypeptides that can be used in the methods provided herein, and systems comprising and methods of using them, are described in PCT / US2021 / 020948, incorporated herein by reference, for example, with respect to genetically modified polypeptides comprising retroviral reverse transcriptase domains. In some embodiments, the genetically modified polypeptide incorporates a sequence into a gene. In some embodiments, the genetically modified polypeptide incorporates a sequence into a sequence external to a gene. "Genetic modification system," as used herein, refers to a system comprising a genetically modified polypeptide and a template nucleic acid.

[0210] As used herein, the term "domain" refers to a structure of a biomolecule that contributes to a specific function of the biomolecule. A domain can include a continuous region (e.g., a contiguous sequence) or discrete, non-contiguous regions (e.g., non-contiguous sequences) of a biomolecule. Examples of protein domains include, but are not limited to, endonuclease domains, DNA-binding domains, and reverse transcription domains; examples of nucleic acid domains include regulatory domains, such as transcription factor binding domains. In some embodiments, a domain (e.g., a Cas domain) can include two or more smaller domains (e.g., a DNA-binding domain and an endonuclease domain).

[0211] As used herein, the term "exogenous," when used in reference to a biomolecule (e.g., a nucleic acid sequence or a polypeptide), means that the biomolecule has been introduced into a host genome, cell, or organism by human intervention. For example, a nucleic acid that is added to an existing genome, cell, tissue, or subject using recombinant DNA technology or other methods is exogenous to the existing nucleic acid sequence, cell, tissue, or subject.

[0212] As used herein, the terms "first strand" and "second strand" used to describe individual DNA strands of a target DNA distinguish between the two DNA strands upon which a reverse transcriptase domain initiates polymerization, e.g., upon which target-primed synthesis is initiated. The first strand refers to the strand of target DNA upon which a reverse transcriptase domain initiates polymerization, e.g., upon which target-primed synthesis is initiated. The second strand refers to the other strand of target DNA. The names first strand and second strand do not otherwise describe target site DNA strands; for example, in some embodiments, the first strand and second strand are nicked by the polypeptides described herein, but the names "first" and "second" strand are independent of the order in which such nicks appear.

[0213] The term "heterologous," when used to refer to a first element with respect to a second element, means that the first and second elements do not naturally exist in the arrangement 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 the 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 modified or mutated relative to its natural state; or (c) a polypeptide or nucleic acid molecule that has altered expression compared to native expression levels under similar conditions. For example, heterologous regulatory sequences (e.g., promoters, enhancers) can be used to regulate expression of a gene or nucleic acid molecule in a manner different from that in which the gene or 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 a nucleic acid encoding a DNA-binding domain of a polypeptide) can be positioned relative to other domains or can be of a different sequence or derived from a different source compared to other domains or portions of a polypeptide or its encoding nucleic acid. In certain embodiments, a heterologous nucleic acid molecule may be present in the native host cell genome, but may have an altered expression level or a different sequence, or both. In other embodiments, a heterologous nucleic acid molecule may not be endogenous to the host cell or host genome, but instead may be introduced into the host cell by transformation (e.g., transfection, electroporation), where the added molecule may be integrated into the host genome or may exist as extrachromosomal genetic material, either transiently (e.g., mRNA) or semi-stable for more than one generation (e.g., episomal viral vectors, plasmids, or other self-replicating vectors).

[0214] As used herein, "insertion" of a sequence into a target site refers to the net addition of a DNA sequence at the target site, e.g., where there is a new nucleotide in the heterologous sequence of interest that does not have a cognate position in the unedited target site. In some embodiments, nucleotide alignment of the PBS sequence and the heterologous sequence of interest to the target nucleic acid sequence will result in an alignment gap in the target nucleic acid sequence.

[0215] As used herein, a "deletion" generated by a heterologous sequence of interest at a target site refers to the net deletion of DNA sequence at the target site, e.g., where there is a nucleotide in the unedited target site that does not have a cognate position in the heterologous sequence of interest. In some embodiments, nucleotide alignment of the PBS sequence and heterologous sequence of interest to the target nucleic acid sequence will result in an alignment gap in the molecule comprising the PBS sequence and the heterologous sequence of interest.

[0216] The term "inverted terminal repeat" or "ITR" as used herein refers to an AAV viral cis-element so named due to its symmetry. This element promotes efficient propagation of the AAV genome. The minimum elements for ITR function are hypothesized to be a Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' for AAV2; SEQ ID NO: 4601) and a terminal separation site (TRS; 5'-AGTTGG-3' for AAV2; SEQ ID NO: 4602), as well as a variable palindromic sequence that allows hairpin formation. According to the present invention, an ITR contains at least these three elements (RBS, TRS, and a sequence that allows hairpin formation). Additionally, in the present invention, the term "ITR" refers to the ITRs of known natural AAV serotypes (e.g., ITRs of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAVs), chimeric ITRs formed by fusing ITR elements from different serotypes, and functional variants thereof. "Functional variant" refers to a sequence that exhibits at least 80%, 85%, 90%, preferably at least 95% sequence identity with a known ITR and allows for the enrichment of sequences containing said ITR in the presence of Rep proteins.

[0217] The term "mutation region," as used herein, refers to a region in a template RNA that has one or more sequence differences compared to the corresponding sequence in a target nucleic acid. Sequence differences can include, for example, substitutions, insertions, frameshifts, or deletions.

[0218] The term "mutant," when applied to a nucleic acid sequence, means that nucleotides within a nucleic acid sequence have been inserted, deleted, or changed relative to a reference (e.g., naturally occurring) nucleic acid sequence. A single alteration may be made at a single locus (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. Nucleic acid sequences can be mutated by any method known in the art.

[0219] "Nucleic acid molecule" refers to both RNA and DNA molecules, including, but not limited to, complementary DNA ("cDNA"), genomic DNA ("gDNA"), and messenger RNA ("mRNA"), and also synthetic nucleic acid molecules, such as those chemically synthesized or recombinantly produced, such as RNA templates, as described herein. Nucleic acid molecules can be double-stranded or single-stranded, circular or linear. If single-stranded, the nucleic acid molecule can be the sense or antisense strand. Unless otherwise specified, and as an example of a nucleic acid containing all sequences described herein in the general format "SEQ ID NO:1," or "SEQ ID NO:1," refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complementary to SEQ ID NO:1. The choice between the two is determined by the context in which SEQ ID NO:1 is used. For example, if the nucleic acid is used as a probe, the choice between the two is determined by the requirement that the probe be complementary to the desired target. The nucleic acid sequences of the present disclosure may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalating agents (e.g., acridines, psoralens, etc.), chelators, alkylating agents, and modified linkages (e.g., α-anomeric nucleic acids, etc.). Chemically modified bases (e.g., see Table 13), backbones (e.g., see Table 14), and modified caps (e.g., see Table 15) are also included. Synthetic molecules that mimic polynucleotides in their ability to bind to designated sequences through hydrogen bonding and other chemical interactions are also included. Such molecules are known in the art and include, for example, those that substitute peptide linkages for phosphate linkages in the backbone of the molecule, eg, peptide nucleic acids (PNAs).Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structures, such as modifications found in "locked" nucleic acids (LNA). In various embodiments, the nucleic acid is operatively associated with additional genetic elements, such as tissue-specific expression-controlling sequences (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, e.g., elements derived from viruses (e.g., AAV ITRs)) and tandem repeats, inverted / direct repeats, homologous regions (segments with varying degrees of homology to the target DNA), untranslated regions (UTRs) (5', 3', or both 5' and 3' UTRs), and various combinations of the foregoing. Nucleic acid elements of the systems provided by the present invention can be provided in various topologies, including single-stranded, double-stranded, circular, linear, open-ended linear, closed-ended linear, and specific versions thereof, e.g., doggybone DNA (dbDNA), closed-ended DNA (ceDNA).

[0220] 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 to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, 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 can be contiguous or non-contiguous. Where necessary to link two protein coding regions, operably linked sequences can be in the same reading frame.

[0221] The term "host genome" or "host cell," as used herein, refers to a cell and / or its genome into which proteins and / or genetic material have been introduced. These terms refer not only to the particular subject cell and / or genome, but also to the progeny of such a cell and / or the genomes of the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, it is understood that 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 comprising a living tissue or organism. In some cases, the host cell may be an animal cell or a plant cell, e.g., as described herein. In certain examples, the host cell may be a mammalian cell, a human cell, an avian cell, a reptilian cell, a bovine cell, an equine cell, a porcine cell, a caprine cell, a ovine cell, a chicken cell, or a turkey cell. In particular examples, the host cell can be a corn cell, a soybean cell, a wheat cell, or a rice cell.

[0222] As used herein, "operably associated" describes the functional relationship between two nucleic acid sequences, e.g., 1) a promoter and 2) a heterologous sequence of interest, and in such instances means that the promoter and heterologous sequence of interest (e.g., a gene of interest) are oriented such that, under appropriate conditions, the promoter drives expression of the heterologous sequence of interest. For example, a template nucleic acid bearing a promoter and a heterologous sequence of interest can be, for example, single-stranded in either a (+) or (-) orientation. The "operably associated" relationship between the promoter and heterologous sequence of interest in this template means that the template nucleic acid will be correctly transcribed when under appropriate conditions (e.g., in the (+) orientation, in the presence of required catalytic factors, NTPs, etc.), regardless of whether the template nucleic acid will be transcribed under a particular condition. Operative association applies similarly to other pairs of nucleic acids, including sequences encoding other tissue-specific expression control sequences (e.g., enhancers, repressors, and microRNA recognition sequences), IR / DR, ITR, UTR, or homologous regions, and a heterologous sequence of interest, or a retroviral RT domain.

[0223] The term "primer binding site sequence" or "PBS sequence," as used herein, refers to a portion of a template RNA that can bind to a region contained in a target nucleic acid sequence. In some cases, a PBS sequence is a nucleic acid sequence that contains at least 3, 4, 5, 6, 7, or 8 bases that are 100% identical to a region contained in a target nucleic acid sequence. In some embodiments, a primer region contains at least 5, 6, 7, or 8 bases that are 100% identical to a region contained in a target nucleic acid sequence. Without intending to be bound by theory, in some embodiments, when a template RNA contains a PBS sequence and a heterologous sequence of interest, the PBS sequence binds to a region contained in the target nucleic acid sequence, enabling the reverse transcriptase domain to use that region as a primer for reverse transcription and to use the heterologous sequence of interest as a template for reverse transcription.

[0224] As used herein, a "stem-loop sequence" refers to a nucleic acid sequence (e.g., an RNA sequence) having a stem containing sufficient self-complementarity to form a stem-loop, e.g., at least 2 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) base pairs, and a loop having at least 3 (e.g., 4) base pairs. The stem may contain mismatches or bulges.

[0225] As used herein, "tissue-specific expression-control sequence" refers to a nucleic acid element that increases or decreases the level of a transcript containing a heterologous sequence of interest in a target tissue in a tissue-specific manner, e.g., preferentially in on-target tissue compared to off-target tissue. In some embodiments, the tissue-specific expression-control sequence preferentially drives or represses the transcription, activity, or half-life of a transcript containing a heterologous sequence of interest in a target tissue in a tissue-specific manner, e.g., preferentially in on-target tissue compared to off-target tissue. Exemplary tissue-specific expression-control sequences include tissue-specific promoters, repressors, enhancers, or combinations thereof, and tissue-specific microRNA recognition sequences. Tissue specificity refers to on-target (tissues in which expression or activity of the template nucleic acid is desired or acceptable) and off-target (tissues in which expression or activity of the template nucleic acid is undesirable or unacceptable). For example, a tissue-specific promoter preferentially drives expression in on-target tissue compared to off-target tissue. In contrast, microRNAs that bind to tissue-specific microRNA recognition sequences are preferentially expressed in off-target tissues compared to on-target tissues, thereby reducing the expression of the template nucleic acid in the off-target tissue. Thus, promoters and microRNA recognition sequences specific to the same tissue, such as target tissues, have contrasting functions with respect to the transcription, activity, or half-life of the associated sequence in the tissue (i.e., matching expression levels, i.e., promoting and suppressing high levels of the microRNA in off-target tissues and low levels in on-target tissues, respectively, while the promoter drives high expression in on-target tissues and low expression in off-target tissues).

[0226] List of Headlines 1) Introduction 2) Genetic modification system a) Polypeptide components of the genetic modification system i) Lighting Domain ii) Endonuclease domain and DNA binding domain (1) A genetically modified polypeptide containing a Cas domain (2) TAL effectors and zinc finger nucleases iii) Linker iv) Localization sequences for gene modification systems v) Evolved variants of genetically modified polypeptides and systems vi) Intein vii) Further domains b) Template nucleic acid i) gRNA spacers and gRNA scaffolds ii) Heterologous sequence of interest iii) PBS sequence iv) Exemplary template sequences c) gRNA with inducible activity d) Circular RNA and ribozymes in gene modification systems e) Target nucleic acid site f) Second Strand Nicking 3) Preparation of compositions and systems 4) Therapeutic use 5) Administration and Delivery a) Tissue-specific activity / administration i) Promoter ii) microRNA b) Viral vectors and their components c) AAV administration d) Lipid nanoparticles 6) Kits, Products, and Pharmaceutical Compositions 7) Chemistry, Manufacturing, and Controls (CMC)

[0227] introduction The present disclosure relates to methods of treating phenylketonuria (PKU) and compositions for targeting, editing, modifying, or manipulating DNA sequences (e.g., inserting a heterologous sequence of interest at a target site in a mammalian genome) at one or more locations within the DNA sequence in a cell, tissue, or subject, e.g., in vivo or in vitro. The heterologous DNA sequence of interest may, for example, include a substitution.

[0228] More particularly, the present disclosure provides methods for treating PKU using a reverse transcriptase-based system for modifying a genomic DNA sequence of interest, for example, by inserting, deleting, or substituting one or more nucleotides into / from the sequence of interest.

[0229] The present disclosure provides, in part, methods for treating PKU using a genetic modification system comprising a genetically modified polypeptide component and a template nucleic acid (e.g., template RNA) component. In some embodiments, the genetic modification system can be used to introduce modifications into a target site in a genome. In some embodiments, the genetically modified 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, the template nucleic acid (e.g., template RNA) comprises a sequence (e.g., a gRNA spacer) that binds to the target site in the genome (e.g., binds to the second strand of the target site), a sequence that binds to the genetically modified polypeptide component (e.g., a gRNA scaffold), a heterologous sequence of interest, and a PBS sequence. Without wishing to be bound by theory, it is believed that the template nucleic acid (e.g., template RNA) binds to the second strand of the target site in the genome and binds to the genetically modified polypeptide component (e.g., localizes the polypeptide component to the target site in the genome). The endonuclease (e.g., nickase) of the genetically modified polypeptide component may cleave the target site (e.g., the first strand of the target site) and, for example, a PBS sequence may bind to the sequence adjacent to the site to be modified on the first strand of the target site. The writing domain (e.g., reverse transcriptase domain) of the polypeptide component may polymerize, for example, a sequence complementary to the heterologous target sequence, using the first strand of the target site bound to the PBS sequence of the template nucleic acid as a primer and a complementary sequence comprising the heterologous target sequence of the template nucleic acid as a template. Without wishing to be bound by theory, it is believed that selection of an appropriate heterologous target sequence may result in the substitution, deletion, and / or insertion of one or more nucleotides at the target site.

[0230] Genetic Modification System In some embodiments, the genetic modification systems described herein include (A) a genetic modification polypeptide or a nucleic acid encoding a genetic modification polypeptide, where the genetic modification polypeptide includes (i) a reverse transcriptase domain and either (x) an endonuclease domain comprising DNA-binding functionality or (y) an endonuclease domain and a separate DNA-binding domain; and (B) a template RNA. In some embodiments, the genetic modification polypeptide acts as a substantially autonomous protein machinery capable of incorporating a template nucleic acid sequence into a target DNA molecule (e.g., within a mammalian host cell, such as a genomic DNA molecule within the host cell) substantially independent of the host machinery. For example, the genetic modification protein may include a DNA-binding domain, a reverse transcriptase domain, and an endonuclease domain. In some embodiments, the DNA-binding function may include an RNA component, e.g., a gRNA spacer, that guides the protein to the DNA sequence. In other embodiments, the genetic modification polypeptide may include a reverse transcriptase domain and an endonuclease domain. The RNA template element of the genetic modification system is typically heterologous to the genetic modification polypeptide element and provides the sequence of interest to be inserted (reverse transcribed) into the host genome. In some embodiments, the genetically modified polypeptide is capable of target-primed reverse transcription. In some embodiments, the genetically modified polypeptide is capable of second strand synthesis.

[0231] In some embodiments, the genetic modification system is combined with a second polypeptide. In some embodiments, the second polypeptide may include an endonuclease domain. In some embodiments, the second polypeptide may include a polymerase domain, such as a reverse transcriptase domain. In some embodiments, the second polypeptide may include a DNA-dependent DNA polymerase domain. In some embodiments, the second polypeptide assists in completing genome editing, for example, by contributing to second strand synthesis or DNA repair recovery.

[0232] A functional gene-modifying polypeptide can be composed of unrelated DNA-binding, reverse transcription, and endonuclease domains. This modular structure allows for the combination of functional domains, such as dCas9 (DNA binding), MMLV reverse transcriptase (reverse transcription), and FokI (endonuclease). In some embodiments, multiple functional domains can occur in a single protein, such as Cas9 or Cas9 nickase (DNA binding, endonuclease).

[0233] In some embodiments, a genetically modified polypeptide comprises one or more domains that collectively: 1) bind to a template nucleic acid; 2) facilitate binding to a target DNA molecule; and 3) facilitate integration of at least a portion of the template nucleic acid into the target DNA. In some embodiments, a genetically modified polypeptide is an altered polypeptide comprising one or more amino acid substitutions relative to the corresponding native sequence. In some embodiments, a genetically modified polypeptide comprises two or more domains that are heterologous to each other, e.g., by heterologous fusion (or other conjugate) of otherwise wild-type domains as well as fusion of modified domains, e.g., by substitution or fusion of heterologous subdomains or other replacement domains. For example, in some embodiments, one or more of the RT domain is heterologous to the DBD; the DBD is heterologous to the endonuclease domain; or the RT domain is heterologous to the endonuclease domain.

[0234] In some embodiments, a template RNA molecule for use in the system comprises, from 5' to 3', (1) a gRNA spacer; (2) a gRNA scaffold; (3) a heterologous sequence of interest; and (4) a primer binding site (PBS) sequence. (1) a gRNA spacer of about 18 to 22 nt, for example, 20 nt; (2) A gRNA scaffold comprising one or more hairpin loops, e.g., one, two, or three loops for associating the template with a Cas domain, e.g., a nickase Cas9 domain. In some embodiments, the gRNA scaffold comprises, from 5' to 3', the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCC (SEQ ID NO: 5008). (3) In some embodiments, the heterologous sequence of interest is, for example, 7 to 74, e.g., 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, or 70 to 80 or 80 to 90 nt in length. In some embodiments, the first (usually 5') base of the sequence is not C. (4) In some embodiments, the PBS sequence that binds to the target priming sequence after nicking is, for example, 3 to 20 nt, for example, 7 to 15 nt, for example, 12 to 14 nt, and has a GC content of 40 to 60%.

[0235] In some embodiments, a second gRNA associated with the system can help drive complete integration. In some embodiments, the second gRNA can target a position 0-200 nt away from the first strand nick, e.g., 0-50, 50-100, or 100-200 nt away from the first strand nick. In some embodiments, the second gRNA can only bind to its target sequence after editing has occurred, e.g., the gRNA binds to a sequence present in the heterologous sequence of interest but not in the initial target sequence.

[0236] In some embodiments, the genetic modification systems described herein are used to perform edits in HEK293, K562, U2OS, or HeLa cells. In some embodiments, the genetic modification systems are used to perform edits in primary cells, such as primary cortical neurons from E18.5 mice.

[0237] In some embodiments, the genetically modified polypeptides described herein comprise a reverse transcriptase or RT domain (e.g., as described herein) comprising a MoMLV RT sequence or a variant thereof. In embodiments, the 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, the MoMLV RT sequence comprises a combination of mutations, such as D200N, L603W, and T330P, optionally further comprising T306K and / or W313F.

[0238] In some embodiments, the endonuclease domain (e.g., as described herein) is nCAS9, e.g., including an N863A mutation (e.g., in spCas9) or an H840A mutation.

[0239] In some embodiments, a heterologous sequence of interest (e.g., in a system 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.

[0240] In some embodiments, the RT and endonuclease domains are linked by a flexible linker, for example, comprising the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 5006).

[0241] In some embodiments, the endonuclease domain is N-terminal to the RT domain. In some embodiments, the endonuclease domain is C-terminal to the RT domain.

[0242] In some embodiments, the system incorporates a heterologous sequence of interest into a target site by TPRT, for example, as described herein.

[0243] In some embodiments, the genetically modified polypeptide comprises a DNA-binding domain. In some embodiments, the genetically modified polypeptide comprises an RNA-binding domain. In some embodiments, the RNA-binding domain comprises an RNA-binding domain of a B-box protein, an MS2 coat protein, dCas, or an element of a sequence in a table herein. In some embodiments, the RNA-binding domain can bind to the template RNA with higher affinity than a standard RNA-binding domain.

[0244] In some embodiments, the genetic modification system is capable of generating an insertion at a target site that is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally up to 500, 400, 300, 200, or 100 nucleotides). In some embodiments, the genetic modification system is capable of generating an insertion at a target site that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally up to 500, 400, 300, 200, or 100 nucleotides). In some embodiments, the genetic modification system can generate insertions at target sites of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 kilobases (and optionally up to 1, 5, 10, or 20 kilobases). In some embodiments, the genetic modification system can generate deletions of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally up to 500, 400, 300, or 200 nucleotides). In some embodiments, the genetic modification system is capable of generating deletions of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally up to 500, 400, 300, or 200 nucleotides). In some embodiments, the genetic modification system is capable of generating deletions of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally up to 500, 400, 300, or 200 nucleotides).In some embodiments, the genetic modification system is capable of generating deletions of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 kilobases (and optionally up to 1, 5, 10, or 20 kilobases). In some embodiments, the genetic modification system is capable of generating substitutions at the target site of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more nucleotides. In some embodiments, the genetic modification system can generate substitutions at 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.

[0245] In some embodiments, the substitution is a transition mutation. In some embodiments, the substitution is a transversion mutation. In some embodiments, the substitution converts adenine to thymine, adenine to guanine, adenine to cytosine, guanine to thymine, guanine to cytosine, guanine to adenine, thymine to cytosine, thymine to adenine, thymine to guanine, cytosine to adenine, cytosine to guanine, or cytosine to thymine.

[0246] In some embodiments, the insertion, deletion, substitution, or a combination thereof increases or decreases expression (e.g., transcription or translation) of a gene. In some embodiments, the insertion, deletion, substitution, or a combination thereof increases or decreases expression (e.g., transcription or translation) of a gene by modifying, adding, or deleting sequences in a promoter or enhancer, such as sequences that bind transcription factors. In some embodiments, the insertion, deletion, substitution, or a combination thereof alters the translation of a gene (e.g., alters the amino acid sequence), inserts or deletes start or stop codons, alters or restores the translation frame of a gene. In some embodiments, the insertion, deletion, substitution, or a combination thereof alters the splicing of a gene, for example, by inserting, deleting, or modifying a splice acceptor or donor site. In some embodiments, the insertion, deletion, substitution, or a combination thereof alters the half-life of a transcript or protein. In some embodiments, the insertion, deletion, substitution, or combination thereof alters protein localization in a cell (e.g., from the cytoplasm to mitochondria, from the cytoplasm to the extracellular space (e.g., adding a secretion tag)). In some embodiments, the insertion, deletion, substitution, or combination thereof alters (e.g., improves) protein folding (e.g., to prevent the accumulation of misfolded proteins). In some embodiments, the insertion, deletion, substitution, or combination thereof alters, increases, decreases the activity of a gene, e.g., a protein encoded by the gene.

[0247] Exemplary genetically modified polypeptides, and systems comprising and methods of using them, are described in PCT / US2021 / 020948, which is incorporated herein by reference, for example, with respect to retroviral RT domains, including amino acid and nucleic acid sequences therein.

[0248] Exemplary genetically modified polypeptide and retroviral RT domain sequences are also described, for example, in International Patent Application No. PCT / US21 / 20948, filed March 4, 2021, e.g., Tables 30, 31, and 44 therein; the entire application is incorporated herein by reference, e.g., with respect to the retroviral RT sequences and tables. Thus, the genetically modified polypeptides described herein can comprise an amino acid sequence according to any of the tables described in this paragraph or a domain thereof (e.g., a retroviral RT domain), or a functional fragment or variant thereof of any of the above, or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0249] In some embodiments, a polypeptide for use in any of the systems described herein can be a molecular or ancestral reconstructor based on aligned polypeptide sequences of multiple homologous proteins. In some embodiments, a reverse transcriptase domain for use in any of the systems described herein can be a molecular or ancestral reconstructor, or can be modified at specific residues based on alignment of reverse transcriptase domains from the same or different sources. Those skilled in the art can align polypeptide or nucleic acid sequences based on the accession numbers provided herein, for example, by using routine sequence analysis tools such as the Basic Local Alignment Search Tool (BLAST) or CD-Search for conserved domain analysis. Molecular reconstructors can be generated based on sequence consensus, for example, using techniques described in Ivics et al., Cell 1997, 501-510; Wagstaff et al., Molecular Biology and Evolution 2013, 88-99.

[0250] Polypeptide components of gene modification systems In some embodiments, the genetically modified polypeptide has the functions of DNA target site binding, template nucleic acid (e.g., RNA) binding, DNA target site cleavage, and template nucleic acid (e.g., RNA) writing, e.g., reverse transcription. In some embodiments, each function is contained within a different domain. In some embodiments, a function can be attributed to two or more domains (e.g., two or more domains together exhibit functionality). In some embodiments, two or more domains can have the same or similar function (e.g., two or more domains each independently have DNA binding functionality, e.g., in two different DNA sequences). In other embodiments, one or more domains can perform one or more functions; for example, a Cas9 domain can perform both DNA binding and target site cleavage. In some embodiments, all of the domains are located within a single polypeptide. In some embodiments, the first domain is present in a first polypeptide and the second domain is present in a second polypeptide. For example, in some embodiments, the sequence may be split between a first polypeptide and a second polypeptide, e.g., the first polypeptide comprises a reverse transcriptase (RT) domain and the second polypeptide comprises a DNA-binding domain and an endonuclease domain, e.g., a nickase domain. By way of further example, in some embodiments, the first polypeptide and the second polypeptide each comprise a DNA-binding domain (e.g., a first DNA-binding domain and a second DNA-binding domain). In some embodiments, the first and second polypeptides may be post-translationally joined via a split intein to form a single genetically modified polypeptide.

[0251] In some embodiments, a genetically modified polypeptide described herein comprises (e.g., a system described herein comprises a genetically modified polypeptide comprising: 1) a Cas domain (e.g., a Cas nickase domain, e.g., a Cas9 nickase domain); 2) a reverse transcriptase (RT) domain of Table D, 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 to the Cas domain; and a linker disposed between the RT domain and the Cas domain, the linker having a sequence from the same row as the RT domain of Table D, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0252] In some embodiments, the RT domain has a sequence 100% identical to an RT domain of Table D, and the linker has a sequence 100% identical to a linker sequence from the same row as the RT domain of Table D. In some embodiments, the Cas domain comprises a sequence of Table 8, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical thereto. In some embodiments, the genetically modified polypeptide comprises an amino acid sequence according to any of SEQ ID NOs: 1-3332 in the Sequence Listing, or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical thereto.

[0253] In some embodiments, the genetically modified polypeptide comprises a GG amino acid sequence between the Cas domain and the linker, an AG amino acid sequence between the RT domain and the second NLS, and / or a GG amino acid sequence between the linker and the RT domain. In some embodiments, the genetically modified polypeptide comprises the sequence of SEQ ID NO: 4000 comprising the first NLS and the Cas domain, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, the genetically modified polypeptide comprises the sequence of SEQ ID NO: 4001 comprising the second NLS, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

[0254] Exemplary N-terminal NLS-Cas9 domains [ka]

[0255] Exemplary C-terminal sequences containing an NLS AGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 4001)

[0256] Lighting Domain (RT Domain) In certain aspects of the present invention, the writing domain of the genetic modification system has reverse transcriptase activity and is also referred to as a reverse transcriptase domain (RT domain). In some embodiments, the RT domain comprises an RT catalytic portion and an RNA binding region (e.g., a region that binds to a template RNA).

[0257] In some embodiments, the nucleic acid encoding the reverse transcriptase is modified from its native sequence to have altered codon usage, e.g., improved for human cells. In some embodiments, the reverse transcriptase domain is a heterologous reverse transcriptase from a retrovirus. In some embodiments, the RT domain comprising the genetically modified polypeptide has been mutated from its original amino acid sequence, e.g., has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions. In some embodiments, the RT domain is derived from a retroviral RT, e.g., HIV-1 RT, Moloney Murine Leukemia Virus (MMLV) RT, avian myeloblastosis virus (AMV) RT, or Rous Sarcoma Virus (RSV) RT.

[0258] In some embodiments, the retroviral reverse transcriptase (RT) domain exhibits increased stringency for target-primed reverse transcription (TPRT) initiation, e.g., compared to the endogenous RT domain. In some embodiments, the RT domain initiates TPRT when 3 nt within the target site immediately upstream of the first-strand nick, e.g., the genomic DNA priming the RNA template, are at least 66% or 100% complementary to the 3 nt of homology in the RNA template. In some embodiments, the RT domain initiates TPRT when there is less than a 5 nt mismatch (e.g., less than a 1, 2, 3, 4, or 5 nt mismatch) between the template RNA and the target DNA primed reverse transcription. In some embodiments, the RT domain is modified to increase the stringency of mismatches in priming the TPRT reaction, e.g., the RT domain tolerates no mismatches or tolerates fewer mismatches within the priming region compared to a wild-type (e.g., unmodified) RT domain. In some embodiments, the RT domain comprises an HIV-1 RT domain. In embodiments, the HIV-1 RT domain initiates synthesis at a lower level, even with three nucleotide mismatches, compared to alternative RT domains (e.g., as described by Jambuluthugoda and Eickbush J Mol Biol 407(5):661-672 (2011), incorporated herein by reference in its entirety). In some embodiments, the RT domain forms a dimer (e.g., a heterodimer or a homodimer). In some embodiments, the RT domain is a monomer. In some embodiments, the RT domain naturally functions as a monomer or a dimer (e.g., a heterodimer or a homodimer). In some embodiments, the RT domain is derived from a virus that naturally functions as a monomer, e.g., functions as a monomer.In embodiments, the RT domain is selected from the group consisting of murine leukemia virus (MLV; also known as MoMLV) (e.g., P03355), porcine endogenous retrovirus (PERV) (e.g., UniProt Q4VFZ2), mouse mammary tumor virus (MMTV) (e.g., UniProt P03365), avian reticuloendotheliosis virus (AVIRE) (e.g., UniProtKB accession: P03360); feline leukemia virus (FLV or FeLV) (e.g., UniProtKB accession: P10273); Mason-Pfizer monkey virus (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 In some embodiments, the RT domain is selected from the RT domain or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity thereto) from a virus that functions as a dimer, such as a simian foamy virus (SFV) (e.g., SFV3L) (e.g., UniProt P23074 or P27401), ... or a bovine foamy / syncytial virus (BFV / BSV) (e.g., UniProt O41894). In some embodiments, the RT domain is a dimer in its native functionality. In some embodiments, the RT domain is derived from a virus that functions as a dimer.In embodiments, the RT domain is selected from the group consisting of avian sarcoma / leukemia virus (ASLV) (e.g., UniProt A0A142BKH1), Rous sarcoma virus (RSV) (e.g., UniProt P03354), avian myeloblastosis virus (AMV) (e.g., UniProt Q83133), human immunodeficiency virus 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 virus (BIV) (e.g., UniProt P19560), equine infectious anemia virus (EIAV) (e.g., UniProt P03371), or feline immunodeficiency virus (FIV) (e.g., UniProt P16088) (Herschhorn 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%, 80%, 90%, 95%, or 99% identity thereto). Naturally, heterodimeric RT domains may, in some embodiments, also be functional as homodimers. In some embodiments, the dimeric RT domain is expressed as a fusion protein, e.g., as a homodimeric fusion protein or a heterodimeric fusion protein. In some embodiments, the RT function of the system is fulfilled by multiple RT domains (e.g., as described herein).In further embodiments, the multiple RT domains can be fused or separate, for example, on the same polypeptide or on different polypeptides.

[0259] In some embodiments, the genetic modification systems described herein include an integrase domain, e.g., the integrase domain can be part of an RT domain. In some embodiments, the RT domain (e.g., as described herein) includes an integrase domain. In some embodiments, the RT domain (e.g., as described herein) lacks an integrase domain or includes an integrase domain that has been inactivated by mutation or deletion. In some embodiments, the genetic modification systems described herein include an RNase H domain, e.g., the RNase H domain can be part of the RT domain. In some embodiments, the RNase H domain is not part of the RT domain but is covalently linked via a flexible linker. In some embodiments, the RT domain (e.g., as described herein) includes an RNase H domain, e.g., an endogenous RNase H domain or a heterologous RNase H domain. In some embodiments, the 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 exchanged for a heterologous RNase H domain. In some embodiments, the polypeptide comprises an inactivated endogenous RNase H domain. In some embodiments, an endogenous RNase H domain from one of the polypeptide's other domains 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 that comprises the domain. In some embodiments, mutation of the RNase H domain produces a polypeptide that exhibits reduced RNase activity, e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less, compared to an otherwise similar domain without the mutation, e.g., as measured by the method in Kotewicz et al. Nucleic Acids Res 16(1):265-277 (1988), incorporated herein by reference in its entirety.In some embodiments, RNase H activity is abolished.

[0260] In some embodiments, the RT domain is mutated to increase fidelity compared to other similar domains that do not have the mutation. For example, in some embodiments, a YADD or YMDD motif in the RT domain (e.g., in a reverse transcriptase) is replaced with YVDD. In some embodiments, the YADD, or YMDD, or YVDD substitution results in higher fidelity in retroviral reverse transcriptase activity (e.g., as described in Jamburuthugoda and Eickbush J Mol Biol 2011; the entire contents of which are incorporated herein by reference).

[0261] In some embodiments, a genetically modified polypeptide described herein comprises an RT domain having an amino acid sequence according to Table 6, or a sequence with at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. In some embodiments, a nucleic acid described herein encodes an RT domain having an amino acid sequence according to Table 6, or a sequence with at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0262] [Table 1]

[0263] [Table 2]

[0264] [Table 3]

[0265] [Table 4]

[0266]

Table 5

[0267]

Table 6

[0268]

Table 7

[0269]

Table 8

[0270]

Table 9

[0271]

Table 10

[0272]

Table 11

[0273]

Table 12

[0274]

Table 13

[0275]

Table 14

[0276]

Table 15

[0277] [Table 16]

[0278] In some embodiments, the reverse transcriptase domain is modified, for example, by site-directed mutagenesis. In some embodiments, the reverse transcriptase domain is engineered to have improved properties, such as the SuperScript IV (SSIV) reverse transcriptase from MMLV RT. In some embodiments, the reverse transcriptase domain may be engineered to have a lower error rate, for example, as described in International Publication No. WO2001068895, incorporated herein by reference. In some embodiments, the reverse transcriptase domain may be engineered to have increased thermostability. In some embodiments, the reverse transcriptase domain may be engineered to have increased processivity. In some embodiments, the reverse transcriptase domain may be engineered to be resistant to inhibitors. In some embodiments, the reverse transcriptase domain may be engineered to be faster. In some embodiments, the reverse transcriptase domain may be engineered to have increased tolerance to modified nucleotides in the RNA template. In some embodiments, the reverse transcriptase domain may be engineered to insert modified DNA nucleotides. In some embodiments, the reverse transcriptase domain is engineered to bind to the template RNA. In some embodiments, the one or more mutations are selected from D200N, L603W, T330P, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L671P, E69K, H8Y, T306K, or D653N in the RT domain of murine leukemia virus reverse transcriptase, or a corresponding mutation at a corresponding position in another RT domain.

[0279] In some embodiments, the genetically modified polypeptide comprises an RT domain from a retroviral reverse transcriptase, such as, for example, wild-type M-MLV RT comprising the following sequence: M-MLV(WT): [ka]

[0280] In some embodiments, the genetically modified polypeptide comprises an RT domain from a retroviral reverse transcriptase, such as, for example, M-MLV RT, comprising the following sequence: [ka]

[0281] In some embodiments, the genetically modified polypeptide comprises an RT domain from a retroviral reverse transcriptase comprising the sequence of amino acids 659 to 1329 of NP_057933. In embodiments, the genetically modified polypeptide further comprises one additional amino acid at the N-terminus of the sequence of amino acids 659 to 1329 of NP_057933, for example, as shown below. [ka] [ka] RNAseH (underlined), annotation as above

[0282] In embodiments, the genetically modified polypeptide further comprises one additional amino acid at the C-terminus of the sequence of amino acids 659-1329 of NP_057933. In some embodiments, the genetically modified polypeptide comprises an RNase H1 domain (e.g., amino acids 1178-1318 of NP_057933).

[0283] In some embodiments, a retroviral reverse transcriptase domain, e.g., M-MLV RT, can contain one or more mutations from the wild-type sequence that can improve characteristics of the RT, such as thermostability, processivity, and / or template binding. In some embodiments, the M-MLV RT domain 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, K103L relative to the M-MLV(WT) sequence above, e.g., a combination of mutations such as D200N, L603W, and T330P, optionally further comprising T306K and W313F. In some embodiments, an M-MLV RT as used herein comprises the mutations D200N, L603W, T330P, T306K, and W313F. In embodiments, the mutant M-MLV RT comprises the following amino acid sequence: M-MLV(PE2): [ka]

[0284] In some embodiments, the writing domain (e.g., the RT domain) comprises an RNA-binding domain that specifically binds to, for example, an RNA sequence. In some embodiments, the template RNA comprises an RNA sequence that is specifically bound by the RNA-binding domain of the writing domain.

[0285] In some embodiments, the reverse transcription domain simply recognizes and reverse transcribes a specific template of the system, e.g., a template RNA. In some embodiments, the template comprises a sequence or structure that allows recognition and reverse transcription by the reverse transcription domain. In some embodiments, the template comprises a sequence or structure that allows association with an RNA-binding domain of a polypeptide component of a genome modification system described herein. In some embodiments, the genome modification system preferentially reverse transcribes a template that includes an association sequence over a template that lacks the association sequence.

[0286] The writing domain may also comprise DNA-dependent DNA polymerase activity, e.g., an enzymatic activity capable of writing DNA into a genome from a template DNA sequence. In some embodiments, DNA-dependent DNA polymerization is used to complete second strand synthesis of target site editing. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a DNA polymerase domain in the polypeptide. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a reverse transcriptase domain that is also capable of DNA-dependent DNA polymerization, e.g., second strand synthesis. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a second polypeptide of the system. In some embodiments, the DNA-dependent DNA polymerase activity is optionally provided by an endogenous host cell polymerase recruited to the target site by a component of the genome modification system.

[0287] In some embodiments, the reverse transcriptase domain exhibits a lower probability of poor termination (P) in vitro compared to a reference reverse transcriptase domain. off In some embodiments, the reference reverse transcriptase domain is a viral reverse transcriptase domain, for example, the RT domain from M-MLV.

[0288] In some embodiments, the reverse transcriptase domain has a nucleotide sequence of about 5×10 in vitro, e.g., as measured on 1094 nt RNA. -3 / nt, 5 × 10 -4 / nt, or 5 x 10 -6 A lower probability of insufficient termination (P off In some embodiments, insufficient termination rates in vitro are determined as described in Bibillo and Eickbush (2002) J Biol Chem 277(38):34836-34845, which is incorporated herein by reference in its entirety.

[0289] In some embodiments, the reverse transcriptase domain can complete at least about 30% or 50% of integrations in the cell. The percentage of complete integrations can be measured by dividing the number of substantially full-length integration events (e.g., genomic sites containing at least 98% of the expected integration sequence) by the total number of integration events (including substantially full-length and partial) in the cell population. In some embodiments, integration in the cell is determined (e.g., through the integration site) using long-read amplicon sequencing, as described, for example, in Karst et al. (2020) bioRxiv doi.org / 10.1101 / 645903 (incorporated herein by reference in its entirety).

[0290] In embodiments, quantifying integration in a cell comprises counting the percentage of integrations that comprise at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the DNA sequence corresponding to the template RNA (e.g., a template RNA having a length of at least 0.05, 0.1, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, or 5 kb, e.g., 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).

[0291] In some embodiments, the reverse transcriptase domain is capable of polymerizing dNTPs in vitro. In embodiments, the reverse transcriptase domain is capable of polymerizing dNTPs in vitro at a rate of 0.1 to 50 nt / sec (e.g., 0.1 to 1, 1 to 10, or 10 to 50 nt / sec). In embodiments, polymerization of dNTPs by the reverse transcriptase domain is measured by a single-molecule assay, e.g., as described in Schwartz and Quake (2009) PNAS 106(48):20294-20299, which is incorporated by reference in its entirety.

[0292] In some embodiments, the reverse transcriptase domain is at least 1×10 nucleotides in length, e.g., as described in Yasukawa et al. (2017) Biochem Biophys Res Commun 492(2):147-153, which is incorporated herein by reference in its entirety. -3 ~1×10 -4 or 1 x 10 -4 ~1×10 -5 In some embodiments, the reverse transcriptase domain has an in vitro error rate (e.g., nucleotide misincorporation) of 1×10 substitutions / nt. In some embodiments, the reverse transcriptase domain is sequenced at 1×10 nucleotides per nucleotide in cells (e.g., HEK293T cells), e.g., by long-read amplicon sequencing, as described, e.g., in Karst et al. (2020) bioRxiv doi.org / 10.1101 / 645903 (incorporated herein by reference in its entirety). -3 ~1×10 -4 or 1 x 10 -4 ~1×10 -5 It has an error rate (e.g., nucleotide misincorporation) of substitutions / nt.

[0293] In some embodiments, the reverse transcriptase domain is capable of performing reverse transcription of the target RNA in vitro. In some embodiments, the reverse transcriptase requires a primer of at least 3 nucleotides to initiate reverse transcription of the template. In some embodiments, reverse transcription of the target RNA is determined by detecting cDNA from the target RNA (e.g., when an ssDNA primer is provided that anneals to the 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).

[0294] In some embodiments, the reverse transcriptase domain performs reverse transcription (e.g., by generating cDNA) at least 5-fold or 10-fold more efficiently, e.g., when converting its RNA template to cDNA, compared to, e.g., an RNA template lacking a protein-binding motif (e.g., a 3'UTR). In embodiments, the efficiency of reverse transcription is measured as described in Yasukawa et al. (2017) Biochem Biophys Res Commun 492(2):147-153, which is incorporated herein by reference in its entirety.

[0295] In some embodiments, the reverse transcriptase domain specifically binds to a particular RNA template at a higher frequency (e.g., about 5-fold or 10-fold higher frequency) than any endogenous cellular RNA, e.g., when expressed in a cell (e.g., HEK293T cell). In embodiments, the frequency of specific binding between the reverse transcriptase domain and the template RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(11):5490-5501, which is incorporated herein by reference in its entirety.

[0296] In some embodiments, an RT domain (e.g., as listed in Table 6) comprises one or more mutations as listed in Table 2 below. In some embodiments, an RT domain as listed in Table 6 comprises 1, 2, 3, 4, 5, or 6 of the mutations listed in the corresponding row of Table 2 below.

[0297] [Table 17]

[0298] [Table 18]

[0299] [Table 19]

[0300] [Table 20]

[0301] template nucleic acid binding domain Genetically modified polypeptides typically have a region that can associate with template nucleic acids (e.g., template RNA). In some embodiments, the template nucleic acid binding domain is an RNA binding domain. In some embodiments, the RNA binding domain is a modular domain that can associate with RNA molecules that contain specific signatures, such as structural motifs. In other embodiments, the template nucleic acid binding domain (e.g., RNA binding domain) is contained within a reverse transcription domain, for example, a component derived from reverse transcriptase has a known signature of RNA preference.

[0302] In other embodiments, the template nucleic acid binding domain (e.g., RNA binding domain) is contained within the target DNA binding domain. For example, in some embodiments, the DNA binding domain is a CRISPR-associated protein that recognizes the structure of a template nucleic acid (e.g., template RNA) that includes a gRNA. In some embodiments, the genetically modified polypeptide comprises a DNA binding domain that includes a CRISPR-associated protein that associates with a gRNA scaffold, allowing the DNA binding domain to bind to a target genomic DNA sequence. In some embodiments, the gRNA scaffold and gRNA spacer are contained within the template nucleic acid (e.g., template RNA), such that the DNA binding domain is also the template nucleic acid binding domain. In some embodiments, the polypeptide has RNA binding function in multiple domains, e.g., it may bind to a gRNA structure within the CRISPR-associated DNA binding domain and an additional sequence or structure within the reverse transcriptase domain.

[0303] In some embodiments, the RNA-binding domain can bind to the template RNA with higher affinity than a standard RNA-binding domain. In some embodiments, the standard RNA-binding domain is an RNA-binding domain from S. pyogenes Cas9. In some embodiments, the RNA-binding domain can bind to the template RNA with an affinity of 100 pM to 10 nM (e.g., 100 pM to 1 nM or 1 nM to 10 nM). In some embodiments, the affinity of the RNA-binding domain for its template RNA is measured in vitro, e.g., by thermophoresis, as described, e.g., in Asmari et al. Methods 146:107-119 (2018), which is incorporated herein by reference in its entirety. In some embodiments, the affinity of the RNA-binding domain for its template RNA is measured in a cell (e.g., by FRET or CLIP-Seq).

[0304] In some embodiments, the RNA-binding domain associates with the template RNA in vitro at least about 5-fold or 10-fold more frequently than scrambled RNA. In some embodiments, the frequency of association between the RNA-binding domain and the template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(11):5490-5501, incorporated herein by reference in its entirety. In some embodiments, the RNA-binding domain associates with the template RNA in cells (e.g., HEK293T cells) at least about 5-fold or 10-fold more frequently than scrambled RNA. In some embodiments, the frequency of association between the RNA-binding domain and the template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019) supra.

[0305] Endonuclease domain and DNA binding domain In some embodiments, the genetically modified polypeptide functions to cleave a DNA target site via an endonuclease domain. In some embodiments, the genetically modified polypeptide comprises, for example, a DNA binding domain for binding to a target nucleic acid. In some embodiments, a domain of the genetically modified polypeptide (e.g., a Cas domain) comprises two or more smaller domains, for example, a DNA binding domain and an endonuclease domain. When a DNA binding domain (e.g., a Cas domain) is described as binding to a target nucleic acid sequence, it is understood that in some embodiments, the binding is mediated by a gRNA.

[0306] In some embodiments, the domain has two functions. For example, in some embodiments, the endonuclease domain is also a DNA-binding domain. In some embodiments, the endonuclease domain is also a template nucleic acid (e.g., template RNA)-binding domain. For example, in some embodiments, the polypeptide comprises a CRISPR-associated endonuclease domain that binds to a template RNA, including a gRNA, binds to a target DNA sequence (e.g., has complementarity to a portion of the gRNA), and cleaves the target DNA sequence. In some embodiments, an endonuclease domain or endonuclease / DNA-binding domain derived from a heterologous source can be used or modified (e.g., by inserting, deleting, or substituting one or more residues) in the genetic modification systems described herein.

[0307] In some embodiments, the nucleic acid encoding the endonuclease domain or endonuclease / DNA-binding domain is modified from its native sequence to have modified 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., Fok1), a meganuclease (e.g., I-SceI), or other endonuclease domain.

[0308] In certain aspects, the DNA-binding domain of the genetically modified polypeptide described herein is selected, designed, or engineered for binding to a desired host DNA target sequence. In certain embodiments, the DNA-binding domain of the polypeptide is a heterologous DNA-binding factor. In some embodiments, the heterologous DNA-binding factor is a zinc finger factor or TAL effector factor, e.g., a zinc finger or TAL polypeptide or a functional fragment thereof. In some embodiments, the heterologous DNA-binding factor is a sequence-guided DNA-binding factor, such as Cas9, Cpfl, or other CRISPR-associated protein, that has been modified to lack endonuclease activity. In some embodiments, the heterologous DNA-binding factor retains endonuclease activity. In some embodiments, the heterologous DNA-binding factor retains partial endonuclease activity, such as cleaving ssDNA, e.g., has nickase activity. In certain embodiments, the heterologous DNA-binding domain can be any one or more of Cas9, a TAL domain, a ZF domain, a Myb domain, a combination thereof, or a complex thereof.

[0309] In some embodiments, the DNA-binding domain is modified, e.g., by site-directed mutagenesis, to increase or decrease DNA-binding factors (e.g., the number and / or specificity of zinc fingers), etc., to alter DNA-binding specificity and affinity. In some embodiments, the nucleic acid sequence encoding the DNA-binding domain is modified from its native sequence to have altered codon usage, e.g., improved for human cells. In several embodiments, the DNA-binding domain includes one or more modifications relative to the wild-type DNA-binding domain, e.g., modifications by directed evolution, e.g., phage-assisted continuous evolution (PACE).

[0310] In some embodiments, the DNA-binding domain comprises a meganuclease domain (e.g., an endonuclease domain portion, e.g., as described herein), or a functional fragment thereof. In some embodiments, the meganuclease domain has endonuclease activity, e.g., double-strand cleavage and / or nickase activity. In other embodiments, the 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 the DNA-binding domain, e.g., as described in Fonfara et al. Nucleic Acids Res 40(2):847-860 (2012), which is incorporated herein by reference in its entirety.

[0311] In some embodiments, the genetically modified polypeptide comprises modifications to the DNA-binding domain, e.g., compared to the wild-type polypeptide. In some embodiments, the DNA-binding domain comprises additions, deletions, substitutions, or modifications to the amino acid sequence of the original DNA-binding domain. In some embodiments, the DNA-binding domain is modified to comprise a heterologous functional domain that specifically binds to a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the functional domain replaces at least a portion (e.g., the entirety) of a previous DNA-binding domain of the polypeptide. In some embodiments, the functional domain comprises a zinc finger (e.g., a zinc finger that specifically binds to a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the 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, the Cas domain comprises Cas9 or a mutant or variant thereof (e.g., as described herein). In embodiments, the Cas domain is associated with a guide RNA (gRNA), e.g., as described herein. In embodiments, the Cas domain is guided to the target nucleic acid (e.g., DNA) sequence of interest by the gRNA. In some embodiments, the Cas domain is encoded in the same nucleic acid (e.g., RNA) molecule as the gRNA. In some embodiments, the Cas domain is encoded in a different nucleic acid (e.g., RNA) molecule than the gRNA.

[0312] In some embodiments, the DNA-binding domain can bind to a target sequence (e.g., a dsDNA target sequence) with higher affinity than a standard DNA-binding domain. In some embodiments, the standard DNA-binding domain is a DNA-binding domain from S. pyogenes Cas9. In some embodiments, the DNA-binding domain can bind to a target sequence (e.g., a dsDNA target sequence) with an affinity of 100 pM to 10 nM (e.g., 100 pM to 1 nM or 1 nM to 10 nM).

[0313] In some embodiments, the affinity of a DNA binding domain for its target sequence (e.g., a dsDNA target sequence) is measured in vitro, e.g., by thermophoresis, as described, e.g., in Asmari et al. Methods 146:107-119 (2018), which is incorporated herein by reference in its entirety.

[0314] In embodiments, the DNA-binding domain can bind to its target sequence (e.g., a dsDNA target sequence) with an affinity of, for example, 100 pM to 10 nM (e.g., 100 pM to 1 nM or 1 nM to 10 nM) in the presence of a molar excess, e.g., about a 100-fold molar excess, of scrambled sequence competitor dsDNA.

[0315] In some embodiments, the DNA-binding domain is found to bind to its target sequence (e.g., a dsDNA target sequence) at a higher frequency than any other sequence in the genome of the target cell, e.g., a human target cell, as measured by, e.g., ChIP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21, which is incorporated herein by reference in its entirety. In some embodiments, the DNA-binding domain is found to bind to its target sequence (e.g., a dsDNA target sequence) at a frequency at least about 5-fold or 10-fold higher than any other sequence in the genome of the target cell, as measured by, e.g., ChIP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010) supra.

[0316] In some embodiments, the endonuclease domain has nickase activity and cleaves one strand of the target DNA. In some embodiments, the nickase activity reduces the formation of double-strand breaks at the target site. In some embodiments, the endonuclease domain generates staggered nicks in the first and second strands of the target DNA. In some embodiments, the staggered nicks generate free 3' overhangs at the target site. In some embodiments, the free 3' overhangs at the target site improve editing efficiency, for example, by enhancing access and annealing of the 3' homologous region of the template nucleic acid. In some embodiments, the staggered nicks reduce the formation of double-strand breaks at the target site.

[0317] In some embodiments, the endonuclease domain cleaves both strands of the target DNA, e.g., resulting in a blunt-end cleavage of the target with no ssDNA overhangs on either side of the cleavage site. The amino acid sequence of the endonuclease domain of the genetic modification system described herein can 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%, or at least about 99% identical to the amino acid sequence of an endonuclease domain described herein, e.g., an endonuclease domain from Table 8.

[0318] In certain embodiments, the heterologous endonuclease is Fok1 or a functional fragment thereof. In certain embodiments, the heterologous endonuclease is a Holliday junction resolvase or a homolog thereof, such as the Holliday junction cleavage enzyme (Ssol Hje) from Sulfolobus solfataricus (Govindaraju et al., Nucleic Acids Research 44:7, 2016). In certain embodiments, the heterologous endonuclease is a large fragment endonuclease of a spliceosomal protein, such as Prp8 (Mahbub et al., Mobile DNA 8:16, 2017). In certain embodiments, the heterologous endonuclease is derived from a CRISPR-associated protein, such as Cas9. In certain embodiments, the heterologous endonuclease is modified to have only ssDNA cleavage activity, e.g., only nickase activity, e.g., a Cas9 nickase, e.g., SpCas9 with a D10A, H840A, or N863A mutation. Table 8 lists exemplary Cas proteins and mutations associated with nickase activity. In yet other embodiments, the homologous endonuclease domain is modified, e.g., by site-directed mutagenesis, to alter DNA endonuclease activity. In yet other embodiments, the endonuclease domain is modified to reduce DNA sequence specificity, e.g., by truncation to remove a domain that confers DNA sequence specificity or mutations to inactivate the region that confers DNA sequence specificity.

[0319] In some embodiments, the endonuclease domain has nickase activity and does not form double-stranded breaks. In some embodiments, the endonuclease domain forms single-stranded breaks more frequently than double-stranded breaks, for example, at least 90%, 95%, 96%, 97%, 98%, or 99% of the cuts are single-stranded breaks, or less than 10%, 5%, 4%, 3%, 2%, or 1% of the cuts are double-stranded breaks. In some embodiments, the endonuclease does not substantially form double-stranded breaks. In some embodiments, the endonuclease does not form detectable levels of double-stranded breaks.

[0320] In some embodiments, the endonuclease domain has a nickase activity that nicks the target site DNA of the first strand; for example, in some embodiments, the endonuclease domain cleaves the genomic DNA of the target site near the modification site on the strand that will be extended by the writing domain. In some embodiments, the endonuclease domain has a nickase activity that nicks the target site DNA of the first strand but does not nick the target site DNA of the second strand. For example, when a polypeptide comprises a CRISPR-associated endonuclease domain with nickase activity, in some embodiments, the CRISPR-associated endonuclease domain nicks the target site DNA strand that contains the PAM site (e.g., does not nick the target site DNA strand that does not contain the PAM site). By way of further example, when a polypeptide comprises a CRISPR-associated endonuclease domain with nickase activity, in some embodiments, the CRISPR-associated endonuclease domain nicks the target site DNA strand that does not contain a PAM site (e.g., and does not nick the target site DNA strand that contains a PAM site).

[0321] In some other embodiments, the endonuclease domain has nickase activity, which creates nicks in the first and second strands of target site DNA. Without intending to be bound by theory, after the writing domain (e.g., RT domain) of a polypeptide described herein polymerizes (e.g., reverse transcribes) from a heterologous target sequence of a template nucleic acid (e.g., template RNA), the cellular DNA repair machinery must repair the nick on the first DNA strand. The target site DNA here contains two distinct sequences relative to the first DNA strand: one corresponding to the original genomic DNA (e.g., with a free 5' end) and the second corresponding to that polymerized from the heterologous target sequence (e.g., with a free 3' end). It is believed that the two distinct sequences equilibrate with each other, with first one hybridizing to the second strand, followed by the other, and the order of incorporation of the cellular DNA repair machinery into its repair target site is a stochastic process. Without intending to be bound by any particular theory, it is believed that the introduction of an additional nick into the second strand can bias cellular DNA repair mechanisms to use sequences based on the heterologous target sequence more frequently than the original genomic sequence (Anzalone et al. Nature 576:149-157 (2019)). In some embodiments, the additional nick is positioned at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides 5' or 3' of the target site modification (e.g., insertion, deletion, or substitution) or relative to the nick on the first strand.

[0322] Alternatively or additionally, without intending to be bound by any particular theory, it is believed that an additional nick in the second strand may facilitate second strand synthesis. In some embodiments, when the genetic modification system inserts or replaces a portion of the first strand, synthesis of a new sequence corresponding to the insertion / substitution in the second strand is required.

[0323] In some embodiments, the polypeptide comprises a single domain having endonuclease activity (e.g., a single endonuclease domain), which nicks both the first strand and the second strand. For example, in such embodiments, the endonuclease domain can be a CRISPR-associated endonuclease domain, and the 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, the polypeptide comprises multiple domains having endonuclease activity, wherein 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., is unable to) nick the second strand and the second endonuclease domain does not (e.g., is unable to) nick the first strand).

[0324] In some embodiments, the endonuclease domain can nick the first and second strands. In some embodiments, the first and second strand nicks occur at the same position in the target site, but not on opposite strands. In some embodiments, the second strand nick occurs at a staggered position, e.g., upstream or downstream from the first nick. In some embodiments, the endonuclease domain generates a deletion of the target site when the second strand nick is upstream of the first strand nick. In some embodiments, the endonuclease domain generates a duplication of the target site when the second strand nick is downstream of the first strand nick. In some embodiments, the endonuclease domain does not generate a duplication and / or deletion when the first and second strand nicks occur at the same position in the target site. In some embodiments, the endonuclease domain has altered activity depending on the protein conformation or RNA binding state, for example, to promote first strand or second strand nicking (e.g., as described in Christensen et al. PNAS 2006; incorporated herein by reference in its entirety).

[0325] In some embodiments, the endonuclease domain comprises a meganuclease, or a functional fragment thereof. In some embodiments, the endonuclease domain comprises a homing endonuclease, or a functional fragment thereof. In some embodiments, the endonuclease domain comprises a meganuclease from the LAGLIDADG, GIY-YIG, HNH, His-Cys Box, or PD-(D / E)XK family, e.g., having a conserved amino acid motif, e.g., as indicated in the family name, or a functional fragment or variant thereof. In some embodiments, the endonuclease domain comprises a meganuclease, or a fragment thereof, selected from, for example, I-SmaMI (Uniprot F7WD42), I-SceI (Uniprot P03882), I-AniI (Uniprot P03880), I-DmoI (Uniprot P21505), I-CreI (Uniprot P05725), I-TevI ​​(Uniprot P13299), I-OnuI (Uniprot Q4VWW5), or I-BmoI (Uniprot Q9ANR6). In some embodiments, the meganuclease is naturally a monomer, e.g., I-SceI, I-TevI, or a dimer, e.g., I-CreI, in its functional form. For example, LAGLIDADG meganucleases with a single copy of the LAGLIDADG motif generally form homodimers, while members with two copies of the LAGLIDADG motif are generally found as monomers. In some embodiments, meganucleases that normally form as dimers are expressed as fusions, e.g., two subunits are expressed as a single ORF, optionally linked by a linker, e.g., an I-CreI dimer fusion (Rodriguez-Fornes et al. Gene Therapy 2020; the entire contents of which are incorporated herein by reference).In some embodiments, meganucleases, or functional fragments thereof, are engineered to preferentially exhibit nickase activity in one strand of a double-stranded DNA molecule, e.g., I-SceI (K122I and / or K223I) (Niu et al. J Mol Biol 2008), I-AniI (K227M) (McConnell Smith et al. PNAS 2009), I-DmoI (Q42A and / or K120M) (Molina et al. J Biol Chem 2015). In some embodiments, meganucleases or functional fragments thereof with this preference for single-strand cleavage are used, for example, as endonuclease domains with nickase activity. In some embodiments, the endonuclease domain comprises a meganuclease, or a functional fragment thereof, that naturally targets or has been engineered to target a safe harbor site, e.g., an SH6 site that targets I-CreI (Rodriguez-Fornes et al., supra). In some embodiments, the endonuclease domain comprises a meganuclease, or a functional fragment thereof, with a sequence-tolerant catalytic domain, e.g., I-TevI, which recognizes the minimal motif CNNNG (Kleinstiver et al. PNAS 2012). In some embodiments, the target sequence-resistant catalytic domain is fused to a DNA-binding domain, e.g., fusion of I-TevI ​​to (i) a Zn finger to create Tev-ZFE (Kleinstiver et al. PNAS 2012), (ii) another meganuclease to create MegaTev (Wolfs et al. Nucleic Acids Res 2014), and / or (iii) Cas9 to create TevCas9 (Wolfs et al. PNAS 2016) induces activity.

[0326] In some embodiments, the endonuclease domain comprises a restriction enzyme, e.g., a Type IIS or Type IIP restriction enzyme. In some embodiments, the endonuclease domain comprises a Type IIS restriction enzyme, e.g., FokI, or a fragment or variant thereof. In some embodiments, the endonuclease domain comprises a Type IIP restriction enzyme, e.g., PvuII, or a fragment or variant thereof. In some embodiments, the dimeric restriction enzyme is expressed as a fusion, e.g., a FokI dimer fusion, such that it functions as a single strand (Minczuk et al. Nucleic Acids Res 36(12):3926-3938 (2008)).

[0327] 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.

[0328] In some embodiments, the genetically modified polypeptide comprises a modification to the endonuclease domain, e.g., compared to a wild-type Cas protein. In some embodiments, the endonuclease domain comprises an addition, deletion, substitution, or modification to the amino acid sequence of a wild-type Cas protein. In some embodiments, the endonuclease domain is modified to comprise a heterologous functional domain that specifically binds to and / or directs endonucleolytic cleavage of a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the endonuclease domain comprises a zinc finger. In several embodiments, the endonuclease domain, including a Cas domain, associates with a guide RNA (gRNA), e.g., as described herein. In some embodiments, the endonuclease domain is modified to comprise a functional domain that does not target a specific target nucleic acid (e.g., DNA) sequence. In several embodiments, the endonuclease domain comprises a Fok1 domain.

[0329] In some embodiments, the endonuclease domain associates with the target dsDNA at least about 5-fold or 10-fold more frequently than scrambled dsDNA in vitro. In some embodiments, the endonuclease domain associates with the target dsDNA at least about 5-fold or 10-fold more frequently than scrambled dsDNA in vitro, e.g., in a cell (e.g., HEK293T cell). In some embodiments, the frequency of association between the endonuclease domain and the target DNA or scrambled DNA is measured by ChIP-seq, e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21, incorporated herein by reference in its entirety.

[0330] In some embodiments, the endonuclease domain may catalyze the formation of nicks at the target sequence, e.g., by at least about a 5-fold or 10-fold increase, 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 nicking is measured using Nick-Seq, e.g., as described in Elacqua et al. (2019) bioRxiv doi.org / 10.1101 / 867937, which is incorporated by reference in its entirety.

[0331] In some embodiments, the endonuclease domain is capable of nicking DNA in vitro. In embodiments, the nick results in an exposed base. In embodiments, the 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 herein by reference in its entirety. In embodiments, the level of exposed bases (e.g., as detected by a nuclease sensitivity assay) is increased by at least 10%, 50%, or more compared to the reference endonuclease domain. In some embodiments, the reference endonuclease domain is an endonuclease domain from Cas9 of Streptococcus pyogenes (S. pyogenes).

[0332] In some embodiments, the endonuclease domain is capable of nicking DNA in a cell. In embodiments, the endonuclease domain is capable of nicking DNA in a HEK293T cell. In embodiments, unrepaired nicks that undergo replication in the absence of Rad51 result in an increased rate of NHEJ at the site of the nick, detectable, for example, by using a Rad51 inhibition assay, e.g., as described in Bothmer et al. (2017) Nat Commun 8:13905 (incorporated herein by reference in its entirety). In embodiments, the NHEJ rate is increased by more than 0-5%. In embodiments, the NHEJ rate is increased, for example, by 20-70% (e.g., 30%-60% or 40-50%) upon Rad51 inhibition.

[0333] In some embodiments, the endonuclease domain releases the target after cleavage. In some embodiments, target release is indicated indirectly by assessing multiple enzymatic turnover, e.g., as described in Yourik at al. RNA 25(1):35-44 (2019) (incorporated herein by reference in its entirety) and as shown in Figure 2. In some embodiments, the k of the endonuclease domain exp is measured by this method and is 1×10 -3 ~1×10 -5 Minute -1.

[0334] In some embodiments, the endonuclease domain is capable of binding to about 1 x 10 8 s -1 M -1 Catalytic efficiency (k cat / K m In some embodiments, the endonuclease domain has a nucleotide sequence of about 1 x 10 in vitro. 5 , 1×10 6 , 1×10 7 , or 1×10 8 s -1 M -1 In some embodiments, the catalytic efficiency is determined as described in Chen et al. (2018) Science 360(6387):436-439, which is incorporated herein by reference in its entirety. In some embodiments, the endonuclease domain has a catalytic efficiency of greater than about 1×10 in a cell. 8 s -1 M -1 Catalytic efficiency (k cat / K m In some embodiments, the endonuclease domain has a denaturing activity of about 1×10 5 , 1×10 6 , 1×10 7 , or 1×10 8 s -1 M -1 It has a catalytic efficiency of more than

[0335] Genetically modified polypeptides containing Cas domains In some embodiments, the genetic modification polypeptide described herein comprises a Cas domain. In some embodiments, the Cas domain can guide the genetic modification polypeptide to a target site specified by a gRNA spacer, thereby modifying the target nucleic acid sequence in "cis." In some embodiments, the genetic modification polypeptide is fused to a Cas domain. In some embodiments, the genetic modification polypeptide comprises a CRISPR / Cas domain (also referred to herein as a CRISPR-associated protein). In some embodiments, the CRISPR / Cas domain comprises a protein involved in the clustered regularly interspaced short palindromic repeats (CRISPR) system, e.g., a Cas protein, and optionally binds to a guide RNA, e.g., a single guide RNA (sgRNA).

[0336] The CRISPR system is an adaptive defense system first discovered in bacteria and archaea. CRISPR systems use RNA-guided nucleases called CRISPR-associated or "Cas" endonucleases (e.g., Cas9 or Cpf1) to cleave foreign DNA. For example, in a typical CRISPR-Cas system, the endonuclease is guided to a target nucleotide sequence (e.g., a site in the genome to be sequence-edited) by a sequence-specific, non-coding "guide RNA" that targets single- or double-stranded DNA sequences. Three classes of CRISPR systems (I-III) have been identified. 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 typically contains a "spacer" sequence (protospacer), an approximately 20-nucleotide RNA sequence that corresponds to the target DNA sequence. In wild-type systems, and in some engineered systems, the crRNA binds to the tracrRNA, forming a partially double-stranded structure that is cleaved by RNase III.cThe crRNA / tracrRNA hybrid molecule also contains a region that results in a rRNA / tracrRNA hybrid molecule. The crRNA / tracrRNA hybrid then guides the Cas endonuclease to recognize and cleave the target DNA sequence. The target DNA sequence is generally flanked by a "protospacer adjacent motif" ("PAM") that is specific for a given Cas endonuclease and required for cleavage activity at the target site that matches the spacer of the crRNA. CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements, for example, as listed for exemplary Cas enzymes in Table 7; example 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, such as the Cas9 endonuclease, associate with G-rich PAM sites, e.g., 5'-NGG; SEQ ID NO: 11,023, and perform blunt-end cleavage of target DNA three nucleotides upstream (5') from the PAM site. Another class II CRISPR system includes the V-type endonuclease Cpf1, which is smaller than Cas9; examples include AsCpf1 (from Acidaminococcus sp.) and LbCpf1 (from Lachnospiraceae sp.). Cpf1-associated CRISPR arrays do not require tracrRNA and are processed into mature crRNA; in other words, the Cpf1 system, in some embodiments, exclusively contains the Cpf1 nuclease and crRNA to cleave the target DNA sequence. The Cpf1 endonuclease typically associates with T-rich PAM sites, e.g., 5'-TTN. Cpf1 can also recognize the 5'-CTA PAM motif.Cpf1 typically cleaves target DNA by introducing an offset or staggered double-stranded break into a 4- or 5-nucleotide 5' overhang, e.g., cleaving the target DNA with a 5-nucleotide offset or staggered break located 18 nucleotides downstream (3') from the PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complementary strand; the 5-nucleotide overhang resulting from such an offset break allows for more precise genome editing by DNA insertion via homologous recombination rather than insertion with blunt-ended cut DNA. See, e.g., Zetsche et al. (2015) Cell, 163:759-771.

[0337] Various CRISPR-associated (Cas) genes or proteins can be used in the techniques provided by the present disclosure, and the choice of Cas protein will depend on the specific requirements of the method. Specific examples of Cas proteins include Class II systems, including Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpf1, C2C1, or C2C3. In some embodiments, the Cas protein, e.g., the Cas9 protein, can be derived from any of a variety of prokaryotic species. In some embodiments, a particular Cas protein, e.g., a particular Cas9 protein, is selected to recognize a particular protospacer adjacent motif (PAM) sequence. In some embodiments, the DNA-binding domain or endonuclease domain comprises a sequence-targeting polypeptide, such as a Cas protein, e.g., Cas9. In certain embodiments, the Cas protein, e.g., the Cas9 protein, can be obtained from bacteria or archaea, or can be synthesized using known methods. In certain embodiments, the Cas protein can be derived from Gram-positive or Gram-negative bacteria. In certain embodiments, the Cas protein is selected from the group consisting of Streptococcus (e.g., S. pyogenes or S. thermophilus), Francisella (e.g., F. novicida), Staphylococcus (e.g., S. aureus), Acidaminococcus (e.g., Acidaminococcus sp. BV3L6), and the like. sp. BV3L6), Neisseria (e.g., N. meningitidis), Cryptococcus, Corynebacterium, Haemophilus, Eubacterium, Pasteurella, Prevotella, Veillonella, or Marinobacter.

[0338] In some embodiments, the genetically modified polypeptide can comprise the amino acid sequence of SEQ ID NO: 4000, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In embodiments, the amino acid sequence of SEQ ID NO: 4000, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is located at the N-terminus of the genetically modified polypeptide. In some embodiments, the amino acid sequence of SEQ ID NO: 4000, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the N-terminus of the genetically modified polypeptide.

[0339] Exemplary N-terminal NLS-Cas9 domains [ka]

[0340] In some embodiments, the genetically modified polypeptide can comprise the amino acid sequence of SEQ ID NO: 4001, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In embodiments, the amino acid sequence of SEQ ID NO: 4001, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is located at the C-terminus of the genetically modified polypeptide. In some embodiments, the amino acid sequence of SEQ ID NO: 4001, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is located within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the C-terminus of the genetically modified polypeptide.

[0341] Exemplary C-terminal sequences containing an NLS AGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 4001)

[0342] Example benchmark sequence [ka] [ka]

[0343] In some embodiments, the genetically modified polypeptide may comprise a Cas domain listed in Tables 7 or 8, or a functional fragment thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

[0344] [Table 21]

[0345] [Table 22]

[0346] [Table 23]

[0347] [Table 24]

[0348] [Table 25]

[0349] [Table 26]

[0350] Table 27

[0351] Table 28

[0352] Table 29

[0353] Table 30

[0354] Table 31

[0355] Table 32

[0356] Table 33

[0357] Table 34

[0358] Table 35

[0359] Table 36

[0360] Table 37

[0361] In some embodiments, Cas proteins require a protospacer adjacent motif (PAM) to be present within or adjacent to the target DNA sequence to which the Cas protein binds and / or functions. In some embodiments, the 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 represents any nucleotide, Y represents C or T, R represents A or G, and V represents A, C, or G. In some embodiments, the Cas protein is a protein listed in Table 7 or 8. In some embodiments, the Cas protein comprises one or more mutations that alter its PAM. In some embodiments, the Cas protein comprises E1369R, E1449H, and R1556A mutations or analogous substitutions for the amino acids corresponding to said positions. In some embodiments, the Cas protein comprises E782K, N968K, and R1015H mutations or analogous substitutions for the amino acids corresponding to said positions. In some embodiments, the Cas protein comprises D1135V, R1335Q, and T1337R mutations or analogous substitutions for the amino acids corresponding to said positions. In some embodiments, the Cas protein comprises S542R and K607R mutations or analogous substitutions for the amino acids corresponding to said positions. In some embodiments, the Cas protein comprises S542R, K548V, and N552R mutations or analogous substitutions for the amino acids corresponding to said positions. Exemplary advances in modifying Cas enzymes to recognize modified PAM sequences are reviewed in Collias et al. Nature Communications 12:555 (2021), the entire contents of which are incorporated herein by reference.

[0362] In some embodiments, the Cas protein is catalytically active and cleaves one or both strands of the target DNA site, and in some embodiments, following cleavage of the target DNA site, a modification, e.g., an insertion or deletion, is formed, e.g., by cellular repair mechanisms.

[0363] In some embodiments, the Cas protein is modified to inactivate or partially inactivate the nuclease, e.g., nuclease-deficient Cas9. While wild-type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by gRNAs, several CRISPR endonucleases with modified functionality are available, e.g., partially inactivated "nickase" versions of Cas9 generate only single-strand breaks; catalytically inactive Cas9 ("dCas9") does not cleave target DNA. In some embodiments, binding of dCas9 to a DNA sequence can interfere with transcription at that site due to steric hindrance. In some embodiments, binding of dCas9 to an anchor sequence can interfere with (e.g., reduce or prevent) the formation and / or maintenance of a genome complex (e.g., ASMC). In some embodiments, the DNA-binding domain comprises a catalytically inactive Cas9, e.g., dCas9. Numerous catalytically inactive Cas9 proteins are known in the art. In some embodiments, dCas9 comprises mutations, e.g., D10A and H840A or N863A mutations, within each endonuclease domain of the Cas protein. 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 7. In some embodiments, a Cas protein listed in a given row of Table 7 comprises one, two, three, or all of the mutations listed in the same row of Table 7. In some embodiments, for example, a Cas protein not listed in Table 7 comprises one, two, three, or all of the mutations listed in a row of Table 7, or corresponding mutations at corresponding sites in the Cas protein.

[0364] In some embodiments, catalytically inactive, e.g., dCas9, or partially inactivated Cas9 proteins comprise a D11 mutation (e.g., a D11A mutation) or an analogous substitution for the amino acid corresponding to said position. In some embodiments, catalytically inactive Cas9 proteins, e.g., dCas9, or partially inactivated Cas9 proteins comprise a H969 mutation (e.g., a H969A mutation) or an analogous substitution for the amino acid corresponding to said position. In some embodiments, catalytically inactive Cas9 proteins, e.g., dCas9, or partially inactivated Cas9 proteins comprise a N995 mutation (e.g., a N995A mutation) or an analogous substitution for the amino acid corresponding to said position. In some embodiments, catalytically inactive Cas9 proteins, e.g., dCas9, comprise mutations at one, two, or three of positions D11, H969, and N995 (e.g., a D11A, H969A, and N995A mutations) or an analogous substitution for the amino acid corresponding to said positions.

[0365] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or a partially inactivated Cas9 protein, comprises a D10 mutation (e.g., a D10A mutation) or an analogous substitution for the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or a partially inactivated Cas9 protein, comprises a H557 mutation (e.g., a H557A mutation) or an analogous substitution for the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D10 mutation (e.g., a D10A mutation) and a H557 mutation (e.g., a H557A mutation) or an analogous substitution for the amino acid corresponding to said position.

[0366] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or a partially inactivated Cas9 protein, comprises a D839 mutation (e.g., a D839A mutation) or an analogous substitution for the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or a partially inactivated Cas9 protein, comprises a H840 mutation (e.g., a H840A mutation) or an analogous substitution for the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or a partially inactivated Cas9 protein, comprises a N863 mutation (e.g., a N863A mutation) or an analogous substitution for the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a D10 mutation (e.g., D10A), a D839 mutation (e.g., D839A), an H840 mutation (e.g., H840A), and an N863 mutation (e.g., N863A) or an analogous substitution for the amino acids corresponding to the positions.

[0367] In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, or partially inactivated Cas9 protein, comprises an E993 mutation (e.g., an E993A mutation) or an analogous substitution for the amino acid corresponding to said position.

[0368] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or a partially inactivated Cas9 protein, comprises a D917 mutation (e.g., a D917A mutation) or an analogous substitution for the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or a partially inactivated Cas9 protein, comprises an E1006 mutation (e.g., an E1006A mutation) or an analogous substitution for the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or a partially inactivated Cas9 protein, comprises a D1255 mutation (e.g., a D1255A mutation) or an analogous substitution for 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), an E1006 mutation (e.g., E1006A), and a D1255 mutation (e.g., D1255A) or an analogous substitution for the amino acid corresponding to said position.

[0369] In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, or partially inactivated Cas9 protein, comprises a D16 mutation (e.g., a D16A mutation) or an analogous substitution for the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, or partially inactivated Cas9 protein, comprises a D587 mutation (e.g., a D587A mutation) or an analogous substitution for the amino acid corresponding to said position. In some embodiments, the partially inactivated Cas domain has nickase activity. In some embodiments, the partially inactivated Cas9 domain is a Cas9 nickase domain. In some embodiments, the catalytically inactive Cas domain or inactive Cas domain does not form a detectable double-stranded break. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, or partially inactivated Cas9 protein, comprises a H588 mutation (e.g., a H588A mutation) or an analogous substitution for the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, or partially inactivated Cas9 protein, comprises an N611 mutation (e.g., an N611A mutation) or an analogous substitution for the amino acid corresponding to said position. In some embodiments, the catalytically inactive Cas9 protein, e.g., dCas9, comprises a D16 mutation (e.g., D16A), a D587 mutation (e.g., D587A), an H588 mutation (e.g., H588A), and an N611 mutation (e.g., N611A) or an analogous substitution for the amino acid corresponding to said position.

[0370] In some embodiments, the DNA binding domain or endonuclease domain can comprise a Cas molecule that includes or is linked (e.g., covalently) to a gRNA (e.g., a template nucleic acid that includes a gRNA, e.g., a template RNA).

[0371] In some embodiments, the endonuclease domain or DNA-binding domain comprises Streptococcus pyogenes Cas9 (SpCas9) or a functional fragment or variant thereof. In some embodiments, the endonuclease domain or DNA-binding domain comprises a modified SpCas9. In several embodiments, the modified SpCas9 comprises a modification that alters protospacer adjacent motif (PAM) specificity. In several embodiments, the PAM has specificity for the nucleic acid sequence 5'-NGT-3'. In several embodiments, the modified SpCas9 comprises one or more amino acid substitutions, e.g., at one or more of the following positions: L1111, D1135, G1218, E1219, A1322, or R1335, e.g., selected from the following: L1111R, D1135V, G1218R, E1219F, A1322R, R1335V. In some embodiments, the modified SpCas9 comprises the amino acid substitution T1337R and one or more additional amino acid substitutions selected from the following: L1111, 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 a corresponding amino acid substitution thereof. In some embodiments, the modified SpCas9 comprises: (i) one or more amino acid substitutions selected from the following: D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, and T1337; and (ii) one or more additional amino acid substitutions selected from the following: L1111R, G1218R, E1219F, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M, or a corresponding amino acid substitution thereof.

[0372] In some embodiments, the endonuclease domain or DNA-binding domain comprises a Cas domain, such as a Cas9 domain. In several embodiments, the endonuclease domain or DNA-binding domain comprises a nuclease-active Cas domain, a Cas nickase (nCas) domain, or a nuclease-inactive Cas (dCas). In several embodiments, the endonuclease domain or DNA-binding domain comprises a nuclease-active Cas9 domain, a Cas9 nickase (nCas9) domain, or a nuclease-inactive Cas9 (dCas). In some embodiments, the endonuclease domain or DNA-binding domain comprises a Cas9 domain of Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, or Cas12i. In some embodiments, the endonuclease domain or DNA-binding domain comprises Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, or Cas12i. In some embodiments, the endonuclease domain or DNA-binding domain comprises S. pyogenes or S. thermophilus Cas9, or a functional fragment thereof. In some embodiments, the 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, the endonuclease domain or DNA binding domain comprises the HNH nuclease subdomain and / or RuvC1 subdomain of a Cas, e.g., Cas9, or a variant thereof, as described herein.In some embodiments, the endonuclease domain or DNA-binding domain comprises Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, or Cas12i. In some embodiments, the endonuclease domain or DNA-binding domain comprises a Cas polypeptide (e.g., an enzyme), or a functional fragment thereof. In several embodiments, the Cas polypeptide (e.g., an enzyme) is selected from the following: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpf l, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Cs y3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1 , Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, C The Cas9 effector protein is selected from sa5, a type II Cas effector protein, a type V Cas effector protein, a type VI Cas effector protein, CARF, DinG, Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, Cas12c / C2c3, SpCas9(K855A), eSpCas9(1.1), SpCas9-HF1, a hyper accurate Cas9 mutant (HypaCas9), a homologue thereof, a modified or engineered version thereof, and / or a functional fragment thereof.In some embodiments, the Cas9 comprises one or more substitutions selected from the following: H840A, D10A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A. In some embodiments, the Cas9 comprises one or more mutations at a position selected from the following: D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987, for example, one or more substitutions selected from the following: D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A. In some embodiments, the endonuclease domain or DNA binding domain is selected from the group consisting of Corynebacterium ulcerans, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquis, Staphylococcus thermophilus, Listeria innocua, Campylobacter jejuni, Neisseria meningitidis, and Escherichia coli. meningitidis, Streptococcus pyogenes, or Staphylococcus aureus, or functional fragments or variants thereof.

[0373] In some embodiments, the endonuclease domain or DNA binding domain comprises a Cpf1 domain comprising one or more substitutions, e.g., at positions D917, E1006A, D1255, or any combination thereof, selected from the following: D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, and D917A / E1006A / D1255A.

[0374] In some embodiments, the 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).

[0375] In some embodiments, the genetically modified polypeptide has an endonuclease domain that includes a Cas9 nickase, e.g., Cas9 H840A. In some embodiments, Cas9 H840A has the following amino acid sequence: Cas9 Nickase (H840A): [ka]

[0376] In some embodiments, the genetically modified polypeptide comprises a dCas9 sequence comprising a D10A and / or H840A mutation, for example, the following sequence: [ka]

[0377] TAL effectors and zinc finger nucleases In some embodiments, the endonuclease domain or DNA-binding domain comprises a TAL effector molecule. A TAL effector molecule, for example, a TAL effector molecule that specifically binds to a DNA sequence, typically comprises multiple TAL effector domains or fragments thereof, and optionally one or more additional portions of a naturally occurring TAL effector (for example, the N-terminus and / or C-terminus of multiple TAL effector domains). Many TAL effectors are known to those skilled in the art and are commercially available, for example, from Thermo Fisher Scientific.

[0378] Naturally occurring TALEs are natural effector proteins secreted by numerous species of bacterial pathogens, including the plant pathogen Xanthomonas, that regulate gene expression in host plants and promote bacterial colonization and survival. The specific binding of TAL effectors is typically based on a central repeat domain (repeated variable dinucleotide, RVD domain) of tandemly arranged, nearly identical repeats of 33 or 34 amino acids.

[0379] Members of the TAL effector family differ primarily in the number and order of their repeats. The number of repeats typically ranges from 1.5 to 33.5 repeats, with the C-terminal repeats usually being shorter in length (e.g., approximately 20 amino acids) and commonly referred to as "half-repeats." Each repeat in a TAL effector is generally characterized by a one-repeat-to-one base-pair correlation (one repeat recognizes one base pair in the target gene sequence), with different repeat types exhibiting different base-pair specificities. Generally, a decrease in the number of repeats weakens the protein-DNA interaction. It has been shown that several 6.5 repeats are sufficient to activate transcription of a reporter gene (Scholze et al., 2010).

[0380] The variation between repeats occurs primarily at amino acid positions 12 and 13, which are therefore termed "hypervariable" and are responsible for the specificity of the interaction with the target DNA promoter sequence, as shown in Table 9, which lists exemplary repeat variable dinucleotides (RVDs) and their correspondence to nucleobase targets.

[0381] [Table 38]

[0382] Therefore, it is possible to modify the repeats of TAL effectors to target specific DNA sequences. Furthermore, studies have shown that RVD NK can target G. Furthermore, the target sites of TAL effectors tend to contain a T adjacent to the 5' base targeted by the first repeat, although the exact mechanism of this recognition is unknown. Over 113 TAL effector sequences are known to date. Non-limiting examples of TAL effectors from Xanthomonas include Hax2, Hax3, Hax4, AvrXa7, AvrXa10, and AvrBs3.

[0383] Thus, the TAL effector domain of the TAL effector molecules described herein can be derived from a TAL effector from any bacterial species (e.g., Xanthomonas species, such as African strains of Xanthomonas oryzae pv. oryzae (Yu et al. 2011), Xanthomonas campestris pv. raphani strain 756C, and Xanthomonas oryzae pv. oryzicola BLS256 (Bogdanove et al. 2011)). In some embodiments, the TAL effector domain also comprises an RVD domain and flanking sequences (sequences N- and / or C-terminal to the RVD domain) from a naturally occurring TAL effector. It may contain more or fewer RVD repeats than the naturally occurring TAL effector. TAL effector molecules can be designed to target a given DNA sequence based on the above codes or others known in the art. The number of TAL effector domains (e.g., repeats (monomers or modules)) and their specific sequences can be selected based on the desired DNA target sequence. For example, TAL effector domains, e.g., repeats, can be removed or added as appropriate for a particular target sequence. In some embodiments, a TAL effector molecule of the invention comprises between 6.5 and 33.5 TAL effector domains, e.g., repeats. In some embodiments, a TAL effector molecule of the invention comprises between 8 and 33.5 TAL effector domains, e.g., repeats, for example, between 10 and 25 TAL effector domains, e.g., repeats, for example, between 10 and 14 TAL effector domains, e.g., repeats.

[0384] In some embodiments, a TAL effector molecule comprises a TAL effector domain that corresponds to a perfect match with the DNA target sequence. In some embodiments, mismatches between repeats and target base pairs on the DNA target sequence are tolerated as long as they allow the polypeptide comprising the TAL effector molecule to function. Generally, TALE binding is inversely correlated with the number of mismatches. In some embodiments, a TAL effector molecule of a polypeptide of the present invention comprises at most seven mismatches, six mismatches, five mismatches, four mismatches, three mismatches, two mismatches, or one mismatch with the target DNA sequence, and optionally no mismatches. While not intending to be bound by a particular theory, generally, as the number of TAL effector domains in a TAL effector molecule decreases, a reduced number of mismatches is not only tolerated but also allows the polypeptide comprising the TAL effector molecule to function. Binding affinity is thought to depend on the sum of matching repeat-DNA combinations. For example, a TAL effector molecule with 25 or more TAL effector domains may be able to tolerate up to seven mismatches.

[0385] In addition to the TAL effector domain, the TAL effector molecules of the present invention may contain additional sequences derived from naturally occurring TAL effectors. The length of the C-terminal and / or N-terminal sequences included on either side of the TAL effector domain portion of the TAL effector molecule can vary and can be selected by those skilled in the art based on, for example, the study of Zhang et al. (2011). Zhang et al. characterized several C-terminal and N-terminal truncation mutants in proteins based on Hax3-derived TAL effectors and identified key elements that contribute to optimal binding to target sequences and, therefore, transcriptional activation. Generally, transcriptional activity was found to be inversely correlated with the length of the N-terminus. Regarding the C-terminus, key elements in the DNA-binding residues within the first 68 amino acids of the Hax3 sequence were identified. Thus, in some embodiments, the first 68 amino acids on the C-terminal side of the TAL effector domain of a naturally occurring TAL effector are included in the TAL effector molecule. Thus, in one embodiment, the TAL effector molecule comprises: 1) one or more TAL effector domains derived from a naturally occurring TAL effector; 2) at least 70, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260, 270, 280 or more amino acids from a naturally occurring TAL effector N-terminal to the TAL effector domain; and / or 3) at least 68, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260 or more amino acids from a naturally occurring TAL effector C-terminal to the TAL effector domain.

[0386] In some embodiments, the endonuclease domain or DNA-binding domain is or comprises a zinc finger molecule. The zinc finger molecule comprises a zinc finger protein, such as a naturally occurring zinc finger protein or a modified zinc finger protein, or a fragment thereof. Many zinc finger proteins are known to those skilled in the art and are commercially available, for example, from Sigma-Aldrich.

[0387] In some embodiments, the zinc finger molecule comprises a non-naturally occurring zinc finger protein engineered to bind to a selected target DNA sequence (see, e.g., 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. al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Patent 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 Nos. 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Application Publication Nos. 2005 / 0064474; 2007 / 0218528; and 2005 / 0267061 (all of which are incorporated by reference in their entirety).

[0388] The engineered zinc finger proteins may have novel binding specificities compared to naturally occurring zinc finger proteins. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design, for example, involves the use of a database containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to a particular triplet or quadruplet sequence. See, for example, U.S. Patent Nos. 6,453,242 and 6,534,261 (incorporated herein by reference in their entireties).

[0389] Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Patent 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 Publications WO 98 / 37186; WO 98 / 53057; WO 00 / 27878; and WO 01 / 88197 and GB 2,338,237. Furthermore, increased binding specificity in zinc finger proteins is described, for example, in International Patent Publication WO 02 / 077227.

[0390] Furthermore, as disclosed in these and other references, zinc finger domains and / or multi-fingered zinc finger proteins can be linked together using any suitable linker sequence, including, for example, linkers of five or more amino acids in length. For exemplary linker sequences of six or more amino acids in length, see also U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949. The proteins described herein can include any combination of suitable linkers between the individual zinc fingers of the protein. Furthermore, increased binding specificity in zinc finger binding domains is described, for example, in co-owned International Patent Publication WO 02 / 077227.

[0391] Zinc finger proteins and methods for the design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and include those disclosed in U.S. Patent 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 / 19432; and WO 03 / 016496.

[0392] Furthermore, as disclosed in these and other references, zinc finger proteins and / or multi-fingered zinc finger proteins can be linked together, e.g., as a fusion protein, using any suitable linker sequence, including, for example, linkers of five or more amino acids in length. For exemplary linker sequences of six or more amino acids in length, see also U.S. Patent Nos. 6,479,626; 6,903,185; and 7,153,949. The zinc finger molecules described herein can include any combination of suitable linkers between the individual zinc finger proteins and / or multi-fingered zinc finger proteins of the zinc finger molecule.

[0393] In certain embodiments, the DNA-binding domain or endonuclease domain comprises a zinc finger molecule comprising an engineered zinc finger protein that binds (in a sequence-specific manner) to a target DNA sequence. In some embodiments, the zinc finger molecule comprises one zinc finger protein or a fragment thereof. In other embodiments, the zinc finger molecule comprises multiple zinc finger proteins (or fragments thereof), for example, 2, 3, 4, 5, 6 or more zinc finger proteins (and optionally at most 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 zinc finger proteins). In some embodiments, the zinc finger molecule comprises at least three zinc finger proteins. In some embodiments, the zinc finger molecule comprises four, five, or six fingers. In some embodiments, the zinc finger molecule comprises eight, nine, ten, eleven, or twelve fingers. In some embodiments, a zinc finger molecule comprising three zinc finger proteins recognizes a target DNA sequence comprising 9 or 10 nucleotides. In some embodiments, a zinc finger molecule comprising four zinc finger proteins recognizes a target DNA sequence comprising 12 to 14 nucleotides, and in some embodiments, a zinc finger molecule comprising six zinc finger proteins recognizes a target DNA sequence comprising 18 to 21 nucleotides.

[0394] In some embodiments, the zinc finger molecule comprises a bimanual zinc finger protein. A bimanual zinc finger protein is a protein in which two clusters of zinc finger proteins are separated by an intervening amino acid, such that the two zinc finger domains bind to two discontinuous target DNA sequences. An example of a bimanual zinc finger binding protein is SIP1, in which a cluster of four zinc finger proteins is located at the amino terminus of the protein and a cluster of three zinc 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 can bind to a unique target sequence, and the spacing between the two target sequences can include multiple nucleotides.

[0395] Linker In some embodiments, the genetically modified polypeptide can include a linker, e.g., a peptide linker, e.g., a linker described in Table 10. In some embodiments, the genetically modified polypeptide includes, from N-terminal to C-terminal, a Cas domain (e.g., a Cas domain in Table 8), a linker in Table 10 (or a sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% identity thereto), and an RT domain (e.g., an RT domain in Table 6). In some embodiments, the genetically modified polypeptide includes a flexible linker between the endonuclease and the RT domain, e.g., a linker comprising the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 11,002). In some embodiments, the RT domain of the genetically modified polypeptide can be positioned C-terminal to the endonuclease domain. In some embodiments, the RT domain of the genetically modified polypeptide can be positioned N-terminal to the endonuclease domain.

[0396] [Table 39]

[0397] [Table 40]

[0398] [Table 41]

[0399] [Table 42]

[0400] [Table 43]

[0401] In some embodiments, the linker of the genetically modified polypeptide is (SGGS) n (SEQ ID NO: 5025), (GGGS) n (SEQ ID NO: 5026), (GGGGS) n (SEQ ID NO: 5027), (G) n , (EAAAK) n (SEQ ID NO: 5028), (GGS) n , or (XP) n The motif is selected from:

[0402] Selection of genetically modified polypeptides by pooled screening Candidate gene modification polypeptide can be screened to evaluate the gene editing ability of candidate.For example, can be used RNA gene modification system designed for the targeted editing of coding sequence in human genome.In certain embodiments, this gene modification system can be used with pool screening method.

[0403] For example, a library of candidate genetically modified polypeptides and template guide RNAs (tgRNAs) can be introduced into mammalian cells to test the gene-editing capabilities of the candidates using a pooled screening approach. In certain embodiments, the library of candidate genetically modified polypeptides is introduced into mammalian cells, followed by introduction of tgRNAs into the cells.

[0404] Representative, non-limiting examples of mammalian cells that can be used in the screen include HEK293T cells, U2OS cells, HeLa cells, HepG2 cells, Huh7 cells, K562 cells, or iPS cells.

[0405] The candidate genetic modification polypeptides can include 1) a Cas-nuclease, e.g., a wild-type Cas nuclease, e.g., a wild-type Cas9 nuclease, a mutant Cas nuclease, e.g., a Cas nickase, e.g., a Cas9 nickase such as Cas9 N863A nickase, or a Cas nuclease selected from Table 7 or Table 8, 2) a peptide linker, e.g., a sequence from Table D or Table 10, which can exhibit varying degrees of length, flexibility, hydrophobicity, and / or secondary structure; and 3) a reverse transcriptase (RT), e.g., an RT domain from Table D or Table 6. The candidate genetic modification polypeptide library includes a plurality of different candidate genetic modification polypeptides that differ from each other with respect to one, two, or all three of the Cas nuclease, peptide linker, or RT domain components, or a plurality of nucleic acid expression vectors encoding such candidate genetic modification polypeptides.

[0406] For screening of candidate genetically modified polypeptides, a two-component system including a genetically modified polypeptide component and a tgRNA component can be used. The genetic modification component can include, for example, an expression vector, such as an expression plasmid or lentiviral vector encoding the candidate genetically modified polypeptide, including, for example, a human codon-optimized nucleic acid encoding the candidate genetically modified polypeptide, such as the above-mentioned Cas-linker-RT fusion. In certain embodiments, a lentiviral cassette is used that includes: (i) a promoter for expression in mammalian cells, such as a CMV promoter; (ii) a candidate genetically modified library, such as a Cas-linker-RT fusion including a Cas nuclease in Table 7 or Table 8, a peptide linker in Table 10, and an RT in Table 6, for example, a Cas-linker-RT fusion as in Table D; (iii) a self-cleaving polypeptide, such as a T2A peptide; (iv) a marker that allows selection in mammalian cells, such as a puromycin resistance gene; and (v) a termination signal, such as a polyA tail.

[0407] The tgRNA component can include a tgRNA or an expression vector, e.g., an expression plasmid that generates the tgRNA and drives expression of the tgRNA using, e.g., a U6 promoter, where the tgRNA is a non-coding RNA sequence that is recognized by Cas, localizing it to the genomic locus of interest, and that templates reverse transcription of the desired edit into the genome via the RT domain.

[0408] To prepare a pool of cells expressing a genetically modified polypeptide library candidate, mammalian cells, e.g., HEK293T or U2OS cells, can be transduced with a pooled genetically modified polypeptide candidate expression vector preparation, e.g., a lentiviral preparation of the genetically modified candidate polypeptide library. In certain embodiments, lentiviral plasmids are used, and HEK293 Lenti-X cells are seeded in 15 cm plates (approximately 12 x 10 cells) prior to lentiviral plasmid transfection. 6In such an embodiment, lentiviral plasmid transfection can be performed using Lentiviral Packaging Mix (Biosettia), and transfection of plasmid DNA for the gene modification candidate library can be performed using Lipofectamine 2000 and Opti-MEM medium according to the manufacturer's protocol. In such an embodiment, extracellular DNA can be removed by a complete medium change the next day, and virus-containing medium can be collected 48 hours later. The lentiviral medium can be concentrated using a Lenti-X Concentrator (TaKaRa Biosciences), and 5 mL lentiviral aliquots can be made and stored at -80°C. Lentiviral titer measurement can be performed after selection, for example, by counting colony-forming units after puromycin selection.

[0409] To monitor gene editing of target DNA, mammalian cells, such as HEK293T or U2OS cells carrying target DNA, can be used. In other embodiments for monitoring gene editing of target DNA, mammalian cells, such as HEK293T or U2OS cells carrying a target DNA genomic landing pad, can be used. In certain embodiments, the target DNA genomic landing pad can contain a gene to be edited for the treatment of a disease or disorder of interest. In other specific embodiments, the target DNA is a genetic sequence that expresses a protein exhibiting a detectable characteristic that can be monitored to determine whether gene editing has occurred. For example, in certain embodiments, blue fluorescent protein (BFP)- or green fluorescent protein (GFP)-expressing genomic landing pads are used. In certain embodiments, mammalian cells, such as HEK293T or U2OS cells carrying target DNA, e.g., a target DNA genomic landing pad, are seeded into culture plates at 500x to 3000x cells per genetic modification library candidate and transduced at a 0.2 to 0.3 multiplicity of infection (MOI) to minimize the number of infections per cell. Puromycin (2.5 μg / mL) can be added 48 hours after infection to allow for selection of infected cells. In such an embodiment, the cells are placed under puromycin selection for at least 7 days and then scaled up for tgRNA introduction, e.g., tgRNA electroporation.

[0410] To confirm whether gene editing occurs, mammalian cells containing the target DNA to be edited can be infected with the candidate gene modification polypeptide library and then transfected with a tgRNA designed for use in editing the target DNA. The cells can then be analyzed, for example, by cell sorting and sequence analysis, to determine whether editing of the target locus occurred according to the designed results, or whether no editing or incomplete editing occurred.

[0411] In certain embodiments, to confirm whether genome editing occurs, BFP- or GFP-expressing mammalian cells, such as HEK293T or U2OS cells, may be infected with a genetically modified library candidate and then transfected or electroporated at 250,000 cells / well with a tgRNA plasmid or RNA, e.g., 200 ng of a tgRNA plasmid designed to convert BFP to GFP or GFP to BFP, at a cell number that ensures >250×-1000× coverage per library candidate. In such embodiments, the genome editing ability of various constructs in this assay may be assessed by sorting cells by fluorescence-activated cell sorting (FACS) for the expression of color-converted fluorescent proteins (FPs) 4-10 days after electroporation. Cells are sorted and collected into distinct populations: non-edited cells (showing the original fluorescent protein signal), edited cells (showing the converted fluorescent protein signal), and incompletely edited cells (showing no fluorescent protein signal). A sample of unsorted cells may also be collected as an input population to determine candidate enrichment during analysis.

[0412] To determine whether the genetically modified library candidates exhibit genome editing capabilities in the assay, genomic DNA (gDNA) is collected from the sorted cell populations and analyzed by sequencing the genetically modified library candidates in each population. Briefly, genetically modified candidates are amplified from the genome using primers specific to the genetically modified polypeptide expression vector, e.g., a lentiviral cassette, and amplified in a second round of PCR to dilute the genomic DNA, which can then be sequenced, for example, by a next-generation sequencing platform. After quality control of the sequencing reads, reads of at least about 1500 nucleotides, and generally no more than about 3200 nucleotides, are mapped to the genetically modified polypeptide library sequence, and those containing a minimum of about 80% match with the library sequence are considered to have successfully aligned with a given candidate for this pooled screen. To identify candidates capable of gene editing in the assay, for example, editing BFP to GFP or GFP to BFP, the read count of each library candidate in the edited population is compared to its read count in the initial unsorted population.

[0413] For pooled screening, genetic modification candidates with genome editing capabilities are identified based on the enrichment of the edited (converted FP) population compared to unsorted (input) cells. In some embodiments, an enrichment of at least 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or at least 100 times the input indicates potentially useful gene editing activity, e.g., at least 2-fold enrichment. In some embodiments, enrichment is converted to a log value by taking the log base 2 of the enrichment ratio. In some embodiments, a log enrichment score of at least 0, 1, 2, 3, 4, 5, 5.5, 6.0, 6.2, 6.3, 6.4, 6.5, or at least 6.6 indicates potentially useful gene editing activity, e.g., a log enrichment score of at least 1.0. In certain embodiments, the enrichment value observed for a genetic modification candidate can be compared to the enrichment value observed under similar conditions using a reference, for example, element ID number: 17380.

[0414] In some embodiments, multiple tgRNAs can be used to screen gene modification candidate libraries.In certain embodiments, multiple tgRNAs can be used to optimize template / Cas-linker-RT fusion pairs, for example, for gene editing of specific target genes, for example, gene targets for disease treatment.In certain embodiments, pooling methods for screening gene modification candidates can be performed using many different tgRNAs in an arrayed format.

[0415] In some embodiments, multiple types of edits, e.g., insertions, substitutions, and / or deletions of different lengths, may be used to screen a library of candidate gene modifications.

[0416] In some embodiments, multiple target sequences, e.g., different fluorescent proteins, may be used to screen a genetic modification candidate library. In some embodiments, multiple target sequences, e.g., different fluorescent proteins, may be used to screen a genetic modification candidate library. In some embodiments, multiple cell types, e.g., HEK293T or U2OS, may be used to screen a genetic modification candidate library. Those skilled in the art will understand that a given candidate may exhibit altered editing capabilities or increased or decreased observable or useful activity across different conditions, including tgRNA sequence (e.g., nucleotide modification, PBS length, RT template length), target sequence, target location, type of editing, location of mutation relative to the first strand nick of the genetically modified polypeptide, or cell type. Thus, in some embodiments, genetic modification library candidates are screened across multiple parameters, e.g., using at least two different tgRNAs in at least two cell types, and gene editing activity is identified by enrichment in any single condition. In other embodiments, candidates with more robust activity across different tgRNAs and cell types are identified by enrichment in at least two conditions, e.g., all conditions screened. For clarity, candidates found to show little to no enrichment under any given condition are not presumed to be inactive across all conditions and can be screened using different parameters or reconstituted at the polypeptide level, for example, by exchanging, shuffling, or altering domains (e.g., RT domains), linkers, or other signals (e.g., NLS).

[0417] Exemplary Cas9-Linker-RT Fusion Sequences In some embodiments, the genetically modified polypeptide comprises a linker sequence and an RT sequence. In some embodiments, the genetically modified polypeptide comprises a linker sequence listed in Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the genetically modified polypeptide comprises an amino acid sequence of an RT domain listed in Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the genetically modified polypeptide comprises a linker sequence listed in Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and an amino acid sequence of an RT domain listed in Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the genetically modified polypeptide comprises (i) a linker sequence listed in a row of Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and (ii) an amino acid sequence of an RT domain listed in the same row of Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0418] Exemplary Genetically Modified Polypeptides In some embodiments, a genetically modified polypeptide (e.g., a genetically modified polypeptide that is part of a system described herein) comprises the amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, a genetically modified polypeptide comprises the 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 genetically modified polypeptide comprises the 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 genetically modified polypeptide comprises the 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 genetically modified polypeptide comprises the 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 genetically modified polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-7743. In some embodiments, the genetically modified polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the genetically modified polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0419] In some embodiments, the genetically modified polypeptide comprises an amino acid sequence as listed in Table A1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0420] In some embodiments, the genetically modified polypeptide comprises an amino acid sequence as listed in Table T1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the genetically modified polypeptide comprises a linker comprising a linker sequence as listed in Table T1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the genetically modified polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table T1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the genetically modified polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table T1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table T1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0421] [Table 44]

[0422] In some embodiments, the genetically modified polypeptide comprises an amino acid sequence as listed in Table T2, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto. In some embodiments, the genetically modified polypeptide comprises a linker comprising a linker sequence as listed in Table T2, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto. In some embodiments, the genetically modified polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table T2, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto. In some embodiments, the genetically modified polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0423] [Table 45]

[0424] [Table 46]

[0425] Subsequences of Exemplary Genetically Modified Polypeptides In some embodiments, the genetically modified polypeptide comprises, in order from N-terminus to C-terminus, one or more (e.g., one, two, three, four, five, or all six) N-terminal methionine residues, a first nuclear localization signal (NLS), a DNA-binding domain, a linker, an RT domain, and / or a second NLS. In some embodiments, the genetically modified polypeptide comprises, in order from N-terminus to C-terminus, an 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 the genetically modified polypeptide of any one of SEQ ID NOs: 1-7743, or amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said linker and RT domain. In some embodiments, the genetically modified polypeptide comprises, in order from N-terminus to C-terminus, a DNA-binding domain, a linker, an RT domain, and an NLS (e.g., a second NLS), wherein the linker and RT domain are amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the linker and RT domain of, or said linker and RT domain of, any one of the genetically modified polypeptides set forth in SEQ ID NOs: 1-7743. In some embodiments, the genetically modified polypeptide comprises, in order from N-terminus to C-terminus, a first NLS, a DNA-binding domain, a linker, an RT domain, and a second NLS, wherein the linker and RT domain are amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the linker and RT domain of, or said linker and RT domain of, any one of the genetically modified polypeptides set forth in SEQ ID NOs: 1-7743. In some embodiments, the genetically modified polypeptide further comprises an N-terminal methionine residue.

[0426] In some embodiments, the genetically modified polypeptide comprises, in order from N-terminus to C-terminus, one or more (e.g., one, two, three, four, five, or all six) N-terminal methionine residues, a first nuclear localization signal (NLS) (e.g., an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto) of any one of SEQ ID NOs: 1-7743 and / or a genetically modified polypeptide as listed in any of Table A1, Table T1, or Table T2, a DNA-binding domain (e.g., a Cas domain, e.g., a SpyCas9 domain, e.g., as listed in Table 8 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; or a DNA-binding domain of any one of SEQ ID NOs: 1-7743 and / or a genetically modified polypeptide as listed in any of Tables A1, T1, or T2 or an amino acid sequence having at least 70%, 75%, 80%, 85%, or 99% identity thereto; , 90%, 95%, or 99% identity thereto), a linker (e.g., an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto of a genetically modified polypeptide as set forth in any one of SEQ ID NOs: 1 to 7743 and / or in any of Tables A1, T1, or T2), a RT domain ... and a second NLS (e.g., that of a genetically modified polypeptide as set forth in SEQ ID NOs: 1-7743 and / or that of a genetically modified polypeptide as set forth in any of Table A1, Table T1, or Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto).In some embodiments, the genetically modified polypeptide further comprises (e.g., C-terminal to the second NLS) a T2A sequence and / or a puromycin sequence (e.g., an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto) of any one of SEQ ID NOs: 1-7743 and / or a genetically modified polypeptide as listed in any of Table A1, Table T1, or Table T2. In some embodiments, a nucleic acid (e.g., as described herein) encoding a genetically modified polypeptide encodes a T2A sequence, e.g., wherein the T2A sequence is located between the region encoding the genetically modified polypeptide and a second region, optionally encoding a selectable marker, e.g., puromycin.

[0427] In certain embodiments, the first NLS comprises a first NLS sequence of a genetically modified polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the first NLS comprises a first NLS sequence of a genetically modified polypeptide as listed in any of Table A1, Table T1, or Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the first NLS sequence comprises a C-myc NLS. In certain embodiments, the first NLS comprises the amino acid sequence PAAKRVKLD (SEQ ID NO: 11,095), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

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

[0429] In certain embodiments, the DNA-binding domain comprises the DNA-binding domain of a genetically modified polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the DNA-binding domain comprises the DNA-binding domain of a genetically modified polypeptide as listed in any of Table A1, Table T1, or Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the DNA-binding domain comprises a Cas domain (e.g., as listed in Table 8). In certain embodiments, the DNA-binding domain comprises the amino acid sequence of a SpyCas9 polypeptide (e.g., as listed in Table 8, e.g., the Cas9 N863A polypeptide), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the DNA binding domain has the amino acid sequence: [ka] or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity thereto.

[0430] In certain embodiments, the genetically modified polypeptide further comprises a spacer sequence between the DNA-binding domain and the linker. In certain embodiments, the spacer sequence between the DNA-binding domain and the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence between the DNA-binding domain and the linker comprises the amino acid sequence GG.

[0431] In certain embodiments, the linker comprises the linker sequence of a genetically modified polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the linker comprises the linker sequence of a genetically modified polypeptide as listed in any of Table A1, Table T1, or Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the linker comprises the amino acid sequence as listed in Table D or Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0432] In certain embodiments, the genetically modified polypeptide further comprises a spacer sequence between the linker and the RT domain. In certain embodiments, the spacer sequence between the linker and the RT domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence between the linker and the RT domain comprises the amino acid sequence GG.

[0433] In certain embodiments, the RT domain comprises the RT domain sequence of a genetically modified polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the RT domain comprises the RT domain sequence of a genetically modified polypeptide as listed in any of Table A1, Table T1, or Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the RT domain comprises the amino acid sequence as listed in Table D or Table 6, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain has a length of about 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 amino acids.

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

[0435] In certain embodiments, the second NLS comprises a second NLS sequence of a genetically modified polypeptide of any one of SEQ ID NOs: 1-7743. In certain embodiments, the second NLS comprises a second NLS sequence of a genetically modified polypeptide as listed in any of Table A1, Table T1, or Table T2. In certain embodiments, the second NLS sequence comprises multiple partial NLS sequences. In embodiments, the NLS sequence, e.g., the second NLS sequence, comprises, for example, a first partial NLS sequence comprising the amino acid sequence KRTADGSEFE (SEQ ID NO: 11,097), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In embodiments, the NLS sequence, e.g., the second NLS sequence, comprises a second partial NLS sequence. In embodiments, the NLS sequence, e.g., the second NLS sequence, comprises an SV40A5 NLS, e.g., a bipartite SV40A5 NLS, comprising the amino acid sequence KRTADGSEFESPKKKAKVE (SEQ ID NO: 11,098), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the NLS sequence, e.g., the second NLS sequence, comprises the amino acid sequence KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 11,099), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

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

[0437] Linker and RT domains In some embodiments, the genetically modified polypeptide comprises a linker (e.g., as described herein) and an RT domain (e.g., as described herein). In certain embodiments, the genetically modified polypeptide comprises, in order from N-terminus to C-terminus, a linker (e.g., as described herein) and an RT domain (e.g., as described herein).

[0438] In certain embodiments, the linker comprises a linker sequence as listed in Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the linker comprises a linker sequence of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the linker comprises a linker sequence of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the linker comprises the linker sequence of an exemplary genetically modified polypeptide listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the RT domain comprises the RT domain sequence as listed in Table 6, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the RT domain comprises the RT domain sequence of an exemplary genetically modified polypeptide listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0439] In some embodiments, the genetically modified polypeptide comprises a portion of a genetically modified polypeptide of any one of SEQ ID NOs: 1-7743, including the linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion.

[0440] In some embodiments, the genetically modified polypeptide comprises a linker or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said linker of a genetically modified polypeptide of any one of SEQ ID NOs: 1-7743. In some embodiments, the genetically modified polypeptide comprises a linker or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said linker of a genetically modified polypeptide of any one of SEQ ID NOs: 6001-7743. In some embodiments, the genetically modified polypeptide comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said linker of a genetically modified polypeptide of any one of SEQ ID NOs: 4501-4541. In some embodiments, the genetically modified polypeptide comprises a linker of a genetically modified polypeptide as listed in any of Table A1, Table T1, or Table T2, or a linker comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0441] In some embodiments, the genetically modified polypeptide comprises an RT domain of a genetically modified polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said RT domain. In some embodiments, the genetically modified polypeptide comprises an RT domain of a genetically modified polypeptide of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said RT domain. In some embodiments, the genetically modified polypeptide comprises an RT domain of a genetically modified polypeptide of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said RT domain. In some embodiments, the genetically modified polypeptide comprises an RT domain of a genetically modified polypeptide as listed in any of Table A1, Table T1, or Table T2, or an RT domain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0442] In certain embodiments, the linker and RT domain of the genetically modified polypeptide comprises the amino acid sequence of the linker and RT domain of the genetically modified polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-7743 (or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto). In certain embodiments, the linker and RT domain of the genetically modified polypeptide comprises the amino acid sequence of the linker and RT domain having at least 80% identity thereto. In certain embodiments, the linker and RT domain of the genetically modified polypeptide comprises the amino acid sequence of the linker and RT domain having at least 90% identity thereto. In certain embodiments, the linker and RT domain of the genetically modified polypeptide comprises the amino acid sequence of the linker and RT domain having at least 95% identity thereto. In certain embodiments, the linker and RT domain of the genetically modified polypeptide comprise an amino acid sequence of the linker and RT domain that is at least 99% identical to the linker and RT domain of any one of SEQ ID NOs: 1-7743. In certain embodiments, the linker and RT domain of the genetically modified polypeptide comprise an amino acid sequence of the linker and RT domain of a genetically modified polypeptide having an amino acid sequence of any one of SEQ ID NOs: 6001-7743 (or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto). In certain embodiments, the linker and RT domain of the genetically modified polypeptide comprise an amino acid sequence of the linker and RT domain of a genetically modified polypeptide having an amino acid sequence of any one of SEQ ID NOs: 4501-4541 (or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto).In certain embodiments, the linker and RT domain of the genetically modified polypeptide comprises the amino acid sequence of (or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to) the linker and RT domain from a single row of any of Table A1, Table T1, or Table T2 (e.g., from a single exemplary genetically modified polypeptide as listed in any of Table A1, Table T1, or Table T2).

[0443] In certain embodiments, the linker and RT domains of the genetically modified polypeptide comprise linker and RT domain amino acid sequences from (or amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to) two different amino acid sequences selected from SEQ ID NOs: 1-7743. In certain embodiments, the linker and RT domains of the genetically modified polypeptide comprise linker and RT domain amino acid sequences from (or amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to) different rows of any of Table A1, Table T1, or Table T2.

[0444] In certain embodiments, the genetically modified polypeptide further comprises a first NLS (e.g., a 5' NLS), e.g., as described herein. In certain embodiments, the genetically modified polypeptide further comprises a second NLS (e.g., a 3' NLS), e.g., as described herein. In certain embodiments, the genetically modified polypeptide further comprises an N-terminal methionine residue.

[0445] RT family and mutants In certain embodiments, the genetically modified 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, XMRV6, BLVAU, BLVJ, HTL1A, HTL1C, HTL1L, HTL32, HTL3P, ​​HTLV2, JSRV, MLVF5, MLVRD, MMTVB, MPMV, SFVCP, SMRVH, SRV1, SRV2, and WDSV. In certain embodiments, the genetically modified 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.

[0446] In certain embodiments, the genetically modified polypeptide comprises the amino acid sequence of an RT domain sequence from an MLVMS RT domain. In embodiments, the amino acid sequence of the RT domain sequence comprises one or more point mutations as listed in column 1 of Table M1 or corresponding point mutations thereto. In embodiments, the amino acid sequence of the RT domain sequence comprises one or more point mutations as listed in column 3 of Table M1 (Gen1 MLVMS) or corresponding point mutations thereto. In embodiments, the amino acid sequence of the RT domain sequence comprises one or more point mutations at amino acid positions of the RT domain as listed in columns 1 and 2 of Table M2 or corresponding amino acid positions thereto.

[0447] In certain embodiments, the genetically modified polypeptide comprises the amino acid sequence of an RT domain sequence from the AVIRE RT domain. In embodiments, the amino acid sequence of the RT domain sequence comprises one or more point mutations as listed in column 2 of Table M1 or corresponding point mutations thereto. In embodiments, the amino acid sequence of the RT domain sequence comprises one or more point mutations as listed in column 4 of Table M1 (Gen2 AVIRE) or corresponding point mutations thereto. In embodiments, the amino acid sequence of the RT domain sequence comprises one or more point mutations at amino acid positions of the RT domain as listed in columns 3 and 4 of Table M2 or corresponding amino acid positions thereto. In certain embodiments, the RT domain comprises an IENSSP (e.g., at the C-terminus).

[0448] [Table 47]

[0449] [Table 48]

[0450] In certain embodiments, the genetically modified polypeptide comprises an RT domain derived from a gammaretrovirus. In certain embodiments, the RT domain derived from a gammaretrovirus of the genetically modified 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. In some embodiments, the RT domain derived from a gammaretrovirus of the genetically modified polypeptide is not derived from a PERV. In some embodiments, the RT comprises 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, L671P, E69K, or D653N in the RT domain of murine leukemia virus reverse transcriptase. In some embodiments, the genetically modified polypeptide further comprises a linker having at least 99% identity to the linker domain of any one of SEQ ID NOs: 1-7743. In some embodiments, the genetically modified polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO: 11,041.

[0451] In embodiments, the RT domain comprises the amino acid sequence of the RT domain of AVIRE RT (e.g., the AVIRE_P03360 sequence, e.g., SEQ ID NO: 8001), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of AVIRE RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, G330P, L605W, T306K, and W313F, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of AVIRE RT further comprising one, two, or three mutations selected from the group consisting of D200N, G330P, and L605W, or corresponding positions in a homologous RT domain.

[0452] In embodiments, the RT domain comprises the amino acid sequence of the RT domain of BAEVM RT (e.g., the BAEVM_P10272 sequence, e.g., SEQ ID NO: 8004), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of BAEVM RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L602W, T304K, and W311F, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of BAEVM RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L602W, or corresponding positions in a homologous RT domain.

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

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

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

[0456] In embodiments, the RT domain comprises the amino acid sequence of the RT domain of GALV RT (e.g., the GALV_P21414 sequence, e.g., SEQ ID NO: 8027), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of GALV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L600W, T304K, and W311F, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of GALV RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L600W, or corresponding positions in a homologous RT domain.

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

[0458] In embodiments, the RT domain comprises the amino acid sequence of the RT domain of MLVAV RT (e.g., the MLVAV_P03356 sequence, e.g., SEQ ID NO: 8053), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of MLVAV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of MLVAV RT further comprising one, two, or three mutations selected from the group consisting of D200N, T330P, and L603W, or corresponding positions in a homologous RT domain.

[0459] In embodiments, the RT domain comprises the amino acid sequence of the RT domain of MLVBM RT (e.g., the MLVBM_Q7SVK7 sequence, e.g., SEQ ID NO: 8056), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of MLVBM RT further comprising one, two, three, four, or five mutations selected from the group consisting of D199N, T329P, L602W, T305K, and W312F, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of MLVBM RT further comprising one, two, and three mutations selected from the group consisting of D200N, T330P, and L603W, or corresponding positions in a homologous RT domain.

[0460] In embodiments, the RT domain comprises the amino acid sequence of the RT domain of MLVCB RT (e.g., the MLVCB_P08361 sequence, e.g., SEQ ID NO: 8062), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of MLVCB RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of MLVCB RT further comprising one, two, and three mutations selected from the group consisting of D200N, T330P, and L603W, or corresponding positions in a homologous RT domain.

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

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

[0463] In embodiments, the RT domain comprises the amino acid sequence of the RT domain of a PERV RT (e.g., the PERV_Q4VFZ2 sequence, e.g., SEQ ID NO: 8099), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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 corresponding positions in a homologous RT domain. In some embodiments, the 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 corresponding positions in a homologous RT domain.

[0464] In some embodiments, the RT domain comprises the amino acid sequence of the RT domain of SFV1 RT (e.g., the SFV1_P23074 sequence, e.g., SEQ ID NO: 8105), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of SFV1 RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, N420P, and L396K, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of SFV1 RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and N420P, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of SFV1 RT further comprising D24N, or corresponding positions in a homologous RT domain.

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

[0466] In embodiments, the RT domain comprises the amino acid sequence of the RT domain of WMSV RT (e.g., the WMSV_P03359 sequence, e.g., SEQ ID NO: 8131), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of WMSV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L600W, T304K, and W311F, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of WMSV RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L600W, or corresponding positions in a homologous RT domain.

[0467] In some embodiments, the RT domain comprises the amino acid sequence of the RT domain of XMRV6 RT (e.g., the XMRV6_A1Z651 sequence, e.g., SEQ ID NO: 8134), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of XMRV6 RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or corresponding positions in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of XMRV6 RT further comprising one, two, or three mutations selected from the group consisting of D200N, T330P, and L603W, or corresponding positions in a homologous RT domain.

[0468] In certain embodiments, the RT domain of the genetically modified polypeptide comprises the amino acid sequence of the RT domain of AVIRE RT, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In embodiments, the RT domain comprises the amino acid sequence of the RT domain contained in a sequence listed in column 1 of Table A5, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the genetically modified 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 certain embodiments, the RT domain of the genetically modified polypeptide comprises the amino acid sequence of the RT domain of MLVMS RT, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In embodiments, the RT domain comprises the amino acid sequence of the RT domain contained in a sequence listed in any of columns 2 through 6 of Table A5, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the genetically modified 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 49]

[0471] [Table 50]

[0472] [Table 51]

[0473] [Table 52]

[0474] [Table 53]

[0475] [Table 54]

[0476] [Table 55]

[0477] system In certain aspects, the present disclosure relates to a system comprising a nucleic acid molecule encoding a genetically modified polypeptide (e.g., as described herein) and a template nucleic acid (e.g., a template RNA, e.g., as described herein). In certain embodiments, the nucleic acid molecule encoding the genetically modified polypeptide comprises one or more silent mutations in the coding region (e.g., in the sequence encoding the RT domain) compared to the nucleic acid molecule as described herein. In certain embodiments, the 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 the target DNA to which the genetically modified polypeptide binds).

[0478] In certain embodiments, a nucleic acid molecule encoding a genetically modified polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto. In certain embodiments, a nucleic acid molecule encoding a genetically modified polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto. In certain embodiments, a nucleic acid molecule encoding a genetically modified polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto. In certain embodiments, the nucleic acid molecule encoding the genetically modified polypeptide encodes a polypeptide as listed in any of Table A1, Table T1, or Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0479] In certain embodiments, a nucleic acid molecule encoding a genetically modified polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 1-7743, the portion comprising the linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion. In certain embodiments, a nucleic acid molecule encoding a genetically modified polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 6001-7743, the portion comprising the linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion. In certain embodiments, a nucleic acid molecule encoding a genetically modified polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 4501-4541, the portion comprising the linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion. In certain embodiments, the nucleic acid molecule encoding the genetically modified polypeptide comprises a sequence encoding a portion of a polypeptide listed in any of Table A1, Table T1, or Table T2, including the linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion.

[0480] In certain embodiments, a nucleic acid molecule encoding a genetically modified 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, a nucleic acid molecule encoding a genetically modified 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, a nucleic acid molecule encoding a genetically modified 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding the genetically modified polypeptide comprises a sequence encoding a polypeptide linker as listed in any of Table A1, Table T1, or Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0481] In certain embodiments, a nucleic acid molecule encoding a genetically modified 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, a nucleic acid molecule encoding a genetically modified 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, a nucleic acid molecule encoding a genetically modified 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding the genetically modified polypeptide comprises a sequence encoding the RT domain of a polypeptide as listed in any of Table A1, Table T1 or Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0482] In certain embodiments, the present disclosure relates to a system comprising a genetically modified polypeptide (e.g., as described herein) and a template nucleic acid (e.g., a template RNA, e.g., as described herein).

[0483] In certain embodiments, the genetically modified polypeptide comprises a polypeptide having an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto. In certain embodiments, the genetically modified polypeptide comprises a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto. In certain embodiments, the genetically modified polypeptide comprises a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical thereto. In certain embodiments, the genetically modified polypeptide comprises a polypeptide as listed in any of Table A1, Table T1 or Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0484] In certain embodiments, the genetically modified polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 1-7743, the portion comprising the linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion. In certain embodiments, the genetically modified polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 6001-7743, the portion comprising the linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion. In certain embodiments, the genetically modified polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 4501-4541, the portion comprising the linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion. In certain embodiments, the genetically modified polypeptide comprises a portion of a polypeptide listed in any of Table A1, Table T1, or Table T2, including the linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion.

[0485] In certain embodiments, the genetically modified 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the genetically modified polypeptide comprises a linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or a sequence encoding an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the genetically modified polypeptide comprises a linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or a sequence encoding an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the genetically modified polypeptide comprises a linker of a polypeptide as listed in any of Table A1, Table T1, or Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0486] In certain embodiments, the genetically modified 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the genetically modified 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the genetically modified 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the genetically modified polypeptide comprises the RT domain of a polypeptide as listed in any of Table A1, Table T1 or Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

[0487] [Table 56]

[0488] [Table 57]

[0489] [Table 58]

[0490] [Table 59]

[0491] [Table 60]

[0492] Table 61

[0493] Table 62

[0494] Table 63

[0495] Table 64

[0496] Table 65

[0497] Table 66

[0498] Table 67

[0499] Table 68

[0500] Table 69

[0501] Table 70

[0502] Table 71

[0503] Table 72

[0504] Table 73

[0505] Table 74

[0506] Table 75

[0507] Table 76

[0508] Table 77

[0509] Table 78

[0510] Table 79

[0511] Table 80

[0512] Table 81

[0513] Table 82

[0514] Table 83

[0515] Table 84

[0516] Table 85

[0517] Table 86

[0518] Table 87

[0519] Table 88

[0520] Table 89

[0521] Table 90

[0522]

Table 91

[0523] Table 92

[0524]

Table 93

[0525] Table 94

[0526]

Table 95

[0527] Table 96

[0528] Table 97

[0529] Table 98

[0530]

Table 99

[0531]

Table 100

[0532] Table 101

[0533] Table 102

[0534] Table 103

[0535] Table 104

[0536] Table 105

[0537] Table 106

[0538] Table 107

[0539] Table 108

[0540] Table 109

[0541] Table 110

[0542] Table 111

[0543] Table 112

[0544] Table 113

[0545] Table 114

[0546] Table 115

[0547] Table 116

[0548] Table 117

[0549] Table 118

[0550] Table 119

[0551] Table 120

[0552] Table 121

[0553] Table 122

[0554] Table 123

[0555] Table 124

[0556] Table 125

[0557] Table 126

[0558] Table 127

[0559] Table 128

[0560] Table 129

[0561] Table 130

[0562] Table 131

[0563] Table 132

[0564] Table 133

[0565] Table 134

[0566] Table 135

[0567] Table 136

[0568] Table 137

[0569] Table 138

[0570] Table 139

[0571] Table 140

[0572] Table 141

[0573] Table 142

[0574] Table 143

[0575] Table 144

[0576] Table 145

[0577] Table 146

[0578] Table 147

[0579] Table 148

[0580] Table 149

[0581] Table 150

[0582] Table 151

[0583] Table 152

[0584] Table 153

[0585] Table 154

[0586] Table 155

[0587] Table 156

[0588] Table 157

[0589] Table 158

[0590] Table 159

[0591] Table 160

[0592] Table 161

[0593] Table 162

[0594] Table 163

[0595] Table 164

[0596] Table 165

[0597] Table 166

[0598] Table 167

[0599] Table 168

[0600] Table 169

[0601] Table 170

[0602] Table 171

[0603] Table 172

[0604] Table 173

[0605] Table 174

[0606] Table 175

[0607] Table 176

[0608] Table 177

[0609] Table 178

[0610] Table 179

[0611] Table 180

[0612] Table 181

[0613] Table 182

[0614] Table 183

[0615] Table 184

[0616] Table 185

[0617] Table 186

[0618] Table 187

[0619] Table 188

[0620] Table 189

[0621] Table 190

[0622] Table 191

[0623] Table 192

[0624] Table 193

[0625] Table 194

[0626] Table 195

[0627] Table 196

[0628] Table 197

[0629] Table 198

[0630] Table 199

[0631] Table 200

[0632] Table 201

[0633] Table 202

[0634] Table 203

[0635] Table 204

[0636] Table 205

[0637] Table 206

[0638] Table 207

[0639] Table 208

[0640] Table 209

[0641] Table 210

[0642] Table 211

[0643] Table 212

[0644] Table 213

[0645] Table 214

[0646] Table 215

[0647] Table 216

[0648] Table 217

[0649] Table 218

[0650] Table 219

[0651] Table 220

[0652] Table 221

[0653] Table 222

[0654] Table 223

[0655] Table 224

[0656] Table 225

[0657] Table 226

[0658] Table 227

[0659] Table 228

[0660] Table 229

[0661] Table 230

[0662] Table 231

[0663] Table 232

[0664] Table 233

[0665] Table 234

[0666] Table 235

[0667] Table 236

[0668] Table 237

[0669] Table 238

[0670] Table 239

[0671] Table 240

[0672] Table 241

[0673] Table 242

[0674] Table 243

[0675] Table 244

[0676] Table 245

[0677] Table 246

[0678] Table 247

[0679] Table 248

[0680] Table 249

[0681] Table 250

[0682] Table 251

[0683] Table 252

[0684] Table 253

[0685] Table 254

[0686] Table 255

[0687] Table 256

[0688] Table 257

[0689] Table 258

[0690] Table 259

[0691] Table 260

[0692] Table 261

[0693] Table 262

[0694] Table 263

[0695] Table 264

[0696] Table 265

[0697] Table 266

[0698] Table 267

[0699] Table 268

[0700] Table 269

[0701] Table 270

[0702] Table 271

[0703] Table 272

[0704] Table 273

[0705] Table 274

[0706] Table 275

[0707] Table 276

[0708] Table 277

[0709] Table 278

[0710] Table 279

[0711] Table 280

[0712] Table 281

[0713] Table 282

[0714] Table 283

[0715] Table 284

[0716] Table 285

[0717] Table 286

[0718] Table 287

[0719] Table 288

[0720] Table 289

[0721] Table 290

[0722] Table 291

[0723] Table 292

[0724] Table 293

[0725] Table 294

[0726] Table 295

[0727] Table 296

[0728] Table 297

[0729] Table 298

[0730] Table 299

[0731] Table 300

[0732]

Table 301

[0733]

Table 302

[0734]

Table 303

[0735] Table 304

[0736] Table 305

[0737] Table 306

[0738] Table 307

[0739] Table 308

[0740] Table 309

[0741] Table 310

[0742] Table 311

[0743] Table 312

[0744] Table 313

[0745] Table 314

[0746] Table 315

[0747] Table 316

[0748] Table 317

[0749] Table 318

[0750] Table 319

[0751] Table 320

[0752] Table 321

[0753] Table 322

[0754] Table 323

[0755] Table 324

[0756] Table 325

[0757] Table 326

[0758] Table 327

[0759] Table 328

[0760] Table 329

[0761] Table 330

[0762] Table 331

[0763] Table 332

[0764] Table 333

[0765] Table 334

[0766] Table 335

[0767] Table 336

[0768] Table 337

[0769] Table 338

[0770] Table 339

[0771] Table 340

[0772] Table 341

[0773] Table 342

[0774] Table 343

[0775] Table 344

[0776] Table 345

[0777] Table 346

[0778] Table 347

[0779] Table 348

[0780] Table 349

[0781] Table 350

[0782] Table 351

[0783] Table 352

[0784] Table 353

[0785] Table 354

[0786] Table 355

[0787] Table 356

[0788] Table 357

[0789] Table 358

[0790] Table 359

[0791] Table 360

[0792] Table 361

[0793] Table 362

[0794] Table 363

[0795] Table 364

[0796] Table 365

[0797] Table 366

[0798] Table 367

[0799] Table 368

[0800] Table 369

[0801] Table 370

[0802] Table 371

[0803] Table 372

[0804] Table 373

[0805] Table 374

[0806] Table 375

[0807] Table 376

[0808] Table 377

[0809] Table 378

[0810] Table 379

[0811] Table 380

[0812] Table 381

[0813] Table 382

[0814] Table 383

[0815] Table 384

[0816] Table 385

[0817] Table 386

[0818] Table 387

[0819] Table 388

[0820] Table 389

[0821] Table 390

[0822] Table 391

[0823] Table 392

[0824]

Table 393

[0825] Table 394

[0826] Table 395

[0827] Table 396

[0828] Table 397

[0829] Table 398

[0830] Table 399

[0831] Table 400

[0832]

Table 401

[0833] Table 402

[0834] Table 403

[0835]

Table 404

[0836] Table 405

[0837] Table 406

[0838] Table 407

[0839] Table 408

[0840] Table 409

[0841] Table 410

[0842] Table 411

[0843] Table 412

[0844] Table 413

[0845] Table 414

[0846] Table 415

[0847] Table 416

[0848] Table 417

[0849] Table 418

[0850] Table 419

[0851] Table 420

[0852] Table 421

[0853] Table 422

[0854] Table 423

[0855] Table 424

[0856] Table 425

[0857] Table 426

[0858] Table 427

[0859] Table 428

[0860] Table 429

[0861] Table 430

[0862] Table 431

[0863] Table 432

[0864] Table 433

[0865] Table 434

[0866] Table 435

[0867] Table 436

[0868] Table 437

[0869] Table 438

[0870] Table 439

[0871] Table 440

[0872] Table 441

[0873] Table 442

[0874] Table 443

[0875] Table 444

[0876] Table 445

[0877] Table 446

[0878] Table 447

[0879] Table 448

[0880] Table 449

[0881] Table 450

[0882] Table 451

[0883] Table 452

[0884] Table 453

[0885] Table 454

[0886] Table 455

[0887] Table 456

[0888] Table 457

[0889] Table 458

[0890] Table 459

[0891] Table 460

[0892] Table 461

[0893] Table 462

[0894] Table 463

[0895] Table 464

[0896] Table 465

[0897] Table 466

[0898] Table 467

[0899] Table 468

[0900] Table 469

[0901] Table 470

[0902] Table 471

[0903] Table 472

[0904] Table 473

[0905] Table 474

[0906] Table 475

[0907] Table 476

[0908] Table 477

[0909] Table 478

[0910] Table 479

[0911] Table 480

[0912] Table 481

[0913] Table 482

[0914]

Table 483

[0915] Table 484

[0916] Table 485

[0917] Table 486

[0918] Table 487

[0919] Table 488

[0920] Table 489

[0921] Table 490

[0922] Table 491

[0923] Table 492

[0924] [Table 493]

[0925] [Table 494]

[0926] [Table 495]

[0927] [Table 496]

[0928] [Table 497]

[0929] [Table 498]

[0930] [Table 499]

[0931] [Table 500]

[0932] [Table 501]

[0933] [Table 502]

[0934] Localization sequences for gene modification systems In certain embodiments, the gene editor system RNA further comprises a subcellul...

Claims

1. From 5' to 3', (i) a gRNA spacer complementary to a first portion of a human PAH gene, wherein the gRNA spacer has a sequence comprising the nucleic acid sequence of SEQ ID NO: 16,011, or a sequence comprising the core nucleotides of a gRNA spacer sequence of Table 1A, Table IB, Table 1C, or Table ID, and optionally comprises one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the gRNA spacer, or has the sequence of a spacer selected from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A; (ii) a gRNA scaffold that binds to a genetically modified polypeptide (e.g., binds to a Cas domain of the genetically modified polypeptide); (iii) a heterologous sequence of interest comprising a mutation region for introducing a mutation into a second portion of the human PAH gene (e.g., for correcting a mutation therein), wherein optionally, the heterologous sequence of interest comprises, from 5' to 3', a post-edited homology region, a mutation region, and a pre-edited homology region; and (iv) a primer binding site (PBS) sequence comprising at least 5, 6, 7, or 8 bases of 100% identity to the third portion of the human PAH gene. A template RNA comprising:

2. 2. The template RNA of claim 1, wherein the heterologous target sequence comprises the core nucleotides of an RT template sequence from Table 3A, Table 3B, Table 3C, or Table 3D, and optionally comprises one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the RT template sequence, or the heterologous target sequence comprises the sequence of an RT template sequence from Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A.

3. From 5' to 3', (i) a gRNA spacer complementary to a first portion of the human PAH gene; (ii) a gRNA scaffold that binds to a genetically modified polypeptide (e.g., binds to a Cas domain of the genetically modified polypeptide); (iii) a heterologous target sequence comprising a mutation region for introducing a mutation into a second portion of the human PAH gene (e.g., for correcting a mutation therein), the heterologous target sequence comprising the core nucleotides of an RT template sequence of Table 3A, Table 3B, Table 3C or Table 3D, and optionally one or more contiguous nucleotides starting from the 3′ end of a flanking nucleotide of the RT template sequence, or comprising an RT template sequence of Tables 5A-5F, 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A; and (iv) a PBS sequence comprising at least 5, 6, 7, or 8 bases that are 100% identical to the third portion of the human PAH gene. A template RNA comprising:

4. 2. The template RNA of claim 1, wherein the PBS sequence has a sequence that includes a core nucleotide of a PBS sequence of Table 3A, Table 3B, Table 3C, or Table 3D corresponding to the RT template sequence, the gRNA spacer sequence, or both, and optionally includes one or more contiguous nucleotides starting from the 5' end of a flanking nucleotide of the PBS sequence, or the PBS sequence has a sequence that includes a PBS sequence of Table 5A-5F, Table 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A corresponding to the RT template sequence, the gRNA spacer sequence, or both.

5. the gRNA scaffold comprises a sequence of a gRNA scaffold in Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto; Optionally, the gRNA scaffold comprises the nucleic acid sequence of SEQ ID NO: 37,627, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

6. 2. The template RNA of claim 1, wherein the mutation is a W408R, Q261R, Q243R, and / or IVS10-11A>G mutation in the PAH gene (e.g., to correct a pathogenic R408W, R261Q, R243Q, and / or IVS10-11G>A mutation). (a) the mutation region comprises a first region (e.g., a first nucleotide) designed to correct a pathogenic mutation in the PAH gene and a second region (e.g., a second nucleotide) designed to inactivate a PAM sequence (e.g., a "PAM-kill" mutation as described herein); (b) the template RNA includes one or more silent mutations (e.g., silent substitutions), e.g., as exemplified in Tables 7A-7C, 8A-8D, E6, or E6A; (c) the mutation region comprises a first region designed to correct a pathogenic mutation in the PAH gene and a second region designed to introduce a silent substitution; and / or (d) the template RNA of claim 1, wherein the template RNA comprises one or more chemically modified nucleotides.

8. 1. A genetic modification system comprising: (i) the template RNA of claim 1, and (ii) a genetically modified polypeptide or a nucleic acid (e.g., RNA) encoding the genetically modified polypeptide. A genetic modification system comprising:

9. The genetically modified polypeptide is (i) a reverse transcriptase (RT) domain (e.g., an RT domain from a retrovirus or a polypeptide domain having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto); (ii) a Cas domain (e.g., a Cas9 domain) that binds to a target DNA molecule and is heterologous to the RT domain; and (iii) optionally, a linker interposed between the RT domain and the Cas domain; and optionally (a) the RT domain is (1) an RT domain of Table 6; or (2) RT domains derived from murine leukemia virus (MMLV), porcine endogenous retrovirus (PERV); avian reticuloendotheliosis virus (AVIRE), feline leukemia virus (FLV), simian foamy virus (SFV) (e.g., SFV3L), bovine leukemia virus (BLV), Mason-Pfizer simian virus (MPMV), human foamy virus (HFV), or bovine foamy / syncytial virus (BFV / BSV). Includes; (b) the Cas domain comprises a Cas domain of Table 7 or Table 8; (c) the Cas domain is a Cas9 domain; (d) the Cas domain is a SpyCas9 domain, a SpCas9 domain, a BlatCas9 domain, a Nme2Cas9 domain, a PnpCas9 domain, a SauCas9 domain, a SauCas9-KKH domain, a SauriCas9 domain, a SauriCas9-KKH domain, a ScaCas9-Sc++ domain, a SpyCas9-NG domain, a SpyCas9-SpRY domain, or a St1Cas9 domain; or (e) the Cas domain is a Cas9 domain comprising an N670A mutation, an N611A mutation, an N605A mutation, an N580A mutation, an N588A mutation, an N872A mutation, an N863 mutation, an N622A mutation, or an H840A mutation; (f) the linker comprises a linker sequence in Table 10 (e.g., any of SEQ ID NOs: 5217, 5106, 5190, and 5218); (g) the genetically modified polypeptide comprises one or two NLS sequences from Table 11 (e.g., any of SEQ ID NOs: 5245, 5290, 5323, 5330, 5349, 5350, 5351, and 4001); and / or (h) the genetic modification system creates a first nick in a first strand of the human PAH gene, and optionally, the genetic modification system further comprises a second strand-targeting gRNA that directs a second nick in a second strand of the human PAH gene. The genetic modification system of claim 8.

10. 9. The genetic modification system of claim 8, wherein the template RNA comprises a template RNA sequence of Tables 5A-5F, Tables 8A-8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6, or Table E6A, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. (a) the second strand targeting gRNA comprises: (i) comprises a sequence comprising the core nucleotides of a left gRNA spacer sequence or a right gRNA spacer sequence from Table 2A, Table 2B, Table 2C or Table 2D, and optionally comprises one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of said left gRNA spacer sequence or right gRNA spacer sequence; or (ii) comprises a second strand targeting gRNA comprising a spacer sequence of Table 6A or a spacer sequence having one, two, or three substitutions thereto; (b) the second strand-targeting gRNA comprises a sequence comprising the core nucleotides of a left gRNA spacer sequence or a right gRNA spacer sequence from Table 2A, Table 2B, Table 2C, or Table 2D corresponding to the gRNA spacer sequence of (i), and optionally one or more contiguous nucleotides starting from the 3′ end of a flanking nucleotide of the left gRNA spacer sequence or right gRNA spacer sequence; (c) the second strand targeting gRNA comprises a sequence comprising the core nucleotides of a second nicked gRNA sequence from Table 4A, Table 4B, Table 4C, or Table 4D, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and optionally one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the second nicked gRNA sequence; (d) the second strand targeting gRNA comprises a spacer sequence from Table 6A, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; (e) the second strand targeting gRNA comprises a sequence comprising the core nucleotides of a second nicked gRNA sequence from Table 4A, Table 4B, Table 4C or Table 4D corresponding to the gRNA spacer sequence of (i), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and optionally one or more contiguous nucleotides starting from the 3' end of a flanking nucleotide of the second nicked gRNA sequence; (f) the second strand targeting gRNA has a "PAM-in orientation" with the template RNA of the genetic modification system, e.g., as illustrated in Tables 2A-2D, 4A-4D, or 6A; or (g) the second strand targeting gRNA targets a sequence that overlaps with a target mutation in the template RNA; and optionally, The second strand targeting gRNA is (1) a sequence complementary to the PAH mutation (e.g., a spacer sequence); (2) a sequence complementary to the wild-type sequence at the target locus (e.g., a spacer sequence); (3) a sequence (e.g., a spacer sequence) complementary to a SNP proximal to the target locus, e.g., a SNP contained in the genomic DNA of a subject (e.g., a patient); or (4) a sequence (e.g., a spacer sequence) that is complementary to or contains one or more silent substitutions proximal to the target locus. The genetic modification system of claim 9, comprising:

12. The template RNA according to any one of claims 1 to 7, wherein the heterologous sequence of interest comprises about 8 to 30, 9 to 25, 10 to 20, 11 to 16, or 12 to 15 (e.g., about 11 to 16) nucleotides. Or the genetic modification system according to any one of claims 8 to 11.

13. The template RNA according to any one of claims 1 to 7, wherein the PBS sequence comprises about 5 to 20, 8 to 16, 8 to 14, 8 to 13, 9 to 13, 9 to 12, or 10 to 12 (e.g., about 9 to 12) nucleotides. Or the genetic modification system according to any one of claims 8 to 11.

14. A template RNA comprising the sequence of a template RNA of Table 4A to Table 4D, Table 5A to Table 5F, Table 8A to Table 8D, Table E3, Table E3A, Table BB, Table E5, Table E5A, Table E6 or Table E6A, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

15. 1. A genetic modification system comprising: (i) a template RNA comprising a template RNA sequence of Table 4A, Table 4B, Table 4C, or Table 4D, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and (ii) a second nicked gRNA sequence from the same row as (i) of Table 4A, Table 4B, Table 4C, or Table 4D, to which a sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity. A genetic modification system comprising:

16. A DNA encoding the template RNA according to any one of claims 1 to 7 or 14, or the gene modification system according to any one of claims 8 to 11 or 15.

17. A pharmaceutical composition comprising the system of any one of claims 8 to 11 or 15 or one or more nucleic acids encoding same, and a pharmaceutically acceptable excipient or carrier, Optionally, 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. Optionally, the viral vector is an adeno-associated virus.

18. A host cell (e.g., a mammalian cell, e.g., a human cell) comprising the template RNA of any one of claims 1 to 7 or 14 or the genetic modification system of any one of claims 8 to 11 or 15.

19. A lipid nanoparticle (LNP) composition comprising a template RNA described in any one of claims 1 to 7 or 14, or a gene modification system described in any one of claims 8 to 11 or 15, or DNA encoding the same.

20. 10. A method for making the template RNA of claim 1, comprising synthesizing the template RNA by in vitro transcription (e.g., solid-state synthesis) or by introducing DNA encoding the template RNA into a host cell under conditions that allow for the production of the template RNA, wherein optionally the cell is a mammalian cell, e.g., a human cell.

21. 10. An in vitro or ex vivo method for modifying a target site in a human PAH gene in a cell, comprising contacting the cell with the genetic modification system of claim 8 or DNA encoding same, thereby modifying the target site in the human PAH gene in the cell; Optionally, the cells are derived from a subject with phenylketonuria (PKU) or hyperphenylalaninemia (e.g., mild or severe hyperphenylalaninemia), and optionally, the cells are mammalian cells, e.g., human cells, in vitro or ex vivo methods.

22. A pharmaceutical composition for use in treating a subject having a disease or condition associated with a mutation in the human PAH gene, comprising the genetic modification system of claim 8 or DNA encoding same. Optionally, (a) the disease or condition is phenylketonuria (PKU) or hyperphenylalaninemia (e.g., mild or severe hyperphenylalaninemia); (b) the subject has an R408W, R261Q, R243Q, and / or IVS10-11G>A mutation; and / or (c) the subject is a human. Pharmaceutical compositions.

23. Use of the genetic modification system of claim 8 or DNA encoding same in the manufacture of a medicament for treating a subject having a disease or condition associated with a mutation in the human PAH gene, comprising: Optionally, (a) the disease or condition is phenylketonuria (PKU) or hyperphenylalaninemia (e.g., mild or severe hyperphenylalaninemia); (b) the subject has an R408W, R261Q, R243Q, and / or IVS10-11G>A mutation; and / or (c) the subject is a human. use.

24. A pharmaceutical composition for use in treating a subject having PKU, comprising the gene modification system of claim 8 or DNA encoding same, and optionally, the subject is a human.

25. 10. Use of the genetic modification system of claim 8 or DNA encoding same in the manufacture of a medicament for treating a subject having PKU, optionally wherein the subject is a human.

26. (a) introduction of the system into a target cell results in correction of a pathogenic mutation in the PAH gene. Optionally, the pathogenic mutation is a R408W, R261Q, R243Q, and / or IVS10-11G>A mutation, and the correction comprises a W408R, Q261R, and / or Q243R amino acid substitution or a IVS10-11A>G nucleotide substitution; (b) introduction of the system into target cells results in a sudden restoration of function of the PAH gene. bring about mutations, Optionally, (1) the mutation correction occurs in at least 30% (e.g., 30%, 40%, 50%, 60%, 70% or more) of the target nucleic acids; and / or (2) correction of the mutation occurs in at least 30% (e.g., 30%, 40%, 50%, 60%, 70% or more) of the target cells; (c) the genetic modification system comprises a second strand-targeting gRNA, and correction of the mutation in the target cell population is increased compared to a target cell population treated with a genetic modification system comprising a template RNA without a second strand-targeting gRNA; and / or (d) the template RNA comprises one or more silent substitutions (e.g., as exemplified in Table 7A, Table X4, and Table X4A), and correction of the mutation in the target cell population is increased compared to a target cell population treated with a genetic modification system comprising template RNA that does not comprise one or more silent substitutions. A genetic modification system according to claims 8 to 11 or 15, a method according to claim 20 or 21, a pharmaceutical composition for use according to claim 22 or 24, or a use according to claim 23 or 25.