Gene editing system containing reverse transcriptase

Abstract

The present disclosure relates generally to gene editing systems comprising reverse transcriptases and fusion proteins of the reverse transcriptase with a nickase or nuclease, methods of making such reverse transcriptases and fusion proteins, and methods of using such reverse transcriptases and fusion proteins for site-specific genome editing in cells.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 380,194, filed October 19, 2022, U.S. Provisional Patent Application No. 63 / 386,658, filed December 8, 2022, U.S. Provisional Patent Application No. 63 / 387,268, filed December 13, 2022, U.S. Provisional Patent Application No. 63 / 491,269, filed March 20, 2023, U.S. Provisional Patent Application No. 63 / 500,228, filed May 4, 2023, U.S. Provisional Patent Application No. 63 / 500,509, filed May 5, 2023, and U.S. Provisional Patent Application No. 63 / 510,861, filed June 28, 2023, each of which is incorporated by reference in its entirety. Summary of the Invention

[0002] The present disclosure is based, in part, on the development of a gene editing system that includes a reverse transcriptase, a nuclease or nickase, and a guide RNA or pegRNA.

[0003] Described herein is a fusion protein comprising a nickase linked to a reverse transcriptase using a linker, wherein the reverse transcriptase comprises at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.

[0004] Described herein is a fusion protein comprising a nuclease linked to a reverse transcriptase using a linker, wherein the reverse transcriptase comprises at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.

[0005] Described herein is a fusion protein comprising a catalytically deficient nuclease linked to a reverse transcriptase using a linker, wherein the reverse transcriptase comprises at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.

[0006] Described herein is a gene editing system comprising: a) a nickase; b) a guide nucleic acid configured to form a complex with the nickase and hybridize to a target nucleic acid sequence; and c) a reverse transcriptase having at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585, and configured to form a complex with the nickase. In some embodiments, the gene editing system further comprises a nucleic acid template. In some embodiments, the nickase is a modified endonuclease. In some embodiments, the modified endonuclease is a Type II CRISPR endonuclease. In some embodiments, the modified endonuclease is a Type V CRISPR endonuclease. In some embodiments, the Type II CRISPR endonuclease or the Type V CRISPR endonuclease has nickase activity. In some embodiments, the modified endonuclease is selected from the group consisting of spCas9(H840A), spCas9(D10A), nMG3-6(D13A), nMG3-6(H586A), nMG3-6(N609A), Cas12a, and MG29-1. In some embodiments, the modified endonuclease comprises at least about 80% sequence identity to any one of SEQ ID NOs: 152-154. In some embodiments, the nickase and reverse transcriptase are linked. In some embodiments, the nickase and reverse transcriptase are linked by a linker. In some embodiments, the linker comprises at least 10, 20, or 30 amino acids. In some embodiments, the linker comprises about 30-35 amino acids. In some embodiments, the linker comprises about 30 amino acids. In some embodiments, the linker comprises at least 80% sequence identity to SEQ ID NO: 103. In some embodiments, the linker comprises at least 80% sequence identity to any one of SEQ ID NOs: 155-160. In some embodiments, the nickase and reverse transcriptase are unlinked. In some embodiments, the guide nucleic acid comprises a spacer sequence and a crRNA.In some embodiments, the guide nucleic acid further comprises a reverse transcriptase template (RTT). In some embodiments, the bases in the RTT comprise a bulk modification selected from the group consisting of complex sugars, complex amino groups, and / or other modifications compatible with RNA. In some embodiments, the guide nucleic acid further comprises a primer binding site. In some embodiments, the primer binding site is at the 3' end of the guide nucleic acid. In some embodiments, the primer binding site comprises at least 2, 4, 6, 8, 10, 13, 16, 20, 24, 28, 32, 36, 40, 45, 50, 55, 60, or 65 nucleotides. In some embodiments, the gene editing system further comprises a transposase, integrase, or homing endonuclease. In some embodiments, the gene editing system further comprises a retrotransposon. In some embodiments, the reverse transcriptase comprises a processivity at least about two-fold higher than Moloney Murine Leukemia Virus (MMLV) reverse transcriptase. In some embodiments, the reverse transcriptase comprises a processivity that is at least about 2-fold lower than Moloney Murine Leukemia Virus (MMLV) reverse transcriptase. In some embodiments, the reverse transcriptase comprises an error rate that is less than about 2.5%, 2.0%, 1.5%, 1%, 0.5%, 0.25%, 0.10%, or 0.05%. In some embodiments, the reverse transcriptase comprises an error rate that is less than about 2.5%, 2.0%, 1.5%, 1%, 0.5%, 0.25%, 0.10%, or 0.05% compared to Moloney Murine Leukemia Virus (MMLV) reverse transcriptase.

[0007]

[0003] Described herein is a gene editing system comprising: a) a nuclease; b) a guide nucleic acid configured to form a complex with the nuclease and hybridize to a target nucleic acid sequence; and c) a reverse transcriptase having at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585, and configured to form a complex with the nuclease. In some embodiments, the gene editing system further comprises a nucleic acid template. In some embodiments, the nuclease is a double-stranded nuclease. In some embodiments, the nuclease is a Type II CRISPR endonuclease. In some embodiments, the CRISPR endonuclease is Cas9. In some embodiments, the Cas9 is a catalytically deficient Cas9 (dCas9). In some embodiments, the nuclease and the reverse transcriptase are linked. In some embodiments, the nuclease and reverse transcriptase are linked by a linker. In some embodiments, the linker comprises at least 10, 20, or 30 amino acids. In some embodiments, the linker comprises about 30-35 amino acids. In some embodiments, the linker comprises about 30 amino acids. In some embodiments, the linker comprises at least 80% sequence identity to SEQ ID NO: 103. In some embodiments, the linker comprises at least 80% sequence identity to any one of SEQ ID NOs: 155-160. In some embodiments, the nuclease and reverse transcriptase are unlinked. In some embodiments, the guide nucleic acid further comprises a primer binding site. In some embodiments, the primer binding site is at the 3' end of the guide nucleic acid. In some embodiments, the primer binding site comprises at least 2, 4, 6, 8, 10, 13, 16, 20, 24, 28, 32, 36, 40, 45, 50, 55, 60, or 65 nucleotides. In some embodiments, the gene editing system further comprises a transposase, an integrase, or a homing endonuclease. In some embodiments, the gene editing system further comprises a retrotransposon.In some embodiments, the reverse transcriptase comprises a processivity that is at least about 2-fold higher than Moloney murine leukemia virus (MMLV) reverse transcriptase. In some embodiments, the reverse transcriptase comprises a processivity that is at least about 2-fold lower than Moloney murine leukemia virus (MMLV) reverse transcriptase. In some embodiments, the reverse transcriptase comprises an error rate that is less than about 2.5%, 2.0%, 1.5%, 1%, 0.5%, 0.25%, 0.10%, or 0.05%. In some embodiments, the reverse transcriptase comprises an error rate that is less than about 2.5%, 2.0%, 1.5%, 1%, 0.5%, 0.25%, 0.10%, or 0.05% compared to Moloney murine leukemia virus (MMLV) reverse transcriptase.

[0008]

[0010] Described herein is a gene editing system comprising: a) a nickase; b) a guide nucleic acid configured to form a complex with the nickase and hybridize to a target nucleic acid sequence; and c) a reverse transcriptase configured to form a complex with the nickase, wherein the reverse transcriptase has an X1X2DD motif, where X1 is F or Y, and if X1 is Y, then X2 is A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, V, W, or Y. In some embodiments, X2 is A or I. In some embodiments, the X1X2DD motif is YADD (SEQ ID NO: 2572) or YIDD (SEQ ID NO: 2573). In some embodiments, the X1X2DD motif is FADD (SEQ ID NO: 2574), FVDD (SEQ ID NO: 2575), FIDD (SEQ ID NO: 2576), or FLDD (SEQ ID NO: 2577). In some embodiments, the reverse transcriptase has at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.

[0009]

[0010] Described herein is a gene editing system comprising: a) a nuclease; b) a guide nucleic acid configured to form a complex with the nuclease and hybridize to a target nucleic acid sequence; and c) a reverse transcriptase configured to form a complex with the nuclease, wherein the reverse transcriptase has an X1X2DD motif, where X1 is F or Y, and if X1 is Y, then X2 is A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, V, W, or Y. In some embodiments, X2 is A or I. In some embodiments, the X1X2DD motif is YADD (SEQ ID NO:2572) or YIDD (SEQ ID NO:2573). In some embodiments, the X1X2DD motif is FADD (SEQ ID NO:2574), FVDD (SEQ ID NO:2575), FIDD (SEQ ID NO:2576), or FLDD (SEQ ID NO:2577). In some embodiments, the reverse transcriptase has at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.

[0010] Described herein is an isolated reverse transcriptase having at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.

[0011] Described herein are nucleic acids encoding the above-described fusion proteins or gene editing systems. In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is mRNA. In some embodiments, the nucleic acid is contained in a vector. In some embodiments, the nucleic acid or vector comprising the nucleic acid is contained in an adeno-associated virus or lipid nanoparticle. In some embodiments, the nucleic acid or vector comprising the nucleic acid is contained in a cell. In some embodiments, the cell is a human cell.

[0012] Described herein are methods for modifying double-stranded and / or single-stranded nucleic acids, comprising contacting a cell with the fusion protein or gene editing system described above.

[0013] Described herein are methods for modifying double-stranded and / or single-stranded nucleic acids in a cell, the methods comprising: a) providing to the cell a guide nucleic acid that binds to a target strand of the nucleic acid; b) providing to the cell a nuclease or nickase to cleave the nucleic acid at the binding site of the guide nucleic acid; and c) providing to the cell a reverse transcriptase to synthesize a modification in the target strand of the nucleic acid at the cleavage site of the nickase and / or nuclease. In some embodiments, the reverse transcriptase has at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585. In some embodiments, the modification is an insertion, deletion, or mutation. In some embodiments, the method further comprises providing to the cell an RNA or DNA template. In some embodiments, the nucleic acid is a genome or vector. In some embodiments, the method further comprises providing to the cell a transposase, integrase, or homing endonuclease. In some embodiments, the method further comprises providing the cell with a retrotransposon. [Brief explanation of the drawings]

[0014] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings:

[0015] [Figure 1A]Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1AA] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1B] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1BB]Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1C] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1CC] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1D]Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1DD] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1E] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1EE]Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1F] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1FF] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1G]Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1GG] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1H] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1HH]Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1I] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1II] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1J]Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1JJ] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1K] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1L]Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1M] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1N] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1O]Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1P] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1Q] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1R]Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1S] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1T] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1U]Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1V] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1W] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1X]Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1Y] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 1Z] Figure 1 shows bar graphs depicting G-to-T transversion editing rates of untethered reverse transcriptase (RT) candidates from the MG151 family with eight different primer binding site (PBS) nucleotides of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG151 candidates 80–85 (Figure 1A–1F), 87–100 (Figure 1G–1T), and 102–117 (Figure 1U–1JJ) are shown, using untreated samples, no RT, wild-type MMLV1, and wild-type MMLV2 as controls. [Figure 2] 1 is a bar graph showing the relative fold change in editing by untethered RT candidates from the MG151 family compared to wild-type MMLV editing normalized to 1. Seven untethered MG151 candidates (candidates 98, 100, 99, 102, 103, 104, and 105) with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) are shown. Bars represent the specific PBS lengths tested for each candidate. [Figure 3A] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3B] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3C] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3D] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3E]Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3F] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3G] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3H] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3I]Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3J] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3K] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3L] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3M]Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3N] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3O] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3P] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3Q]Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3R] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3S] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3T] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3U]Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3V] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 3W] Bar graphs showing G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG153 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells for MG153 candidates 1-5, 7-13, 15, 16, and 21 (Figures 3A-3O), and 14, 17-20, and 25-27 (Figures 3P-3W), using untreated samples and wild-type MMLV1 as controls. [Figure 4A] Figure 4 is a bar graph showing the G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG160 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG160 candidates 1-6 and 8 (Figures 4A-4G) are shown, with untreated samples and wild-type MMLV1 as controls. [Figure 4B]Figure 4 is a bar graph showing the G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG160 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG160 candidates 1-6 and 8 (Figures 4A-4G) are shown, with untreated samples and wild-type MMLV1 as controls. [Figure 4C] Figure 4 is a bar graph showing the G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG160 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG160 candidates 1-6 and 8 (Figures 4A-4G) are shown, with untreated samples and wild-type MMLV1 as controls. [Figure 4D] Figure 4 is a bar graph showing the G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG160 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG160 candidates 1-6 and 8 (Figures 4A-4G) are shown, with untreated samples and wild-type MMLV1 as controls. [Figure 4E] Figure 4 is a bar graph showing the G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG160 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG160 candidates 1-6 and 8 (Figures 4A-4G) are shown, with untreated samples and wild-type MMLV1 as controls. [Figure 4F] Figure 4 is a bar graph showing the G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG160 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG160 candidates 1-6 and 8 (Figures 4A-4G) are shown, with untreated samples and wild-type MMLV1 as controls. [Figure 4G] Figure 4 is a bar graph showing the G to T transediting rates of untethered reverse transcriptase (RT) candidates from the MG160 family with eight different PBS nucleotides of varying lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. MG160 candidates 1-6 and 8 (Figures 4A-4G) are shown, with untreated samples and wild-type MMLV1 as controls. [Figure 5A] Figure 5 shows bar graphs depicting the G-to-T transition editing rates of RT candidates from the MG160 family tethered with spCas9(H840A). MG160 candidates 1-5 (Figures 5A-5G) tethered with spCas9(H840A) were tested for G-to-T transitions in HEK293T cells. Candidates are shown with untreated samples, wild-type MMLV1, wild-type MMLV2, spCas9(H840A)-MMLV1, and spCas9(H840A)-MMLV2 as controls. [Figure 5B] Figure 5 shows bar graphs depicting the G-to-T transition editing rates of RT candidates from the MG160 family tethered with spCas9(H840A). MG160 candidates 1-5 (Figures 5A-5G) tethered with spCas9(H840A) were tested for G-to-T transitions in HEK293T cells. Candidates are shown with untreated samples, wild-type MMLV1, wild-type MMLV2, spCas9(H840A)-MMLV1, and spCas9(H840A)-MMLV2 as controls. [Figure 5C] Figure 5 shows bar graphs depicting the G-to-T transition editing rates of RT candidates from the MG160 family tethered with spCas9(H840A). MG160 candidates 1-5 (Figures 5A-5G) tethered with spCas9(H840A) were tested for G-to-T transitions in HEK293T cells. Candidates are shown with untreated samples, wild-type MMLV1, wild-type MMLV2, spCas9(H840A)-MMLV1, and spCas9(H840A)-MMLV2 as controls. [Figure 5D]Figure 5 shows bar graphs depicting the G-to-T transition editing rates of RT candidates from the MG160 family tethered with spCas9(H840A). MG160 candidates 1-5 (Figures 5A-5G) tethered with spCas9(H840A) were tested for G-to-T transitions in HEK293T cells. Candidates are shown with untreated samples, wild-type MMLV1, wild-type MMLV2, spCas9(H840A)-MMLV1, and spCas9(H840A)-MMLV2 as controls. [Figure 5E] Figure 5 shows bar graphs depicting the G-to-T transition editing rates of RT candidates from the MG160 family tethered with spCas9(H840A). MG160 candidates 1-5 (Figures 5A-5G) tethered with spCas9(H840A) were tested for G-to-T transitions in HEK293T cells. Candidates are shown with untreated samples, wild-type MMLV1, wild-type MMLV2, spCas9(H840A)-MMLV1, and spCas9(H840A)-MMLV2 as controls. [Figure 5F] Figure 5 shows bar graphs depicting the G-to-T transition editing rates of RT candidates from the MG160 family tethered with spCas9(H840A). MG160 candidates 1-5 (Figures 5A-5G) tethered with spCas9(H840A) were tested for G-to-T transitions in HEK293T cells. Candidates are shown with untreated samples, wild-type MMLV1, wild-type MMLV2, spCas9(H840A)-MMLV1, and spCas9(H840A)-MMLV2 as controls. [Figure 5G] Figure 5 shows bar graphs depicting the G-to-T transition editing rates of RT candidates from the MG160 family tethered with spCas9(H840A). MG160 candidates 1-5 (Figures 5A-5G) tethered with spCas9(H840A) were tested for G-to-T transitions in HEK293T cells. Candidates are shown with untreated samples, wild-type MMLV1, wild-type MMLV2, spCas9(H840A)-MMLV1, and spCas9(H840A)-MMLV2 as controls. [Figure 6A]6A is a bar graph showing a blot of the percentage of indels after targeting the endogenous target AAVS1 (FIG. 6A) with nuclease MG3-6 conjugated with pegRNA containing PBSs of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. [Figure 6B] 6B is a bar graph showing a blot of the percentage of indels after targeting the endogenous target B2M (FIG. 6B) with nuclease MG3-6 conjugated with pegRNA containing PBSs of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. [Figure 6C] 6C is a bar graph showing a blot of the percentage of indels after targeting the endogenous target CD5 (FIG. 6C) with nuclease MG3-6 conjugated with pegRNA containing PBSs of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. [Figure 6D] A bar graph showing a blot of the percentage of indels after targeting the endogenous target CD38 (Figure 6D) with nuclease MG3-6 conjugated with pegRNA containing PBSs of various lengths (PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) in HEK293T cells. [Figure 7]A schematic diagram of an exemplary DNA construct for a GFP-based retrotransposition assay is shown. The construct carries a cytomegalovirus promoter (CMVp) followed by a reverse transcriptase (RT-NLS) with an N-terminal tag (Flag-HA-NLS-MCP-linker). The reverse-oriented EF1 alpha promoter (EF1α) drives expression of GFP (GFP exon 2 and GFP exon 1) only upon successful retrotransposition to the target site specified by the construct's nuclease (inverted intron). Target-primed reverse transcription is initiated after binding of the primer binding site (PBS) to the 3' overhang generated by the nuclease. NLS = nuclear localization signal; MCP = MS2 coat protein; GFP = green fluorescent protein; pA = poly(A) sequence; MS2 loop = MS2 coat protein binding site. [Figure 8] A diagram of the mechanism for targeted integration of retron-derived ssDNA by TnpA is shown. Retron ncRNAs (msr in gray, msd in black) contain the desired cargo flanked by structural motifs recognized by TnpA (top left, dashed box). The excised cargo (top right) is circularized by TnpA and finds the targeting motif on the ssDNA target, which becomes available for binding of the RNA-guided effector (bottom right, gray). TnpA mediates integration of the ssDNA donor by cleavage of the target, and the host repair machinery repairs the integrative edit (bottom left, dashed box). [Figure 9-1] Editing with untethered MG151 candidates MG151-118 to MG151-135 for G to T conversions across eight different PBS lengths is shown. [Figure 9-2] Editing with untethered MG151 candidates MG151-118 to MG151-135 for G to T conversions across eight different PBS lengths is shown. [Figure 9-3] Editing with untethered MG151 candidates MG151-118 to MG151-135 for G to T conversions across eight different PBS lengths is shown. [Figure 9-4]Editing with untethered MG151 candidates MG151-118 to MG151-135 for G to T conversions across eight different PBS lengths is shown. [Figure 9-5] Editing with untethered MG151 candidates MG151-118 to MG151-135 for G to T conversions across eight different PBS lengths is shown. [Figure 10] Editing using untethered MG151 candidates MG151-123 to MG151-126 for G to T conversions at PBS lengths of 6, 8, 10, and 13 nucleotides is shown. Two biological replicates were performed for each candidate. [Figure 11-1] Editing using untethered MG151 family mutations for G to T transversion is shown. Figure 11-1A: MG151-98 wild-type is shown in green bars along with MG151-98 point mutations, MG151-98 combination mutations, and MG151-98 trimming mutations. A single replicate is shown in Figure 11-1A, and additional replicates containing various MG151-98 mutations are shown in Figure 11-1B. Mutations K297P and H171N significantly improve wild-type MG151-98 activity. MG151-99, with 152 AA trimmed, significantly improved G to T transversion activity, while trimming 136 AA inhibited editing activity. MMLV1 wild-type is shown in gold bars, and MMLV2 (pentamutation) served as a control for each experiment. [Figure 11-2] Editing using an untethered MG151 family mutation for a G to T transversion is shown. Figure 11-2C: MG151-99 mutant and wild-type MG151-99 have a G to T transversion, and some mutations increase wild-type activity. Figure 11-2D: MG151-99 wild-type was compared to a trimmed version of MG151-99. Trimming 152 AA of MG151-99 significantly improved the activity of the G to T transversion, while trimming 136 AA inhibited editing activity. MMLV1 wild-type is shown as a gold bar, and MMLV2 (pentamutation) served as a control for each experiment. [Figure 12A]Untethered MG151 candidates (MG151-80 to MG151-135) tested for G to T transitions are shown. Editing rates for G to T transitions (Figure 12A) and fold changes compared to MMLV wild-type at PBS13 (Figure 12B). Each dot represents a different PBS length ranging from 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides. [Figure 12B] Untethered MG151 candidates (MG151-80 to MG151-135) tested for G to T transitions are shown. Editing rates for G to T transitions (Figure 12A) and fold changes compared to MMLV wild-type at PBS13 (Figure 12B). Each dot represents a different PBS length, ranging from 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides. [Figure 13A] 1 shows editing with untethered MG153 candidates tested for G to T transitions across eight different PBS lengths for different MG153 candidates. [Figure 13B] 1 shows editing with untethered MG153 candidates tested for G to T transitions across eight different PBS lengths for different MG153 candidates. [Figure 13C] 1 shows editing with untethered MG153 candidates tested for G to T transitions across eight different PBS lengths for different MG153 candidates. [Figure 13D] 1 shows editing with untethered MG153 candidates tested for G to T transitions across eight different PBS lengths for different MG153 candidates. [Figure 13E] 1 shows editing with untethered MG153 candidates tested for G to T transitions across eight different PBS lengths for different MG153 candidates. [Figure 13F] 1 shows editing with untethered MG153 candidates tested for G to T transitions across eight different PBS lengths for different MG153 candidates. [Figure 13G] 1 shows editing with untethered MG153 candidates tested for G to T transitions across eight different PBS lengths for different MG153 candidates. [Figure 13H]Figure 13H shows editing with untethered MG153 candidates tested for G to T transitions across eight different PBS lengths for different MG153 candidates. Figure 13H shows MG153-53 editing when fused with Cas9. [Figure 14A] Untethered MG153 candidates tested for G to T transitions are shown. Editing rates for G to T transitions (Figure 14A) and fold change relative to MMLV wild type for PBS13 (Figure 14B). Each dot represents a different PBS length ranging from 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides. [Figure 14B] Untethered MG153 candidates tested for G to T transitions are shown. Editing rates for G to T transitions (Figure 14A) and fold change relative to MMLV wild type for PBS13 (Figure 14B). Each dot represents a different PBS length ranging from 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides. [Figure 15A] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15B] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15C] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15D] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15E] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15F] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15G]Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15H] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15I] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15J] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15K] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15L] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15M] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15N] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15O] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15P] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15Q] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15R]Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15S] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15T] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 15U] Editing using spCas9(H840A) tethered to the MG160 candidate for G to T transversions across eight different PBS lengths is shown. [Figure 16A] MG160 candidates tethered with spCas9(H840A) tested for G to T transitions are shown. Editing rates for G to T transitions (Figure 16A) and fold change over MMLV wild type at PBS13 (Figure 16B). Each dot represents a different PBS length ranging from 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides. [Figure 16B] MG160 candidates tethered with spCas9(H840A) tested for G to T transitions are shown. Editing rates for G to T transitions (Figure 16A) and fold change over MMLV wild type at PBS13 (Figure 16B). Each dot represents a different PBS length ranging from 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides. [Figure 17A] Non-tethered candidates MG151-98 and MG151-99 are shown. RT candidates MG151-98 (FIG. 17A) and MG151-99 (FIG. 17B) were tested to carry a 24 nt insertion. [Figure 17B] Non-tethered candidates MG151-98 and MG151-99 are shown. RT candidates MG151-98 (FIG. 17A) and MG151-99 (FIG. 17B) were tested to carry a 24 nt insertion. [Figure 17C]Non-tethered candidates MG151-98 and MG151-99 are shown. RT candidates MG151-98 (FIG. 17C) and MG151-99 (FIG. 17D) were tested to perform a 15 nt deletion. [Figure 17D] Non-tethered candidates MG151-98 and MG151-99 are shown. RT candidates MG151-98 (FIG. 17C) and MG151-99 (FIG. 17D) were tested to perform a 15 nt deletion. [Figure 18-1] MG151 candidates including MG151-123 (Figures 18-1A and 18-2E), MG151-124 (Figures 18-1B and 18-2F), MG151-125 (Figures 18-1C and 18-2G), and MG151-126 (Figures 18-1D and 18-2H), which have completed 24-nt insertions (Figures 18-1A to 18-1D) and 15-nt deletions (Figures 18-2E to 18-2H) spanning the length of four PBSs, are shown. [Figure 18-2] MG151 candidates including MG151-123 (Figures 18-1A and 18-2E), MG151-124 (Figures 18-1B and 18-2F), MG151-125 (Figures 18-1C and 18-2G), and MG151-126 (Figures 18-1D and 18-2H), which have completed 24-nt insertions (Figures 18-1A to 18-1D) and 15-nt deletions (Figures 18-2E to 18-2H) spanning the length of four PBSs, are shown. [Figure 19A] Rational engineering of MG151-98 is shown. MG151-98 wild-type is shown as a green bar, along with point mutations, combination mutations, and trimmings of MG151-98. The performance of these mutations for a 24-nt insertion (Figures 19A-19B) and a 15-nt deletion (Figures 19C-19D) is shown above along with the controls MMLV1 and MMLV2. [Figure 19B] Rational engineering of MG151-98 is shown. MG151-98 wild-type is shown as a green bar, along with point mutations, combination mutations, and trimmings of MG151-98. The performance of these mutations for a 24-nt insertion (Figures 19A-19B) and a 15-nt deletion (Figures 19C-19D) is shown above along with the controls MMLV1 and MMLV2. [Figure 19C] Rational engineering of MG151-98 is shown. MG151-98 wild-type is shown as a green bar, along with point mutations, combination mutations, and trimmings of MG151-98. The performance of these mutations for a 24-nt insertion (Figures 19A-19B) and a 15-nt deletion (Figures 19C-19D) is shown above along with the controls MMLV1 and MMLV2. [Figure 19D] Rational engineering of MG151-98 is shown. MG151-98 wild-type is shown as a green bar, along with point mutations, combination mutations, and trimmings of MG151-98. The performance of these mutations for a 24-nt insertion (Figures 19A-19B) and a 15-nt deletion (Figures 19C-19D) is shown above along with the controls MMLV1 and MMLV2. [Figure 20A] Rational engineering of MG151-99 is shown. MG151-99 wild-type is shown as a green bar, along with point mutations, combinatorial mutations, and trimmings of MG151-99. The performance of these mutations for a 24-nt insertion (Figures 20A-20B) and a 15-nt deletion (Figures 20C-20D) is shown above along with the controls MMLV1 and MMLV2. [Figure 20B] Rational engineering of MG151-99 is shown. MG151-99 wild-type is shown as a green bar, along with point mutations, combinatorial mutations, and trimmings of MG151-99. The performance of these mutations for a 24-nt insertion (Figures 20A-20B) and a 15-nt deletion (Figures 20C-20D) is shown above along with the controls MMLV1 and MMLV2. [Figure 20C] Rational engineering of MG151-99 is shown. MG151-99 wild-type is shown as a green bar, along with point mutations, combinatorial mutations, and trimmings of MG151-99. The performance of these mutations for a 24-nt insertion (Figures 20A-20B) and a 15-nt deletion (Figures 20C-20D) is shown above along with the controls MMLV1 and MMLV2. [Figure 20D]Rational engineering of MG151-99 is shown. MG151-99 wild-type is shown as a green bar, along with point mutations, combinatorial mutations, and trimmings of MG151-99. The performance of these mutations for a 24-nt insertion (Figures 20A-20B) and a 15-nt deletion (Figures 20C-20D) is shown above along with the controls MMLV1 and MMLV2. [Figure 21A] MG153 candidates tested for 24 nt Flag insertions spanning 4 to 8 different PBS lengths are shown. [Figure 21B] MG153 candidates tested for 24 nt Flag insertions spanning 4 to 8 different PBS lengths are shown. [Figure 21C] MG153 candidates tested for 24 nt Flag insertions spanning 4 to 8 different PBS lengths are shown. [Figure 21D] MG153 candidates tested for 24 nt Flag insertions spanning 4 to 8 different PBS lengths are shown. [Figure 21E] MG153 candidates tested for 24 nt Flag insertions spanning 4 to 8 different PBS lengths are shown. [Figure 21F] MG153 candidates tested for 24 nt Flag insertions spanning 4 to 8 different PBS lengths are shown. [Figure 21G] MG153 candidates tested for 24 nt Flag insertions spanning 4 to 8 different PBS lengths are shown. [Figure 21H] MG153 candidates tested for 24 nt Flag insertions spanning 4 to 8 different PBS lengths are shown. [Figure 22A] MG153 candidates tested for 15 nt deletions spanning the length of 4-8 different PBSs are shown. [Figure 22B] MG153 candidates tested for 15 nt deletions spanning the length of 4-8 different PBSs are shown. [Figure 22C] MG153 candidates tested for 15 nt deletions spanning the length of 4-8 different PBSs are shown. [Figure 22D]MG153 candidates tested for 15 nt deletions spanning the length of 4-8 different PBSs are shown. [Figure 22E] MG153 candidates tested for 15 nt deletions spanning the length of 4-8 different PBSs are shown. [Figure 22F] MG153 candidates tested for 15 nt deletions spanning the length of 4-8 different PBSs are shown. [Figure 22G] MG153 candidates tested for 15 nt deletions spanning the length of 4-8 different PBSs are shown. [Figure 22H] MG153 candidates tested for 15 nt deletions spanning the length of 4-8 different PBSs are shown. [Figure 23A] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 24 nt insertion at 4–8 different PBS lengths. [Figure 23B] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 24 nt insertion at 4–8 different PBS lengths. [Figure 23C] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 24 nt insertion at 4–8 different PBS lengths. [Figure 23D] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 24 nt insertion at 4–8 different PBS lengths. [Figure 23E] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 24 nt insertion at 4–8 different PBS lengths. [Figure 23F] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 24 nt insertion at 4–8 different PBS lengths. [Figure 23G] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 24 nt insertion at 4–8 different PBS lengths. [Figure 23H]Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 24 nt insertion at 4–8 different PBS lengths. [Figure 24A] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 15 nt insertion at 4–8 different PBS lengths. [Figure 24B] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 15 nt insertion at 4–8 different PBS lengths. [Figure 24C] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 15 nt insertion at 4–8 different PBS lengths. [Figure 24D] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 15 nt insertion at 4–8 different PBS lengths. [Figure 24E] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 15 nt insertion at 4–8 different PBS lengths. [Figure 24F] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 15 nt insertion at 4–8 different PBS lengths. [Figure 24G] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 15 nt insertion at 4–8 different PBS lengths. [Figure 24H] Figure 1 shows the editing efficiency of spCas9(H840A) tethered with the MG160 candidate for a 15 nt insertion at 4–8 different PBS lengths. [Figure 25-1] Figure 25 shows GT transversions using RTs combined with MG nickase MG3-6. Untethered (Figures 25-1A-25-1B) and tethered (Figures 25-1C-25-1D) systems were tested. RTs tested included MG151-98, MG151-24, MG153-53, MG160-4, and MG151-99. [Figure 25-2]Figure 25 shows GT transversions using RTs combined with MG nickase MG3-6. Untethered (Figures 25-2A to 25-2B) and tethered (Figures 25-2C to 25-2D) systems were tested. RTs tested included MG151-98, MG151-24, MG153-53, MG160-4, and MG151-99. [Figure 26A] Figure 1 shows the screening of the indicated control RT and candidate RTs for their ability to retrotranspose an RNA cargo containing GFP in mammalian cells at targets specified by Cas9. Successful retrotransposition was detected by measuring the percentage of GFP-positive cells by flow cytometry in the indicated samples 3, 6, and 8 days after transfection of cells with the RT-containing plasmid, Cas9-containing plasmid, and chemically synthesized guide RNA. Strain-WT (WT-strain-1RT), Strain-Loss (D702Y-strain-1RT, RT-Loss), NT (non-targeted guide), and VEGFA (VEGFA-targeted guide). [Figure 26B] Figure 1 shows the screening of the indicated control RT and candidate RTs for their ability to retrotranspose an RNA cargo containing GFP in mammalian cells at targets specified by Cas9. Successful retrotransposition was detected by measuring the percentage of GFP-positive cells by flow cytometry in the indicated samples 3, 6, and 8 days after transfection of cells with the RT-containing plasmid, Cas9-containing plasmid, and chemically synthesized guide RNA. Strain-WT (WT-strain-1RT), Strain-Loss (D702Y-strain-1RT, RT-Loss), NT (non-targeted guide), and VEGFA (VEGFA-targeted guide). [Figure 26C]Figure 1 shows the screening of the indicated control RT and candidate RTs for their ability to retrotranspose an RNA cargo containing GFP in mammalian cells at targets specified by Cas9. Successful retrotransposition was detected by measuring the percentage of GFP-positive cells by flow cytometry in the indicated samples 3, 6, and 8 days after transfection of cells with the RT-containing plasmid, Cas9-containing plasmid, and chemically synthesized guide RNA. Strain-WT (WT-strain-1RT), Strain-Loss (D702Y-strain-1RT, RT-Loss), NT (non-targeted guide), and VEGFA (VEGFA-targeted guide). [Figure 27A] Figure 27A shows the prime editing capacity of engineered RTs. Figure 27A shows the prime editing percentage (y-axis) of MG160-4RT across different PBS lengths (x-axis). [Figure 27B] Figure 27B shows the prime editing capacity of engineered RTs. Figure 27B shows the prime editing percentage (y-axis) of MG151-98 across different PBS lengths (x-axis). [Figure 27C] Figure 27C shows the prime editing capacity of engineered RTs. Figure 27C shows the prime editing percentage (y-axis) of MG153-3RT across different PBS lengths (x-axis). [Figure 28-1] The ability of the RT candidates to efficiently generate full-length cDNA from large RNA templates in mammalian cells is demonstrated. [Figure 28-2] The ability of the RT candidates to efficiently generate full-length cDNA from large RNA templates in mammalian cells is demonstrated. [Figure 29A]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29AA] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29B]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29BB] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29C]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29CC] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29D]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29DD] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29E]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29F] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29G]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29H] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29I]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29J] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29K]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29L] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29M]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29N] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29O]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29P] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29Q]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29R] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29S]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29T] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29U]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29V] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29W]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29X] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29Y]Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 29Z] Figure 29 shows the editing rates of MG160 family candidates tethered with spCas9(H840A). Candidates from the MG160 family were tethered with spCas9(H840A) and transfected into HEK293T cells to determine G-to-T editing of the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG160-473 (Figure 29A), MG160-283 (Figure 29G), MG160-379 (Figure 29L), MG160-395 (Figure 29O), MG160-9 (Figure 29P), and MG160-107 (Figure 29CC) had G-to-T editing levels (across multiple PBS lengths) comparable to or better than spCas9(H840A) tethered with MMLV WT. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 30A]The editing rates of G-to-T transversions, insertions, and deletions for selected MG160 candidates are shown. Using spCas9(H840A)-tethered MG160 candidates, RTs were loaded to incorporate G-to-T transversions (Figure 30A), 24-nucleotide insertions (Figure 30B), and 15-nucleotide deletions (Figure 30C) into the VEGFA target. MG160-107, MG160-473, MG160-283, MG160-379, and MG160-395 showed comparable or improved editing levels compared with spCas9(H840A) tethered to MMLV WT for all types of editing at various PBS lengths. MG160-473 showed comparable editing levels to spCas9(H840A) tethered to MMLV2 (high-activity mutant). spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 30B] The editing rates of G-to-T transversions, insertions, and deletions for selected MG160 candidates are shown. Using spCas9(H840A)-tethered MG160 candidates, RTs were loaded to incorporate G-to-T transversions (Figure 30A), 24-nucleotide insertions (Figure 30B), and 15-nucleotide deletions (Figure 30C) into the VEGFA target. MG160-107, MG160-473, MG160-283, MG160-379, and MG160-395 showed comparable or improved editing levels compared with spCas9(H840A) tethered to MMLV WT for all types of editing at various PBS lengths. MG160-473 showed comparable editing levels to spCas9(H840A) tethered to MMLV2 (high-activity mutant). spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 30C]The editing rates of G-to-T transversions, insertions, and deletions for selected MG160 candidates are shown. Using spCas9(H840A)-tethered MG160 candidates, RTs were loaded to incorporate G-to-T transversions (Figure 30A), 24-nucleotide insertions (Figure 30B), and 15-nucleotide deletions (Figure 30C) into the VEGFA target. MG160-107, MG160-473, MG160-283, MG160-379, and MG160-395 showed comparable or improved editing levels compared with spCas9(H840A) tethered to MMLV WT for all types of editing at various PBS lengths. MG160-473 showed comparable editing levels to spCas9(H840A) tethered to MMLV2 (high-activity mutant). spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 31A] Figure 31 shows the editing rates of unique reverse transcriptase candidates from the MG retron family untethered with spCas9(H840A). Candidates from various MG retron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG173-1 (Figure 31J) and MG173-2 (Figure 31K) were active and demonstrated above background levels of G-to-T editing across multiple PBS lengths. Controls MMLV1 and MMLV2 were untethered and transfected along with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 31B]Figure 31 shows the editing rates of unique reverse transcriptase candidates from the MG retron family untethered with spCas9(H840A). Candidates from various MG retron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG173-1 (Figure 31J) and MG173-2 (Figure 31K) were active and demonstrated above background levels of G-to-T editing across multiple PBS lengths. Controls MMLV1 and MMLV2 were untethered and transfected along with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 31C] Figure 31 shows the editing rates of unique reverse transcriptase candidates from the MG retron family untethered with spCas9(H840A). Candidates from various MG retron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG173-1 (Figure 31J) and MG173-2 (Figure 31K) were active and demonstrated above background levels of G-to-T editing across multiple PBS lengths. Controls MMLV1 and MMLV2 were untethered and transfected along with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 31D]Figure 31 shows the editing rates of unique reverse transcriptase candidates from the MG retron family untethered with spCas9(H840A). Candidates from various MG retron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG173-1 (Figure 31J) and MG173-2 (Figure 31K) were active and demonstrated above background levels of G-to-T editing across multiple PBS lengths. Controls MMLV1 and MMLV2 were untethered and transfected along with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 31E] Figure 31 shows the editing rates of unique reverse transcriptase candidates from the MG retron family untethered with spCas9(H840A). Candidates from various MG retron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG173-1 (Figure 31J) and MG173-2 (Figure 31K) were active and demonstrated above background levels of G-to-T editing across multiple PBS lengths. Controls MMLV1 and MMLV2 were untethered and transfected along with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 31F]Figure 31 shows the editing rates of unique reverse transcriptase candidates from the MG retron family untethered with spCas9(H840A). Candidates from various MG retron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG173-1 (Figure 31J) and MG173-2 (Figure 31K) were active and demonstrated above background levels of G-to-T editing across multiple PBS lengths. Controls MMLV1 and MMLV2 were untethered and transfected along with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 31G] Figure 31 shows the editing rates of unique reverse transcriptase candidates from the MG retron family untethered with spCas9(H840A). Candidates from various MG retron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG173-1 (Figure 31J) and MG173-2 (Figure 31K) were active and demonstrated above background levels of G-to-T editing across multiple PBS lengths. Controls MMLV1 and MMLV2 were untethered and transfected along with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 31H]Figure 31 shows the editing rates of unique reverse transcriptase candidates from the MG retron family untethered with spCas9(H840A). Candidates from various MG retron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG173-1 (Figure 31J) and MG173-2 (Figure 31K) were active and demonstrated above background levels of G-to-T editing across multiple PBS lengths. Controls MMLV1 and MMLV2 were untethered and transfected along with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 31I] Figure 31 shows the editing rates of unique reverse transcriptase candidates from the MG retron family untethered with spCas9(H840A). Candidates from various MG retron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG173-1 (Figure 31J) and MG173-2 (Figure 31K) were active and demonstrated above background levels of G-to-T editing across multiple PBS lengths. Controls MMLV1 and MMLV2 were untethered and transfected along with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 31J]Figure 31 shows the editing rates of unique reverse transcriptase candidates from the MG retron family untethered with spCas9(H840A). Candidates from various MG retron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG173-1 (Figure 31J) and MG173-2 (Figure 31K) were active and demonstrated above background levels of G-to-T editing across multiple PBS lengths. Controls MMLV1 and MMLV2 were untethered and transfected along with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 31K] Figure 31 shows the editing rates of unique reverse transcriptase candidates from the MG retron family untethered with spCas9(H840A). Candidates from various MG retron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidates MG173-1 (Figure 31J) and MG173-2 (Figure 31K) were active and demonstrated above background levels of G-to-T editing across multiple PBS lengths. Controls MMLV1 and MMLV2 were untethered and transfected along with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 32A]The editing rates of reverse transcriptase candidates from the MG group II intron family untethered with spCas9(H840A) are shown. Candidates from various MG group II intron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidate MG169-1 (Figure 32D) showed slightly above background editing levels for G-to-T editing across multiple PBS lengths. Other MG candidates, MG164-5 (Figure 32A), MG166-2 (Figure 32B), and MG167-4 (Figure 32C), did not show editing levels above background. Control MMLV1 and MMLV2 were untethered and transfected with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 32B] The editing rates of reverse transcriptase candidates from the MG group II intron family untethered with spCas9(H840A) are shown. Candidates from various MG group II intron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidate MG169-1 (Figure 32D) showed slightly above background editing levels for G-to-T editing across multiple PBS lengths. Other MG candidates, MG164-5 (Figure 32A), MG166-2 (Figure 32B), and MG167-4 (Figure 32C), did not show editing levels above background. Control MMLV1 and MMLV2 were untethered and transfected with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 32C]The editing rates of reverse transcriptase candidates from the MG group II intron family untethered with spCas9(H840A) are shown. Candidates from various MG group II intron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidate MG169-1 (Figure 32D) showed slightly above background editing levels for G-to-T editing across multiple PBS lengths. Other MG candidates, MG164-5 (Figure 32A), MG166-2 (Figure 32B), and MG167-4 (Figure 32C), did not show editing levels above background. Control MMLV1 and MMLV2 were untethered and transfected with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 32D] The editing rates of reverse transcriptase candidates from the MG group II intron family untethered with spCas9(H840A) are shown. Candidates from various MG group II intron families were transfected into HEK293T cells in an untethered format along with the nickase spCas9(H840A) to determine G-to-T editing on the VEGFA target. Chemically synthesized guides have primer binding sites ranging in length from 2 to 20 nucleotides. Candidate MG169-1 (Figure 32D) showed slightly above background editing levels for G-to-T editing across multiple PBS lengths. Other MG candidates, MG164-5 (Figure 32A), MG166-2 (Figure 32B), and MG167-4 (Figure 32C), did not show editing levels above background. Control MMLV1 and MMLV2 were untethered and transfected with spCas9(H840A) and chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 33A]Figure 33A shows the editing percentages of WT MG160-4 and engineered mutants tethered with spCas9(H840A). Figure 33B shows the editing percentages for 17 engineered MG160-4 constructs tethered with spCas9(H840A) tested in HEK293T cells for G-to-T transversions on the VEGFA target. Transversions were tested using chemically synthesized guides with PBS lengths ranging from 6 to 13 nucleotides. Point mutations H230K and H230R showed neutral changes in G-to-T editing activity, but combining multiple mutations significantly reduced editing efficiency. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 33B] Figure 33B shows the editing rates of WT MG160-4 and engineered mutants tethered with spCas9(H840A). Figure 33B shows a G-to-T transversion with selected point mutations, which shows similar editing levels to WT MG160-4. MG160-4(H230K) and MG160-4(H230R) were then tested for a 24-nucleotide insertion (Figure 33C) and a 15-nucleotide deletion (Figure 33D). MG160-4(H230R) showed slightly better editing levels than MG160-4WT and MG160-4(H230K) at various desired editing sites. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNAs with a PBS length of 13 nucleotides. [Figure 33C] The editing rates of WT MG160-4 and engineered mutants tethered with spCas9(H840A) are shown. MG160-4(H230K) and MG160-4(H230R) were tested for a 24-nucleotide insertion (Figure 33C) and a 15-nucleotide deletion (Figure 33D). MG160-4(H230R) showed slightly better editing levels than MG160-4WT and MG160-4(H230K) at various desired editing sites. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 33D] The editing rates of WT MG160-4 and engineered mutants tethered with spCas9(H840A) are shown. MG160-4(H230K) and MG160-4(H230R) were tested for a 24-nucleotide insertion (Figure 33C) and a 15-nucleotide deletion (Figure 33D). MG160-4(H230R) showed slightly better editing levels than MG160-4WT and MG160-4(H230K) at various desired editing sites. spCas9-PE1 and spCas9-PE2 were transfected with chemically synthesized pegRNA with a PBS length of 13 nucleotides. [Figure 34] The editing rates of WT MG153-53 and engineered mutants are shown. Six engineered MG153-53 constructs, untethered and co-transfected with spCas9(H840A), were tested in HEK293T cells for G-to-T transversions on the VEGFA target. Transversions were tested using chemically synthesized guides ranging in length from 6 to 13 nucleotides. The point mutation V200R showed increased G-to-T editing activity comparable to WT MG153-53, but combining multiple mutations significantly reduced editing efficiency. MG153-53WT and engineered constructs had levels of editing comparable to or higher than untethered controls TGIRT, Marathon, and Marathon mutants, but dramatically lower than untethered MMLV WT (MMLV1) and MMLV hyperactive mutants (MMLV2). [Figure 35]The editing rate of MG3-6(H586A) using selected RT candidates is shown. MG3-6(H586A) nickase was combined with selected reverse transcriptases to achieve the desired correction in the AAVS1 target. The reverse transcriptase was either untethered (UT) and co-transfected with MG3-6(H586A) or tethered to MG3-6(H586A) at either the C-terminus of nickase I or the N-terminus of nickase (N). PEG RNAs were varied in PBS length of 8, 10, 13, and 20 nucleotides. Background editing was demonstrated with less than 0.1% editing. All selected RTs, except for MG153-53, demonstrated above-background editing with MG3-6(H586A). MG160-4 showed improved activity when tethered to MG3-6(H586A) compared to the untethered state. The engineered candidates for MG151-98 had a slight preference for either being untethered or tethered to the N-terminus of MG3-6(H586A). WT MMLV (MMLV1) and hyperactive mutant MMLV (MMLV2) had the highest editing levels when tethered to the C-terminus of MG3-6(H586A). Each data point represents a single biological replicate at different PBS lengths for each selected RT. [Figure 36A] Figure 36A shows the editing rate of untethered MG71-2 (H883A) using selected RT candidates on the AAVS1 target. Figure 36A shows biological triplicate data for untethered MG71-2n and selected RT candidates implementing five nucleotide changes on the AAVS1 target using chemically synthesized pegRNAs with PBS lengths of 4, 6, 8, 10, 13, and 16 nucleotides. Editing above background levels was defined as greater than 0.1%. Each graph represents a single biological replicate, except for Figure 36A, which shows data from biological triplicates. [Figure 36B]Figure 36 shows the editing rate of untethered MG71-2(H883A) using selected RT candidates on the AAVS1 target. The selected RT candidates were then tested for five nucleotide changes (Figure 36B), five nucleotide changes in the modified scaffold in the pegRNA (Figure 36C), a G to T transversion (Figure 36D), a 24 nucleotide insertion (Figure 36E), and a 15 nucleotide deletion (Figure 36F). Additional engineered MG151-98 candidates, MG151-98(166AA), MG151-98(166AA, H171N), and MG151-98(166AA, K297P), were tested for G to T changes (Figure 36G), 15 nucleotide deletions (Figure 36H), 24 nucleotide insertions (Figure 36I), and 5 nucleotide changes (Figure 36J) across PBS lengths of 4, 6, 8, 10, 13, and 16 nucleotides. Editing above background levels was defined as >0.1%. Each graph represents a single biological replicate, except for Figure 36A, which represents data from biological triplicates. [Figure 36C] Figure 36 shows the editing rate of untethered MG71-2(H883A) using selected RT candidates on the AAVS1 target. The selected RT candidates were then tested for five nucleotide changes (Figure 36B), five nucleotide changes in the modified scaffold in the pegRNA (Figure 36C), a G to T transversion (Figure 36D), a 24 nucleotide insertion (Figure 36E), and a 15 nucleotide deletion (Figure 36F). Additional engineered MG151-98 candidates, MG151-98(166AA), MG151-98(166AA, H171N), and MG151-98(166AA, K297P), were tested for G to T changes (Figure 36G), 15 nucleotide deletions (Figure 36H), 24 nucleotide insertions (Figure 36I), and 5 nucleotide changes (Figure 36J) across PBS lengths of 4, 6, 8, 10, 13, and 16 nucleotides. Editing above background levels was defined as >0.1%. Each graph represents a single biological replicate, except for Figure 36A, which represents data from biological triplicates. [Figure 36D]Figure 36 shows the editing rate of untethered MG71-2(H883A) using selected RT candidates on the AAVS1 target. The selected RT candidates were then tested for five nucleotide changes (Figure 36B), five nucleotide changes in the modified scaffold in the pegRNA (Figure 36C), a G to T transversion (Figure 36D), a 24 nucleotide insertion (Figure 36E), and a 15 nucleotide deletion (Figure 36F). Additional engineered MG151-98 candidates, MG151-98(166AA), MG151-98(166AA, H171N), and MG151-98(166AA, K297P), were tested for G to T changes (Figure 36G), 15 nucleotide deletions (Figure 36H), 24 nucleotide insertions (Figure 36I), and 5 nucleotide changes (Figure 36J) across PBS lengths of 4, 6, 8, 10, 13, and 16 nucleotides. Editing above background levels was defined as >0.1%. Each graph represents a single biological replicate, except for Figure 36A, which represents data from biological triplicates. [Figure 36E] Figure 36 shows the editing rate of untethered MG71-2(H883A) using selected RT candidates on the AAVS1 target. The selected RT candidates were then tested for five nucleotide changes (Figure 36B), five nucleotide changes in the modified scaffold in the pegRNA (Figure 36C), a G to T transversion (Figure 36D), a 24 nucleotide insertion (Figure 36E), and a 15 nucleotide deletion (Figure 36F). Additional engineered MG151-98 candidates, MG151-98(166AA), MG151-98(166AA, H171N), and MG151-98(166AA, K297P), were tested for G to T changes (Figure 36G), 15 nucleotide deletions (Figure 36H), 24 nucleotide insertions (Figure 36I), and 5 nucleotide changes (Figure 36J) across PBS lengths of 4, 6, 8, 10, 13, and 16 nucleotides. Editing above background levels was defined as >0.1%. Each graph represents a single biological replicate, except for Figure 36A, which represents data from biological triplicates. [Figure 36F]Figure 36 shows the editing rate of untethered MG71-2(H883A) using selected RT candidates on the AAVS1 target. The selected RT candidates were then tested for five nucleotide changes (Figure 36B), five nucleotide changes in the modified scaffold in the pegRNA (Figure 36C), a G to T transversion (Figure 36D), a 24 nucleotide insertion (Figure 36E), and a 15 nucleotide deletion (Figure 36F). Additional engineered MG151-98 candidates, MG151-98(166AA), MG151-98(166AA, H171N), and MG151-98(166AA, K297P), were tested for G to T changes (Figure 36G), 15 nucleotide deletions (Figure 36H), 24 nucleotide insertions (Figure 36I), and 5 nucleotide changes (Figure 36J) across PBS lengths of 4, 6, 8, 10, 13, and 16 nucleotides. Editing above background levels was defined as >0.1%. Each graph represents a single biological replicate, except for Figure 36A, which represents data from biological triplicates. [Figure 36G] Figure 36 shows the editing rate of untethered MG71-2(H883A) using selected RT candidates on the AAVS1 target. The selected RT candidates were then tested for five nucleotide changes (Figure 36B), five nucleotide changes in the modified scaffold in the pegRNA (Figure 36C), a G to T transversion (Figure 36D), a 24 nucleotide insertion (Figure 36E), and a 15 nucleotide deletion (Figure 36F). Additional engineered MG151-98 candidates, MG151-98(166AA), MG151-98(166AA, H171N), and MG151-98(166AA, K297P), were tested for G to T changes (Figure 36G), 15 nucleotide deletions (Figure 36H), 24 nucleotide insertions (Figure 36I), and 5 nucleotide changes (Figure 36J) across PBS lengths of 4, 6, 8, 10, 13, and 16 nucleotides. Editing above background levels was defined as >0.1%. Each graph represents a single biological replicate, except for Figure 36A, which represents data from biological triplicates. [Figure 36H]Figure 36 shows the editing rate of untethered MG71-2(H883A) using selected RT candidates on the AAVS1 target. The selected RT candidates were then tested for five nucleotide changes (Figure 36B), five nucleotide changes in the modified scaffold in the pegRNA (Figure 36C), a G to T transversion (Figure 36D), a 24 nucleotide insertion (Figure 36E), and a 15 nucleotide deletion (Figure 36F). Additional engineered MG151-98 candidates, MG151-98(166AA), MG151-98(166AA, H171N), and MG151-98(166AA, K297P), were tested for G to T changes (Figure 36G), 15 nucleotide deletions (Figure 36H), 24 nucleotide insertions (Figure 36I), and 5 nucleotide changes (Figure 36J) across PBS lengths of 4, 6, 8, 10, 13, and 16 nucleotides. Editing above background levels was defined as >0.1%. Each graph represents a single biological replicate, except for Figure 36A, which represents data from biological triplicates. [Figure 36I] Figure 36 shows the editing rate of untethered MG71-2(H883A) using selected RT candidates on the AAVS1 target. The selected RT candidates were then tested for five nucleotide changes (Figure 36B), five nucleotide changes in the modified scaffold in the pegRNA (Figure 36C), a G to T transversion (Figure 36D), a 24 nucleotide insertion (Figure 36E), and a 15 nucleotide deletion (Figure 36F). Additional engineered MG151-98 candidates, MG151-98(166AA), MG151-98(166AA, H171N), and MG151-98(166AA, K297P), were tested for G to T changes (Figure 36G), 15 nucleotide deletions (Figure 36H), 24 nucleotide insertions (Figure 36I), and 5 nucleotide changes (Figure 36J) across PBS lengths of 4, 6, 8, 10, 13, and 16 nucleotides. Editing above background levels was defined as >0.1%. Each graph represents a single biological replicate, except for Figure 36A, which represents data from biological triplicates. [Figure 36J]Figure 36 shows the editing rate of untethered MG71-2(H883A) using selected RT candidates on the AAVS1 target. The selected RT candidates were then tested for five nucleotide changes (Figure 36B), five nucleotide changes in the modified scaffold in the pegRNA (Figure 36C), a G to T transversion (Figure 36D), a 24 nucleotide insertion (Figure 36E), and a 15 nucleotide deletion (Figure 36F). Additional engineered MG151-98 candidates, MG151-98(166AA), MG151-98(166AA, H171N), and MG151-98(166AA, K297P), were tested for G to T changes (Figure 36G), 15 nucleotide deletions (Figure 36H), 24 nucleotide insertions (Figure 36I), and 5 nucleotide changes (Figure 36J) across PBS lengths of 4, 6, 8, 10, 13, and 16 nucleotides. Editing above background levels was defined as >0.1%. Each graph represents a single biological replicate, except for Figure 36A, which represents data from biological triplicates. [Figure 37] The editing rates of G-to-T transversions, insertions, and deletions for engineered MG151-98 mutants are shown. Using spCas9(H840A) and untethered engineered MG151-98 candidates, RTs were loaded to incorporate G-to-T transversions (Figure 37A), 24-nucleotide insertions (Figure 37B), and 15-nucleotide deletions (Figure 37C) into the VEGFA target. MG151-98(Δ166AA) enhanced editing levels for most PBS lengths in all conditions. Specifically, combining the trimmed MG151-98 construct with the point mutations H171N or K297P further increased editing levels, achieving levels comparable to or better than those of MMLV WT. MMLV WT and untethered spCas9(H840A) with MMLV2 were transfected with chemically synthesized PEG-RNAs with PBS lengths of 13 nucleotides. [Figure 38] An overview of the mechanism for achieving programmable genome editing using Cas9, retron reverse transcriptase, and ssDNA transposase TnpA is presented. [Figure 39A]Figure 39A shows an overview of the design principles used to generate engineered ncRNAs of Ec96. Figure 39A shows an overview of three insert sequences of three different lengths flanked by the LE / RE recognition motif of Hp TnpA. [Figure 39B] Figure 39B shows an overview of the design principles used to generate the engineered ncRNA of Ec96. Figure 39B shows a diagram from the indicated paper (Wang et al., Nature Microbiology (2022)) showing regions of msdDNA that are not resolved in the cryo-EM structure of the complex between Ec86 and its product. [Figure 39C] Figure 39C shows an overview of the design principles used to generate engineered ncRNAs for Ec96. Figure 39C shows three different exchangeable regions of the msd stem-loop identified for Ec86 ncRNA. [Figure 40-1] The predicted secondary structure of engineered Ec86 ncRNAs containing a 200-nt or 500-nt partial kanamycin gene insertion flanked by the reverse complement (rc) LE / RE motifs of Hp TnpA is shown. The motifs required for priming of reverse transcription, the msr, and the inverted repeat (IR) are highlighted. [Figure 40-2] The predicted secondary structure of engineered Ec86 ncRNAs containing a 200-nt or 500-nt partial kanamycin gene insertion flanked by the reverse complement (rc) LE / RE motifs of Hp TnpA is shown. The motifs required for priming of reverse transcription, the msr, and the inverted repeat (IR) are highlighted. [Figure 40-3] The predicted secondary structure of engineered Ec86 ncRNAs containing a 200-nt or 500-nt partial kanamycin gene insertion flanked by the reverse complement (rc) LE / RE motifs of Hp TnpA is shown. The motifs required for priming of reverse transcription, the msr, and the inverted repeat (IR) are highlighted. [Figure 41]Quantification of msdDNA production by qPCR in reactions with or without Ec86 reverse transcriptase is shown. WT is wild-type ncRNA. LE40RE_v1-v3, LE200RE_v1 and v3, and LE500RE v1-v3 are engineered ncRNA designs. [Figure 42] PCR confirmation of insertion of chimeric products generated by the TnpA / retron system. PCR products are indicated by arrows. Lane numbers correspond to the following: Lane 1: LE200RE_v1 ncRNA, +RT, +TnpA; Lane 2: LE200RE_v1 ncRNA, ~RT, +TnpA; Lane 3: LE200RE_v3 ncRNA, +RT, +TnpA; Lane 4: LE200RE_v3 ncRNA, ~RT, +TnpA; Lane 5: LE500RE_v1 ncRNA, +RT, +TnpA; Lane 6: LE500RE_v1 ncRNA, ~RT, +TnpA; Lane 7: LE500RE_v2 ncRNA, +RT, +TnpA; Lane 8: LE500RE_v2 ncRNA, ~RT, +TnpA; Lane 9: LE500RE_v3 ncRNA, +RT, +TnpA; Lane 10: LE500RE_v3 Lane 11: LE200RE_v1 ncRNA, +RT, ~TnpA; Lane 12: LE200RE_v1 ncRNA, ~RT, ~TnpA; Lane 13: LE200RE_v3 ncRNA, +RT, ~TnpA; Lane 14: LE200RE_v3 ncRNA, ~RT, ~TnpA; Lane 15: LE500RE_v1 ncRNA, +RT, ~TnpA; Lane 16: LE500RE_v1 ncRNA, ~RT, ~TnpA; Lane 17: LE500RE_v2 ncRNA, +RT, ~TnpA; Lane 18: LE500RE_v2 ncRNA, ~RT, ~TnpA; Lane 19: LE500RE_v3 ncRNA, +RT, ~TnpA; Lane 20: LE500RE_v3 ncRNA, ~RT, ~TnpA. [Figure 43]Figure 43 shows Sanger sequencing results of the inserted ssDNA product generated by TnpA, whose substrate is generated by the Ec86 retron. The highlighted region of the Sanger sequencing chromatogram indicates the junction of the chimeric product, with the 5' sequence corresponding to the right end (RE) motif of Hp TnpA incorporated with the cargo, and the 3' sequence corresponding to the ssDNA target provided in the reaction mixture. Figure 43 discloses SEQ ID NOs: 2578 and 2578, respectively, in order of appearance. [Figure 44] We show how to confirm ncRNA predictions and retron msd insertion tolerance. [Figure 45] The secondary structure prediction of retron ncRNA from the MG154 family is shown, highlighting the 5' and 3' inverted repeats (IRs) and msrs required for priming reverse transcription, along with the msd stem-loop. The region of the msd stem-loop replaced with the engineered sequence is indicated. [Figure 46-1] The secondary structure prediction of retron ncRNA from the MG155 family is shown, highlighting the 5' and 3' inverted repeats (IRs) and msrs required for priming reverse transcription, along with the msd stem-loop. The region of the msd stem-loop replaced with the engineered sequence is indicated. [Figure 46-2] The secondary structure prediction of retron ncRNA from the MG155 family is shown, highlighting the 5' and 3' inverted repeats (IRs) and msrs required for priming reverse transcription, along with the msd stem-loop. The region of the msd stem-loop replaced with the engineered sequence is indicated. [Figure 47] The secondary structure prediction of retron ncRNA from the MG156 family is shown, highlighting the 5' and 3' inverted repeats (IRs) and msrs required for priming reverse transcription, along with the msd stem-loop. The region of the msd stem-loop replaced with the engineered sequence is indicated. [Figure 48-1]The secondary structure prediction of retron ncRNA from the MG157 family is shown, highlighting the 5' and 3' inverted repeats (IRs) and msrs required for priming reverse transcription, along with the msd stem-loop. The region of the msd stem-loop replaced with the engineered sequence is indicated. [Figure 48-2] The secondary structure prediction of retron ncRNA from the MG157 family is shown, highlighting the 5' and 3' inverted repeats (IRs) and msrs required for priming reverse transcription, along with the msd stem-loop. The region of the msd stem-loop replaced with the engineered sequence is indicated. [Figure 49] The secondary structure prediction of retron ncRNA from the MG158 family is shown, highlighting the 5' and 3' inverted repeats (IRs) and msrs required for priming reverse transcription, along with the msd stem-loop. The region of the msd stem-loop replaced with the engineered sequence is indicated. [Figure 50] The secondary structure prediction of retron ncRNA from the MG159 family is shown, highlighting the 5' and 3' inverted repeats (IRs) and msrs required for priming reverse transcription, along with the msd stem-loop. The region of the msd stem-loop replaced with the engineered sequence is indicated. [Figure 51] Figure 1 shows the secondary structure prediction of retron ncRNA from the MG173 family, highlighting the 5' and 3' inverted repeats (IRs) and msrs required for priming reverse transcription, along with the msd stem-loop. The region of the msd stem-loop replaced with the engineered sequence is indicated. [Figure 52] Detection of msdDNA production by qPCR is shown. Ec86 was a positive control retron RT, and the corresponding ncRNA tested contained an approximately 200-nt insertion sequence at the previously described substitutable position, version 1. ncRNAs whose activity was identified using the corresponding retron RT are colored black (msdDNA production >10X higher than the no-RT control). ncRNAs whose activity was not identified using the corresponding retron RT are colored light gray. [Figure 53]Figure 53A shows the percentage of editing of a 5-nt change on the AAVS1 target using MG RT and MG71-2(H883A). RT was tested in either untethered or tethered format (RT at the C-terminus of MG71-2(H883A) designated Nickase-RT, and RT at the N-terminus of MG71-2(H883A) designated RT-Nickase). Figure 53A: MMLV2-RT was tested untethered and tethered with MG71-2(H883A), with the highest editing levels in untethered PBS13, Nickase-RT PBS16, and RT-Nickase PBS13. Figure 53B: Engineered MG151-98 (K297P, Δ166AA) was tested untethered and tethered with MG71-2 (H883A), with the highest editing levels seen in the RT-nickase configuration, with the highest levels seen in the untethered, nickase-RT, and RT-nickase configurations in PBS13. Figure 53C: MG160-4 (H230R) was tested only in the tethered format, with the highest editing levels seen in the nickase-RT configuration in PBS10 and RT-nickase in PBS13. The highest editing levels were seen in the RT-nickase configuration. Figure 53D: MG160-473 was tested in the tethered format, with the highest editing levels seen in the RT-nickase configuration in PBS13. The nickase-RT configuration for MG160-473 had low read counts from NGS processing, so the editing rate could not be determined. A precise edit indicates an intended correction with no errors found in the NGS amplicon. An imprecise edit refers to the incorporation of the intended edit, but also includes errors in the NGS amplicon and the incorporation of the pegRNA scaffold. [Figure 54A] Figure 54A shows the editing rates of G to T transversions for spCas9(H840A) and untethered MG retron family candidates. Figure 54A shows a summary of the editing rates of untethered MG retron candidates from the MG173 and MG192 families for G to T transversions across eight different PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides. The MG173-8 candidate showed the highest level of editing compared to the nine other retron candidates. [Figure 54B]The percentage of G to T transversion edits for spCas9(H840A) and untethered MG retron family candidates is shown. The editing levels labeled "correct edits" in Figures 54B-54J represent intended edits without errors in the NGS amplicon, while the bars labeled "incorrect edits" represent intended edits that incorporate errors in the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. [Figure 54C] The percentage of G to T transversion edits for spCas9(H840A) and untethered MG retron family candidates is shown. The editing levels labeled "correct edits" in Figures 54B-54J represent intended edits without errors in the NGS amplicon, while the bars labeled "incorrect edits" represent intended edits that incorporate errors in the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. [Figure 54D] The percentage of G to T transversion edits for spCas9(H840A) and untethered MG retron family candidates is shown. The editing levels labeled "correct edits" in Figures 54B-54J represent intended edits without errors in the NGS amplicon, while the bars labeled "incorrect edits" represent intended edits that incorporate errors in the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. [Figure 54E]The percentage of G-to-T transversion edits for spCas9(H840A) and untethered MG retron family candidates is shown. The MG173-8 candidate exhibited the highest level of editing compared to the nine other retron candidates. The editing levels labeled "correct edits" in Figures 54B-54J represent intended edits without errors within the NGS amplicon, while the bars labeled "incorrect edits" represent intended edits that incorporate errors within the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. [Figure 54F] The percentage of G-to-T transversion edits for spCas9(H840A) and untethered MG retron family candidates is shown. The MG173-8 candidate exhibited the highest level of editing compared to the nine other retron candidates. The editing levels labeled "correct edits" in Figures 54B-54J represent intended edits without errors within the NGS amplicon, while the bars labeled "incorrect edits" represent intended edits that incorporate errors within the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. [Figure 54G] The percentage of G to T transversion edits for spCas9(H840A) and untethered MG retron family candidates is shown. The editing levels labeled "correct edits" in Figures 54B-54J represent intended edits without errors in the NGS amplicon, while the bars labeled "incorrect edits" represent intended edits that incorporate errors in the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. [Figure 54H]The percentage of G to T transversion edits for spCas9(H840A) and untethered MG retron family candidates is shown. The editing levels labeled "correct edits" in Figures 54B-54J represent intended edits without errors in the NGS amplicon, while the bars labeled "incorrect edits" represent intended edits that incorporate errors in the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. [Figure 54I] The percentage of G-to-T transversion edits for spCas9(H840A) and untethered MG retron family candidates is shown. The MG173-8 candidate exhibited the highest level of editing compared to the nine other retron candidates. The editing levels labeled "correct edits" in Figures 54B-54J represent intended edits without errors within the NGS amplicon, while the bars labeled "incorrect edits" represent intended edits that incorporate errors within the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. [Fig. 54J] The percentage of G-to-T transversion edits for spCas9(H840A) and untethered MG retron family candidates is shown. The MG173-8 candidate exhibited the highest level of editing compared to the nine other retron candidates. The editing levels labeled "correct edits" in Figures 54B-54J represent intended edits without errors within the NGS amplicon, while the bars labeled "incorrect edits" represent intended edits that incorporate errors within the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. [Figure 54K]Figure 5 shows the G-to-T transversion editing rate for spCas9(H840A) and untethered MG retron family candidates. The MG173-8 candidate exhibited the highest level of editing compared to the nine other retron candidates. The editing levels displayed in Figures 54K-54S represent the editing levels across the lengths of eight different PBSs. The bars labeled "Editing" represent intended editing, including errors within the NGS amplicon (excluding pegRNA scaffold incorporation), while the bars labeled "Scaffold incorporation" represent intended editing and scaffold incorporation of pegRNA. [Figure 54L] Figure 54 shows the percentage of G-to-T transversion edits for spCas9(H840A) and untethered MG retron family candidates. Editing levels displayed in Figures 54K-54S show the editing levels across the lengths of eight different PBSs. The bars labeled "Editing" represent intended edits, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bars labeled "Scaffold incorporation" represent intended edits and scaffold incorporation of pegRNA. [Figure 54M] Figure 54 shows the percentage of G-to-T transversion edits for spCas9(H840A) and untethered MG retron family candidates. Editing levels displayed in Figures 54K-54S show the editing levels across the lengths of eight different PBSs. The bars labeled "Editing" represent intended edits, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bars labeled "Scaffold incorporation" represent intended edits and scaffold incorporation of pegRNA. [Figure 54N] Figure 54 shows the percentage of G-to-T transversion edits for spCas9(H840A) and untethered MG retron family candidates. Editing levels displayed in Figures 54K-54S show the editing levels across the lengths of eight different PBSs. The bars labeled "Editing" represent intended edits, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bars labeled "Scaffold incorporation" represent intended edits and scaffold incorporation of pegRNA. [Figure 54O]Figure 54 shows the percentage of G-to-T transversion edits for spCas9(H840A) and untethered MG retron family candidates. Editing levels displayed in Figures 54K-54S show the editing levels across the lengths of eight different PBSs. The bars labeled "Editing" represent intended edits, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bars labeled "Scaffold incorporation" represent intended edits and scaffold incorporation of pegRNA. [Figure 54P] Figure 54 shows the percentage of G-to-T transversion edits for spCas9(H840A) and untethered MG retron family candidates. Editing levels displayed in Figures 54K-54S show the editing levels across the lengths of eight different PBSs. The bars labeled "Editing" represent intended edits, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bars labeled "Scaffold incorporation" represent intended edits and scaffold incorporation of pegRNA. [Figure 54Q] Figure 54 shows the percentage of G-to-T transversion edits for spCas9(H840A) and untethered MG retron family candidates. Editing levels displayed in Figures 54K-54S show the editing levels across the lengths of eight different PBSs. The bars labeled "Editing" represent intended edits, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bars labeled "Scaffold incorporation" represent intended edits and scaffold incorporation of pegRNA. [Figure 54R] Figure 54 shows the percentage of G-to-T transversion edits for spCas9(H840A) and untethered MG retron family candidates. Editing levels displayed in Figures 54K-54S show the editing levels across the lengths of eight different PBSs. The bars labeled "Editing" represent intended edits, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bars labeled "Scaffold incorporation" represent intended edits and scaffold incorporation of pegRNA. [Figure 54S]Figure 54 shows the percentage of G-to-T transversion edits for spCas9(H840A) and untethered MG retron family candidates. Editing levels displayed in Figures 54K-54S show the editing levels across the lengths of eight different PBSs. The bars labeled "Editing" represent intended edits, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bars labeled "Scaffold incorporation" represent intended edits and scaffold incorporation of pegRNA. [Figure 55A] Figure 55A shows the editing rates of tethered MG160 family candidates for G to T transversions across eight different PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides. MG160-45, MG160-121, MG160-136, MG160-193, MG160-232, and MG160-358 demonstrated editing levels of 5% or higher across the various PBS lengths. [Figure 55AA] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AB] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AC]Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Fig. 55AD] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AE] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AF] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AG]Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AH] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AI] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AJ] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AK]Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AL] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AM] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AN] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AO]Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AP] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AQ] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AR] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55AS]Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55B] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55C] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55D] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55E] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55F] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55G] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55H]Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55I] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55J] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55K]Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55L] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55M] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55N]Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55O] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55P] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55Q]Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55R] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55S] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55T]Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55U] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55V] Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55W]Figures 55B-55W show the editing rate of G to T transversions for MG160 family candidates tethered with spCas9(H840A). Figures 55B-55W show editing levels across the length of eight different PBSs. Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. [Figure 55X] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55Y] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 55Z] Figure 55 shows the percentage of G to T transversion editing for MG160 family candidates tethered with spCas9(H840A). Editing levels displayed in Figures 55X-55AS show editing levels across eight different PBSs, with the bar labeled "Editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 56A]Figure 56 shows the editing rates for various edits using spCas9(H840A) and untethered MG151-98 mutants on the VEGFA target. MG151-98 wild-type and mutant MG151-98(D166AA, H171N) and MG151-98(D166AA, K297P) were evaluated for correction of a G to T transversion (Figures 56A and 56D), a 24-nucleotide insertion (Figures 56B and 56E), and a 15-nucleotide deletion (Figures 56C and 56F) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, and 13 nucleotides. Figures 56A-56C: Bars labeled "correct edits" represent intended edits without errors within the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were integrated and contained errors within the NGS amplicon due to misintegration by RT and / or pegRNA scaffold integration. Controls MMLV1 and MMLV2 represent untethered spCas9 (H840A), pegRNA in PBS13, and RT plasmids encoding MMLV1 or MMLV2, respectively. [Figure 56B]Figure 56 shows the editing rates for various edits using spCas9(H840A) and untethered MG151-98 mutants on the VEGFA target. MG151-98 wild-type and mutant MG151-98(D166AA, H171N) and MG151-98(D166AA, K297P) were evaluated for correction of a G to T transversion (Figures 56A and 56D), a 24-nucleotide insertion (Figures 56B and 56E), and a 15-nucleotide deletion (Figures 56C and 56F) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, and 13 nucleotides. Figures 56A-56C: Bars labeled "correct edits" represent intended edits without errors within the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were integrated and contained errors within the NGS amplicon due to misintegration by RT and / or pegRNA scaffold integration. Controls MMLV1 and MMLV2 represent untethered spCas9 (H840A), pegRNA in PBS13, and RT plasmids encoding MMLV1 or MMLV2, respectively. [Figure 56C]Figure 56 shows the editing rates for various edits using spCas9(H840A) and untethered MG151-98 mutants on the VEGFA target. MG151-98 wild-type and mutant MG151-98(D166AA, H171N) and MG151-98(D166AA, K297P) were evaluated for correction of a G to T transversion (Figures 56A and 56D), a 24-nucleotide insertion (Figures 56B and 56E), and a 15-nucleotide deletion (Figures 56C and 56F) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, and 13 nucleotides. Figures 56A-56C: Bars labeled "correct edits" represent intended edits without errors within the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were integrated and contained errors within the NGS amplicon due to misintegration by RT and / or pegRNA scaffold integration. Controls MMLV1 and MMLV2 represent untethered spCas9 (H840A), pegRNA in PBS13, and RT plasmids encoding MMLV1 or MMLV2, respectively. [Figure 56D]Figure 56 shows the editing rates for various edits using spCas9(H840A) and untethered MG151-98 mutants on the VEGFA target. MG151-98 wild-type and mutant MG151-98(D166AA, H171N) and MG151-98(D166AA, K297P) were evaluated for correction of a G to T transversion (Figures 56A and 56D), a 24-nucleotide insertion (Figures 56B and 56E), and a 15-nucleotide deletion (Figures 56C and 56F) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, and 13 nucleotides. The editing levels displayed in Figures 56D-56F show the editing levels across the lengths of four different PBSs. The bar labeled "Editing" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. Controls MMLV1 and MMLV2 represent untethered spCas9 (H840A), pegRNA in PBS13, and RT plasmids encoding MMLV1 or MMLV2, respectively. [Figure 56E]Figure 56 shows the editing rates for various edits using spCas9(H840A) and untethered MG151-98 mutants on the VEGFA target. MG151-98 wild-type and mutant MG151-98(D166AA, H171N) and MG151-98(D166AA, K297P) were evaluated for correction of a G to T transversion (Figures 56A and 56D), a 24-nucleotide insertion (Figures 56B and 56E), and a 15-nucleotide deletion (Figures 56C and 56F) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, and 13 nucleotides. The editing levels displayed in Figures 56D-56F show the editing levels across the lengths of four different PBSs. The bar labeled "Editing" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. Controls MMLV1 and MMLV2 represent untethered spCas9 (H840A), pegRNA in PBS13, and RT plasmids encoding MMLV1 or MMLV2, respectively. [Figure 56F]Figure 56 shows the editing rates for various edits using spCas9(H840A) and untethered MG151-98 mutants on the VEGFA target. MG151-98 wild-type and mutant MG151-98(D166AA, H171N) and MG151-98(D166AA, K297P) were evaluated for correction of a G to T transversion (Figures 56A and 56D), a 24-nucleotide insertion (Figures 56B and 56E), and a 15-nucleotide deletion (Figures 56C and 56F) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, and 13 nucleotides. The editing levels displayed in Figures 56D-56F show the editing levels across the lengths of four different PBSs. The bar labeled "Editing" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. Controls MMLV1 and MMLV2 represent untethered spCas9 (H840A), pegRNA in PBS13, and RT plasmids encoding MMLV1 or MMLV2, respectively. [Figure 57A]57A and 57E, the editing rates for G to T transversions on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, and 13 nucleotides were measured for MG151-123 wild-type and mutants (M304R, H287F, H178R, H178N, G279R, or G279N) (Figures 57A and 57E), MG151-126 wild-type and mutants (H287F, G179R, or G279N) (Figures 57A and 57E). The NGS amplicon was evaluated in the following mutants: G119R, G179N, A280R, A280K, or A276R (Figures 57B and 57F), MG153-18 wild-type and mutant (G119R, P242R, or double mutant G119R and P242R) (Figures 57C and 57G), and MG153-20 wild-type and mutant (N55R, P226R, or double mutant N55R and P226R) (Figures 57D and 57H). Figures 57A-57D: Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 57B]57A and 57E, the editing rates for G to T transversions on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, and 13 nucleotides were measured for MG151-123 wild-type and mutants (M304R, H287F, H178R, H178N, G279R, or G279N) (Figures 57A and 57E), MG151-126 wild-type and mutants (H287F, G179R, or G279N) (Figures 57A and 57E). The NGS amplicon was evaluated in the following mutants: G119R, G179N, A280R, A280K, or A276R (Figures 57B and 57F), MG153-18 wild-type and mutant (G119R, P242R, or double mutant G119R and P242R) (Figures 57C and 57G), and MG153-20 wild-type and mutant (N55R, P226R, or double mutant N55R and P226R) (Figures 57D and 57H). Figures 57A-57D: Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 57C]57A and 57E, the editing rates for G to T transversions on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, and 13 nucleotides were measured for MG151-123 wild-type and mutants (M304R, H287F, H178R, H178N, G279R, or G279N) (Figures 57A and 57E), MG151-126 wild-type and mutants (H287F, G179R, or G279N) (Figures 57A and 57E). The NGS amplicon was evaluated in the following mutants: G119R, G179N, A280R, A280K, or A276R (Figures 57B and 57F), MG153-18 wild-type and mutant (G119R, P242R, or double mutant G119R and P242R) (Figures 57C and 57G), and MG153-20 wild-type and mutant (N55R, P226R, or double mutant N55R and P226R) (Figures 57D and 57H). Figures 57A-57D: Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 57D]57A and 57E, the editing rates for G to T transversions on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, and 13 nucleotides were measured for MG151-123 wild-type and mutants (M304R, H287F, H178R, H178N, G279R, or G279N) (Figures 57A and 57E), MG151-126 wild-type and mutants (H287F, G179R, or G279N) (Figures 57A and 57E). The NGS amplicon was evaluated in the following mutants: G119R, G179N, A280R, A280K, or A276R (Figures 57B and 57F), MG153-18 wild-type and mutant (G119R, P242R, or double mutant G119R and P242R) (Figures 57C and 57G), and MG153-20 wild-type and mutant (N55R, P226R, or double mutant N55R and P226R) (Figures 57D and 57H). Figures 57A-57D: Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 57E]57A and 57E, the editing rates for G to T transversions on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, and 13 nucleotides were measured for MG151-123 wild-type and mutants (M304R, H287F, H178R, H178N, G279R, or G279N) (Figures 57A and 57E), MG151-126 wild-type and mutants (H287F, G179R, or G279N) (Figures 57A and 57E). Editing levels were assessed in MG153-18 wild-type and mutants (G119R, P242R, or double mutant G119R and P242R) (Figures 57B and 57F), MG153-18 wild-type and mutants (N55R, P226R, or double mutant N55R and P226R) (Figures 57D and 57H). Editing levels displayed in Figures 57E-57H show the percentage of editing levels across four different PBS lengths. The bar labeled "edit" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. Controls including "no RT" represent untethered spCas9(H840A) with pegRNA in PBS13, and MMLV1 and MMLV2 represent untethered spCas9(H840A), pegRNA in PBS13, and RT plasmids encoding MMLV1 or MMLV2, respectively. [Figure 57F]57A and 57E, the editing rates for G to T transversions on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, and 13 nucleotides were measured for MG151-123 wild-type and mutants (M304R, H287F, H178R, H178N, G279R, or G279N) (Figures 57A and 57E), MG151-126 wild-type and mutants (H287F, G179R, or G279N) (Figures 57A and 57E). Editing levels were assessed in MG153-18 wild-type and mutants (G119R, P242R, or double mutant G119R and P242R) (Figures 57B and 57F), MG153-18 wild-type and mutants (N55R, P226R, or double mutant N55R and P226R) (Figures 57D and 57H). Editing levels displayed in Figures 57E-57H show the percentage of editing levels across four different PBS lengths. The bar labeled "edit" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. Controls including "no RT" represent untethered spCas9(H840A) with pegRNA in PBS13, and MMLV1 and MMLV2 represent untethered spCas9(H840A), pegRNA in PBS13, and RT plasmids encoding MMLV1 or MMLV2, respectively. [Figure 57G]57A and 57E, the editing rates for G to T transversions on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, and 13 nucleotides were measured for MG151-123 wild-type and mutants (M304R, H287F, H178R, H178N, G279R, or G279N) (Figures 57A and 57E), MG151-126 wild-type and mutants (H287F, G179R, or G279N) (Figures 57A and 57E). Editing levels were assessed in MG153-18 wild-type and mutants (G119R, P242R, or double mutant G119R and P242R) (Figures 57B and 57F), MG153-18 wild-type and mutants (N55R, P226R, or double mutant N55R and P226R) (Figures 57D and 57H). Editing levels displayed in Figures 57E-57H show the percentage of editing levels across four different PBS lengths. The bar labeled "edit" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. Controls including "no RT" represent untethered spCas9(H840A) with pegRNA in PBS13, and MMLV1 and MMLV2 represent untethered spCas9(H840A), pegRNA in PBS13, and RT plasmids encoding MMLV1 or MMLV2, respectively. [Figure 57H]57A and 57E, the editing rates for G to T transversions on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, and 13 nucleotides were measured for MG151-123 wild-type and mutants (M304R, H287F, H178R, H178N, G279R, or G279N) (Figures 57A and 57E), MG151-126 wild-type and mutants (H287F, G179R, or G279N) (Figures 57A and 57E). Editing levels were assessed in MG153-18 wild-type and mutants (G119R, P242R, or double mutant G119R and P242R) (Figures 57B and 57F), MG153-18 wild-type and mutants (N55R, P226R, or double mutant N55R and P226R) (Figures 57D and 57H). Editing levels displayed in Figures 57E-57H show the percentage of editing levels across four different PBS lengths. The bar labeled "edit" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. Controls including "no RT" represent untethered spCas9(H840A) with pegRNA in PBS13, and MMLV1 and MMLV2 represent untethered spCas9(H840A), pegRNA in PBS13, and RT plasmids encoding MMLV1 or MMLV2, respectively. [Figure 58A]Figure 58 shows the editing rates for various editing sequences using spCas9(H840A)-tethered MG160-473 mutants on the VEGFA target. MG160-473 wild-type and mutant MG160-473(F231K) and MG160-473(F231R) were evaluated for correction of a G-to-T transversion (Figures 58A, 58D, 58G, and 58J), a 24-nucleotide insertion (Figures 58B, 58E, 58H, and 58K), and a 15-nucleotide deletion (Figures 58C, 58F, 58I, and 58L) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, 13, and 16 nucleotides. Figures 58A-C and 58G-I: Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" indicate that the intended edits were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 58B] Figure 58 shows the editing rates for various editing sequences using spCas9(H840A)-tethered MG160-473 mutants on the VEGFA target. MG160-473 wild-type and mutant MG160-473(F231K) and MG160-473(F231R) were evaluated for correction of a G-to-T transversion (Figures 58A, 58D, 58G, and 58J), a 24-nucleotide insertion (Figures 58B, 58E, 58H, and 58K), and a 15-nucleotide deletion (Figures 58C, 58F, 58I, and 58L) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, 13, and 16 nucleotides. Figures 58A-C and 58G-I: Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" indicate that the intended edits were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 58C]Figure 58 shows the editing rates for various editing sequences using spCas9(H840A)-tethered MG160-473 mutants on the VEGFA target. MG160-473 wild-type and mutant MG160-473(F231K) and MG160-473(F231R) were evaluated for correction of a G-to-T transversion (Figures 58A, 58D, 58G, and 58J), a 24-nucleotide insertion (Figures 58B, 58E, 58H, and 58K), and a 15-nucleotide deletion (Figures 58C, 58F, 58I, and 58L) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, 13, and 16 nucleotides. Figures 58A-C and 58G-I: Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" indicate that the intended edits were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 58D]Figure 58 shows the editing rates for various editing sequences using spCas9(H840A)-tethered MG160-473 mutants on the VEGFA target. MG160-473 wild-type and mutant MG160-473(F231K) and MG160-473(F231R) were evaluated for correction of a G-to-T transversion (Figures 58A, 58D, 58G, and 58J), a 24-nucleotide insertion (Figures 58B, 58E, 58H, and 58K), and a 15-nucleotide deletion (Figures 58C, 58F, 58I, and 58L) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, 13, and 16 nucleotides. The editing levels displayed in Figures 58D-58F and 58J-L show the percentage of editing levels across different PBS lengths. The bar labeled "Editing" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold integration), and the bar labeled "Scaffold Integration" represents intended editing and scaffold integration of pegRNA. Controls, including "Untreated," represent cells untreated during transfection. cas-PE1 and cas-PE2 represent spCas9(H840A) tethered to MMLV1 or MMLV2 with pegRNA in PBS13, respectively. Asterisks indicate NGS samples with fewer than 1,000 reads. [Figure 58E]Figure 58 shows the editing rates for various editing sequences using spCas9(H840A)-tethered MG160-473 mutants on the VEGFA target. MG160-473 wild-type and mutant MG160-473(F231K) and MG160-473(F231R) were evaluated for correction of a G-to-T transversion (Figures 58A, 58D, 58G, and 58J), a 24-nucleotide insertion (Figures 58B, 58E, 58H, and 58K), and a 15-nucleotide deletion (Figures 58C, 58F, 58I, and 58L) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, 13, and 16 nucleotides. The editing levels displayed in Figures 58D-58F and 58J-L show the percentage of editing levels across different PBS lengths. The bar labeled "Editing" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold integration), and the bar labeled "Scaffold Integration" represents intended editing and scaffold integration of pegRNA. Controls, including "Untreated," represent cells untreated during transfection. cas-PE1 and cas-PE2 represent spCas9(H840A) tethered to MMLV1 or MMLV2 with pegRNA in PBS13, respectively. Asterisks indicate NGS samples with fewer than 1,000 reads. [Figure 58F]Figure 58 shows the editing rates for various editing sequences using spCas9(H840A)-tethered MG160-473 mutants on the VEGFA target. MG160-473 wild-type and mutant MG160-473(F231K) and MG160-473(F231R) were evaluated for correction of a G-to-T transversion (Figures 58A, 58D, 58G, and 58J), a 24-nucleotide insertion (Figures 58B, 58E, 58H, and 58K), and a 15-nucleotide deletion (Figures 58C, 58F, 58I, and 58L) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, 13, and 16 nucleotides. The editing levels displayed in Figures 58D-58F and 58J-L show the percentage of editing levels across different PBS lengths. The bar labeled "Editing" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold integration), and the bar labeled "Scaffold Integration" represents intended editing and scaffold integration of pegRNA. Controls, including "Untreated," represent cells untreated during transfection. cas-PE1 and cas-PE2 represent spCas9(H840A) tethered to MMLV1 or MMLV2 with pegRNA in PBS13, respectively. Asterisks indicate NGS samples with fewer than 1,000 reads. [Figure 58G]Figure 58 shows the editing rates for various editing sequences using spCas9(H840A)-tethered MG160-473 mutants on the VEGFA target. MG160-473 wild-type and mutant MG160-473(F231K) and MG160-473(F231R) were evaluated for correction of a G-to-T transversion (Figures 58A, 58D, 58G, and 58J), a 24-nucleotide insertion (Figures 58B, 58E, 58H, and 58K), and a 15-nucleotide deletion (Figures 58C, 58F, 58I, and 58L) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, 13, and 16 nucleotides. The editing levels displayed in Figures 58D-58F and 58J-L show the percentage of editing levels across different PBS lengths. The bar labeled "Editing" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold integration), and the bar labeled "Scaffold Integration" represents intended editing and scaffold integration of pegRNA. Controls, including "Untreated," represent cells untreated during transfection. cas-PE1 and cas-PE2 represent spCas9(H840A) tethered to MMLV1 or MMLV2 with pegRNA in PBS13, respectively. Asterisks indicate NGS samples with fewer than 1,000 reads. [Figure 58H]Figure 58 shows the editing rates for various editing sequences using spCas9(H840A)-tethered MG160-473 mutants on the VEGFA target. MG160-473 wild-type and mutant MG160-473(F231K) and MG160-473(F231R) were evaluated for correction of a G-to-T transversion (Figures 58A, 58D, 58G, and 58J), a 24-nucleotide insertion (Figures 58B, 58E, 58H, and 58K), and a 15-nucleotide deletion (Figures 58C, 58F, 58I, and 58L) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, 13, and 16 nucleotides. The editing levels displayed in Figures 58D-58F and 58J-L show the percentage of editing levels across different PBS lengths. The bar labeled "Editing" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold integration), and the bar labeled "Scaffold Integration" represents intended editing and scaffold integration of pegRNA. Controls, including "Untreated," represent cells untreated during transfection. cas-PE1 and cas-PE2 represent spCas9(H840A) tethered to MMLV1 or MMLV2 with pegRNA in PBS13, respectively. Asterisks indicate NGS samples with fewer than 1,000 reads. [Figure 58I]Figure 58 shows the editing rates for various editing sequences using spCas9(H840A)-tethered MG160-473 mutants on the VEGFA target. MG160-473 wild-type and mutant MG160-473(F231K) and MG160-473(F231R) were evaluated for correction of a G-to-T transversion (Figures 58A, 58D, 58G, and 58J), a 24-nucleotide insertion (Figures 58B, 58E, 58H, and 58K), and a 15-nucleotide deletion (Figures 58C, 58F, 58I, and 58L) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, 13, and 16 nucleotides. The editing levels displayed in Figures 58D-58F and 58J-L show the percentage of editing levels across different PBS lengths. The bar labeled "Editing" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold integration), and the bar labeled "Scaffold Integration" represents intended editing and scaffold integration of pegRNA. Controls, including "Untreated," represent cells untreated during transfection. cas-PE1 and cas-PE2 represent spCas9(H840A) tethered to MMLV1 or MMLV2 with pegRNA in PBS13, respectively. Asterisks indicate NGS samples with fewer than 1,000 reads. [Figure 58J]Figure 58 shows the editing rates for various editing sequences using spCas9(H840A)-tethered MG160-473 mutants on the VEGFA target. MG160-473 wild-type and mutant MG160-473(F231K) and MG160-473(F231R) were evaluated for correction of a G-to-T transversion (Figures 58A, 58D, 58G, and 58J), a 24-nucleotide insertion (Figures 58B, 58E, 58H, and 58K), and a 15-nucleotide deletion (Figures 58C, 58F, 58I, and 58L) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, 13, and 16 nucleotides. The editing levels displayed in Figures 58D-58F and 58J-L show the percentage of editing levels across different PBS lengths. The bar labeled "Editing" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold integration), and the bar labeled "Scaffold Integration" represents intended editing and scaffold integration of pegRNA. Controls, including "Untreated," represent cells untreated during transfection. cas-PE1 and cas-PE2 represent spCas9(H840A) tethered to MMLV1 or MMLV2 with pegRNA in PBS13, respectively. Asterisks indicate NGS samples with fewer than 1,000 reads. [Figure 58K]Figure 58 shows the editing rates for various editing sequences using spCas9(H840A)-tethered MG160-473 mutants on the VEGFA target. MG160-473 wild-type and mutant MG160-473(F231K) and MG160-473(F231R) were evaluated for correction of a G-to-T transversion (Figures 58A, 58D, 58G, and 58J), a 24-nucleotide insertion (Figures 58B, 58E, 58H, and 58K), and a 15-nucleotide deletion (Figures 58C, 58F, 58I, and 58L) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, 13, and 16 nucleotides. The editing levels displayed in Figures 58D-58F and 58J-L show the percentage of editing levels across different PBS lengths. The bar labeled "Editing" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold integration), and the bar labeled "Scaffold Integration" represents intended editing and scaffold integration of pegRNA. Controls, including "Untreated," represent cells untreated during transfection. cas-PE1 and cas-PE2 represent spCas9(H840A) tethered to MMLV1 or MMLV2 with pegRNA in PBS13, respectively. Asterisks indicate NGS samples with fewer than 1,000 reads. [Figure 58L]Figure 58 shows the editing rates for various editing sequences using spCas9(H840A)-tethered MG160-473 mutants on the VEGFA target. MG160-473 wild-type and mutant MG160-473(F231K) and MG160-473(F231R) were evaluated for correction of a G-to-T transversion (Figures 58A, 58D, 58G, and 58J), a 24-nucleotide insertion (Figures 58B, 58E, 58H, and 58K), and a 15-nucleotide deletion (Figures 58C, 58F, 58I, and 58L) on the VEGFA target using pegRNAs with various PBS lengths of 6, 8, 10, 13, and 16 nucleotides. The editing levels displayed in Figures 58D-58F and 58J-L show the percentage of editing levels across different PBS lengths. The bar labeled "Editing" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold integration), and the bar labeled "Scaffold Integration" represents intended editing and scaffold integration of pegRNA. Controls, including "Untreated," represent cells untreated during transfection. cas-PE1 and cas-PE2 represent spCas9(H840A) tethered to MMLV1 or MMLV2 with pegRNA in PBS13, respectively. Asterisks indicate NGS samples with fewer than 1,000 reads. [Figure 59A]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59B]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59C]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59D]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59E]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59F]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59G]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59H]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59I]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59J]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59K]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59L]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59M]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59N]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59O]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 59P]Figure 1 shows the editing rates of five nucleotide changes on the AAVS1 target using tethered MG reverse transcriptase and MG71-2n. Reverse transcriptase was tested either untethered to MG71-2n, tethered to the C-terminus of MG71-2n (nickase-RT), or tethered to the N-terminus of MG71-2n (RT-nickase), across six different PBS lengths (6, 8, 10, 13, 16, or 20 nucleotides), targeting five nucleotide changes on the AAVS1 target. Reverse transcriptases tested for this modification include: MMLV1 (Figures 59A and 59D), MMLV2 (Figures 59B and 59E), MG160-4 (Figures 59C and 59F), MG151-98(D166AA) (Figures 59G and 59J), MG151-98(D166AA, H171N) (Figures 59H and 59K), MG151-98(D166AA, K297P) (Figures 59I and 59L), MG160-4(H230R) (Figures 59M and 59O), and MG160-473 (Figures 59N and 59P). Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. Low read counts indicate NGS samples with fewer than 1,000 reads. [Figure 60A]Figure 6 shows the editing rates of various edits on the AAVS1 target using MG reverse transcriptase tethered to the N-terminus of MG71-2n. Reverse transcriptases MMLV1, MMLV2, MG160-4 wild-type, or MG160-4(H230R) were tethered to the N-terminus of MG71-2n by a 32-amino acid linker, and pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides were used to introduce either a G-to-T transversion (Figures 60A and 60E), a 24-nucleotide insertion (Figures 60B and 60F), a 15-nucleotide deletion (Figures 60C and 60G), or a 5-nucleotide change (Figures 60D and 60H) into the AAVS1 target. An untethered (UT) MG160 candidate was also tested using pegRNA with a PBS length of 13 nucleotides. Figures 60A-60D: Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" indicate that the intended edits were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 60B]Figure 6 shows the editing rates of various edits on the AAVS1 target using MG reverse transcriptase tethered to the N-terminus of MG71-2n. Reverse transcriptases MMLV1, MMLV2, MG160-4 wild-type, or MG160-4(H230R) were tethered to the N-terminus of MG71-2n by a 32-amino acid linker, and pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides were used to introduce either a G-to-T transversion (Figures 60A and 60E), a 24-nucleotide insertion (Figures 60B and 60F), a 15-nucleotide deletion (Figures 60C and 60G), or a 5-nucleotide change (Figures 60D and 60H) into the AAVS1 target. An untethered (UT) MG160 candidate was also tested using pegRNA with a PBS length of 13 nucleotides. Figures 60A-60D: Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" indicate that the intended edits were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 60C]Figure 6 shows the editing rates of various edits on the AAVS1 target using MG reverse transcriptase tethered to the N-terminus of MG71-2n. Reverse transcriptases MMLV1, MMLV2, MG160-4 wild-type, or MG160-4(H230R) were tethered to the N-terminus of MG71-2n by a 32-amino acid linker, and pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides were used to introduce either a G-to-T transversion (Figures 60A and 60E), a 24-nucleotide insertion (Figures 60B and 60F), a 15-nucleotide deletion (Figures 60C and 60G), or a 5-nucleotide change (Figures 60D and 60H) into the AAVS1 target. An untethered (UT) MG160 candidate was also tested using pegRNA with a PBS length of 13 nucleotides. Figures 60A-60D: Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" indicate that the intended edits were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 60D]Figure 6 shows the editing rates of various edits on the AAVS1 target using MG reverse transcriptase tethered to the N-terminus of MG71-2n. Reverse transcriptases MMLV1, MMLV2, MG160-4 wild-type, or MG160-4(H230R) were tethered to the N-terminus of MG71-2n by a 32-amino acid linker, and pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides were used to introduce either a G-to-T transversion (Figures 60A and 60E), a 24-nucleotide insertion (Figures 60B and 60F), a 15-nucleotide deletion (Figures 60C and 60G), or a 5-nucleotide change (Figures 60D and 60H) into the AAVS1 target. An untethered (UT) MG160 candidate was also tested using pegRNA with a PBS length of 13 nucleotides. Figures 60A-60D: Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" indicate that the intended edits were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 60E]Figure 6 shows the editing rates of various edits on the AAVS1 target using MG reverse transcriptase tethered to the N-terminus of MG71-2n. Reverse transcriptases MMLV1, MMLV2, MG160-4 wild-type, or MG160-4(H230R) were tethered to the N-terminus of MG71-2n by a 32-amino acid linker, and pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides were used to introduce either a G-to-T transversion (Figures 60A and 60E), a 24-nucleotide insertion (Figures 60B and 60F), a 15-nucleotide deletion (Figures 60C and 60G), or a 5-nucleotide change (Figures 60D and 60H) into the AAVS1 target. An untethered (UT) MG160 candidate was also tested using pegRNA with a PBS length of 13 nucleotides. The editing levels displayed in Figures 60E-60H show the percentage of editing levels across the lengths of four different PBSs. The bar labeled "Edit" represents intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. An asterisk indicates that the NGS sample had fewer than 1,000 reads. [Figure 60F]Figure 6 shows the editing rates of various edits on the AAVS1 target using MG reverse transcriptase tethered to the N-terminus of MG71-2n. Reverse transcriptases MMLV1, MMLV2, MG160-4 wild-type, or MG160-4(H230R) were tethered to the N-terminus of MG71-2n by a 32-amino acid linker, and pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides were used to introduce either a G-to-T transversion (Figures 60A and 60E), a 24-nucleotide insertion (Figures 60B and 60F), a 15-nucleotide deletion (Figures 60C and 60G), or a 5-nucleotide change (Figures 60D and 60H) into the AAVS1 target. An untethered (UT) MG160 candidate was also tested using pegRNA with a PBS length of 13 nucleotides. The editing levels displayed in Figures 60E-60H show the percentage of editing levels across the lengths of four different PBSs. The bar labeled "Edit" represents intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. An asterisk indicates that the NGS sample had fewer than 1,000 reads. [Figure 60G]Figure 6 shows the editing rates of various edits on the AAVS1 target using MG reverse transcriptase tethered to the N-terminus of MG71-2n. Reverse transcriptases MMLV1, MMLV2, MG160-4 wild-type, or MG160-4(H230R) were tethered to the N-terminus of MG71-2n by a 32-amino acid linker, and pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides were used to introduce either a G-to-T transversion (Figures 60A and 60E), a 24-nucleotide insertion (Figures 60B and 60F), a 15-nucleotide deletion (Figures 60C and 60G), or a 5-nucleotide change (Figures 60D and 60H) into the AAVS1 target. An untethered (UT) MG160 candidate was also tested using pegRNA with a PBS length of 13 nucleotides. The editing levels displayed in Figures 60E-60H show the percentage of editing levels across the lengths of four different PBSs. The bar labeled "Edit" represents intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. An asterisk indicates that the NGS sample had fewer than 1,000 reads. [Figure 60H]Figure 6 shows the editing rates of various edits on the AAVS1 target using MG reverse transcriptase tethered to the N-terminus of MG71-2n. Reverse transcriptases MMLV1, MMLV2, MG160-4 wild-type, or MG160-4(H230R) were tethered to the N-terminus of MG71-2n by a 32-amino acid linker, and pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides were used to introduce either a G-to-T transversion (Figures 60A and 60E), a 24-nucleotide insertion (Figures 60B and 60F), a 15-nucleotide deletion (Figures 60C and 60G), or a 5-nucleotide change (Figures 60D and 60H) into the AAVS1 target. An untethered (UT) MG160 candidate was also tested using pegRNA with a PBS length of 13 nucleotides. The editing levels displayed in Figures 60E-60H show the percentage of editing levels across the lengths of four different PBSs. The bar labeled "Edit" represents intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. An asterisk indicates that the NGS sample had fewer than 1,000 reads. [Figure 61A]Figure 6 shows the editing rates for various edits on the AAVS1 target using MG71-2n and untethered MG151-98 mutants. Reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n were used to introduce either a G to T transversion (Figures 61A and 61E), a 24-nucleotide insertion (Figures 61B and 61F), a 15-nucleotide deletion (Figures 61C and 61G), or a 5-nucleotide change (Figures 61D and 61H) into the AAVS1 target using pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides. Figures 61A-61D: Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" indicate that the intended edits were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 61B] Figure 6 shows the editing rates for various edits on the AAVS1 target using MG71-2n and untethered MG151-98 mutants. Reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n were used to introduce either a G to T transversion (Figures 61A and 61E), a 24-nucleotide insertion (Figures 61B and 61F), a 15-nucleotide deletion (Figures 61C and 61G), or a 5-nucleotide change (Figures 61D and 61H) into the AAVS1 target using pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides. Figures 61A-61D: Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" indicate that the intended edits were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 61C] Figure 6 shows the editing rates for various edits on the AAVS1 target using MG71-2n and untethered MG151-98 mutants. Reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n were used to introduce either a G to T transversion (Figures 61A and 61E), a 24-nucleotide insertion (Figures 61B and 61F), a 15-nucleotide deletion (Figures 61C and 61G), or a 5-nucleotide change (Figures 61D and 61H) into the AAVS1 target using pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides. Figures 61A-61D: Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" indicate that the intended edits were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 61D]Figure 6 shows the editing rates for various edits on the AAVS1 target using MG71-2n and untethered MG151-98 mutants. Reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n were used to introduce either a G to T transversion (Figures 61A and 61E), a 24-nucleotide insertion (Figures 61B and 61F), a 15-nucleotide deletion (Figures 61C and 61G), or a 5-nucleotide change (Figures 61D and 61H) into the AAVS1 target using pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides. Figures 61A-61D: Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" indicate that the intended edits were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 61E] Figure 6 shows the editing rates for various edits on the AAVS1 target using MG71-2n and untethered MG151-98 mutants. Reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n were used to introduce either a G to T transversion (Figures 61A and 61E), a 24-nucleotide insertion (Figures 61B and 61F), a 15-nucleotide deletion (Figures 61C and 61G), or a 5-nucleotide change (Figures 61D and 61H) into the AAVS1 target using pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides. The editing levels displayed in Figures 61E-61H show the percentage of editing levels across the lengths of four different PBSs, where the bar labeled "Editing" represents intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. [Figure 61F] Figure 6 shows the editing rates for various edits on the AAVS1 target using MG71-2n and untethered MG151-98 mutants. Reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n were used to introduce either a G to T transversion (Figures 61A and 61E), a 24-nucleotide insertion (Figures 61B and 61F), a 15-nucleotide deletion (Figures 61C and 61G), or a 5-nucleotide change (Figures 61D and 61H) into the AAVS1 target using pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides. The editing levels displayed in Figures 61E-61H show the percentage of editing levels across the lengths of four different PBSs, where the bar labeled "Editing" represents intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. [Figure 61G]Figure 6 shows the editing rates for various edits on the AAVS1 target using MG71-2n and untethered MG151-98 mutants. Reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n were used to introduce either a G to T transversion (Figures 61A and 61E), a 24-nucleotide insertion (Figures 61B and 61F), a 15-nucleotide deletion (Figures 61C and 61G), or a 5-nucleotide change (Figures 61D and 61H) into the AAVS1 target using pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides. The editing levels displayed in Figures 61E-61H show the percentage of editing levels across the lengths of four different PBSs, where the bar labeled "Editing" represents intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. [Figure 61H] Figure 6 shows the editing rates for various edits on the AAVS1 target using MG71-2n and untethered MG151-98 mutants. Reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n were used to introduce either a G to T transversion (Figures 61A and 61E), a 24-nucleotide insertion (Figures 61B and 61F), a 15-nucleotide deletion (Figures 61C and 61G), or a 5-nucleotide change (Figures 61D and 61H) into the AAVS1 target using pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides. The editing levels displayed in Figures 61E-61H show the percentage of editing levels across the lengths of four different PBSs, where the bar labeled "Editing" represents intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended editing and scaffold incorporation of pegRNA. [Figure 61I] Figure 6 shows the editing rates for various edits on the AAVS1 target using MG71-2n and untethered MG151-98 mutants. Reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n were used to introduce either a G to T transversion (Figures 61A and 61E), a 24-nucleotide insertion (Figures 61B and 61F), a 15-nucleotide deletion (Figures 61C and 61G), or a 5-nucleotide change (Figures 61D and 61H) into the AAVS1 target using pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides. Figures 61I-61K show the editing levels of each specific modification across four different PBS lengths (8, 10, 13, and 16 nucleotides) for each reverse transcriptase, with the lines representing the average median percentage of editing. [Figure 61J] Figure 6 shows the editing rates for various edits on the AAVS1 target using MG71-2n and untethered MG151-98 mutants. Reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n were used to introduce either a G to T transversion (Figures 61A and 61E), a 24-nucleotide insertion (Figures 61B and 61F), a 15-nucleotide deletion (Figures 61C and 61G), or a 5-nucleotide change (Figures 61D and 61H) into the AAVS1 target using pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides. Figures 61I-61K show the editing levels of each specific modification across four different PBS lengths (8, 10, 13, and 16 nucleotides) for each reverse transcriptase, with the lines representing the average median percentage of editing. [Figure 61K]Figure 6 shows the editing rates for various edits on the AAVS1 target using MG71-2n and untethered MG151-98 mutants. Reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n were used to introduce either a G to T transversion (Figures 61A and 61E), a 24-nucleotide insertion (Figures 61B and 61F), a 15-nucleotide deletion (Figures 61C and 61G), or a 5-nucleotide change (Figures 61D and 61H) into the AAVS1 target using pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides. Figures 61I-61K show the editing levels of each specific modification across four different PBS lengths (8, 10, 13, and 16 nucleotides) for each reverse transcriptase, with the lines representing the average median percentage of editing. [Figure 61L] Figure 6 shows the editing rates for various edits on the AAVS1 target using MG71-2n and untethered MG151-98 mutants. Reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n were used to introduce either a G to T transversion (Figures 61A and 61E), a 24-nucleotide insertion (Figures 61B and 61F), a 15-nucleotide deletion (Figures 61C and 61G), or a 5-nucleotide change (Figures 61D and 61H) into the AAVS1 target using pegRNAs with PBS lengths of 8, 10, 13, and 16 nucleotides. Figures 61I-61K show the editing levels of each specific modification across four different PBS lengths (8, 10, 13, and 16 nucleotides) for each reverse transcriptase, with the lines representing the average median percentage of editing. [Figure 62A]Modifications to the MG71-2 scaffold that result in improved editing rates for five nucleotide changes to the AAVS1 target are shown. The MG71-2 scaffold contains 107 nucleotides and two modified forms, scaffolds D2 and D2C2, resulting in shortened scaffold lengths of 85 and 79 nucleotides, respectively. The D2 scaffold removes the final hairpin from the MG71-2 scaffold, while the D2C2 scaffold removes the final hairpin in combination with a small bulge in the MG71-2 scaffold. Editing levels for five nucleotide changes to the AAVS1 target were tested with wild-type and modified scaffolds over PBS lengths of 8, 10, 13, and 16 nucleotides using reverse transcriptases MMLV2 or MG160-4(H230R) tethered to the N-terminus of MG71-2n. Figure 62A: The bar labeled "correct edit" represents an intended edit without errors in the NGS amplicon, while the bar labeled "incorrect edit" indicates that the intended edit was incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. [Figure 62B]Modifications to the MG71-2 scaffold that result in improved editing rates for five nucleotide changes to the AAVS1 target are shown. The MG71-2 scaffold contains 107 nucleotides and two modified forms, scaffolds D2 and D2C2, resulting in shortened scaffold lengths of 85 and 79 nucleotides, respectively. The D2 scaffold removes the final hairpin from the MG71-2 scaffold, while the D2C2 scaffold removes the final hairpin in combination with a small bulge in the MG71-2 scaffold. Editing levels for five nucleotide changes to the AAVS1 target were tested with wild-type and modified scaffolds over PBS lengths of 8, 10, 13, and 16 nucleotides using reverse transcriptases MMLV2 or MG160-4(H230R) tethered to the N-terminus of MG71-2n. The editing levels displayed in Figure 62B show the percentage of editing levels across the lengths of eight different PBSs, with the bar labeled "editing" representing intended editing including errors in the NGS amplicon (excluding pegRNA scaffold incorporation) and the bar labeled "scaffold incorporation" representing intended editing and scaffold incorporation of pegRNA. [Figure 63A] Guide RNA optimization to improve the editing level of MG71-2n is shown. Figures 63A-63D show reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n, all of which were loaded with five nucleotide changes on the AAVS1 target. PBS lengths of 8, 10, 13, and 16 nucleotides in Figures 63A, 63C, 63E, and 63G had perfect complementarity to the target region. The bar labeled "Edit" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold integration), while the bar labeled "Scaffold Integration" represents intended editing and scaffold integration of pegRNA. [Figure 63B]63A-63D show guide RNA optimization to improve the editing level of MG71-2n. Figures 63A-63D show reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n loaded with five nucleotide changes on the AAVS1 target. In Figures 63B, 63D, 63F, and 63H, PBS lengths of 10, 13, 16, and 20 nucleotides were achieved with perfect complementarity of 8 nucleotides in the region flanking the reverse transcription template (RTT) and various mismatches (mm), resulting in PBS lengths of 10 (2 mismatches), 13 (5 mismatches), 16 (8 mismatches), and 20 (12 mismatches) nucleotides. The bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while the bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 63C]Guide RNA optimization to improve the editing level of MG71-2n is shown. Figures 63A-63D show reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n, all of which were loaded with five nucleotide changes on the AAVS1 target. PBS lengths of 8, 10, 13, and 16 nucleotides in Figures 63A, 63C, 63E, and 63G had perfect complementarity to the target region. The bar labeled "Edit" represents intended editing, including errors in the NGS amplicon (excluding pegRNA scaffold integration), while the bar labeled "Scaffold Integration" represents intended editing and scaffold integration of pegRNA. [Figure 63D]63A-63D show guide RNA optimization to improve the editing level of MG71-2n. Figures 63A-63D show reverse transcriptases MMLV1, MMLV2, MG151-98(D166AA, H171N), MG151-98(D166AA, K297P), MG151-98(D166AA, H171N, K297P), and untethered MG71-2n loaded with five nucleotide changes on the AAVS1 target. In Figures 63B, 63D, 63F, and 63H, PBS lengths of 10, 13, 16, and 20 nucleotides were achieved with perfect complementarity of 8 nucleotides in the region flanking the reverse transcription template (RTT) and various mismatches (mm), resulting in PBS lengths of 10 (2 mismatches), 13 (5 mismatches), 16 (8 mismatches), and 20 (12 mismatches) nucleotides. The bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while the bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 63E]Guide RNA optimization to improve the editing level of MG71-2n is shown. Figures 63E-63H show reverse transcriptases MMLV1, MMLV2, MG160-4, and MG160-4(H230R) tethered to the N-terminus of MG71-2n, which contain five nucleotide changes on the AAVS1 target, as well as untethered (UT) MG160-4 and MG160-4(H230R) MG71-2n. Various mismatches in the pegRNA across the PBS region were tested to determine whether improved editing could be achieved. PBS lengths of 8, 10, 13, and 16 nucleotides in Figures 63A, 63C, 63E, and 63G had perfect complementarity to the target region. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 63F]Guide RNA optimization to improve the editing level of MG71-2n is shown. Figures 63E-63H show reverse transcriptases MMLV1, MMLV2, MG160-4, and MG160-4(H230R) tethered to the N-terminus of MG71-2n, which contain five nucleotide changes on the AAVS1 target, as well as untethered (UT) MG160-4 and MG160-4(H230R) to MG71-2n. Various mismatches in the pegRNA across the PBS region were tested to determine whether improved editing could be achieved. In Figures 63B, 63D, 63F, and 63H, PBS lengths of 10, 13, 16, and 20 nucleotides were achieved with perfect complementarity of 8 nucleotides in the region flanking the reverse transcription template (RTT) and various mismatches (mm), resulting in PBS lengths of 10 (2 mismatches), 13 (5 mismatches), 16 (8 mismatches), and 20 (12 mismatches) nucleotides. The bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while the bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 63G]Guide RNA optimization to improve the editing level of MG71-2n is shown. Figures 63E-63H show reverse transcriptases MMLV1, MMLV2, MG160-4, and MG160-4(H230R) tethered to the N-terminus of MG71-2n, which contain five nucleotide changes on the AAVS1 target, as well as untethered (UT) MG160-4 and MG160-4(H230R) MG71-2n. Various mismatches in the pegRNA across the PBS region were tested to determine whether improved editing could be achieved. PBS lengths of 8, 10, 13, and 16 nucleotides in Figures 63A, 63C, 63E, and 63G had perfect complementarity to the target region. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 63H]Guide RNA optimization to improve the editing level of MG71-2n is shown. Figures 63E-63H show reverse transcriptases MMLV1, MMLV2, MG160-4, and MG160-4(H230R) tethered to the N-terminus of MG71-2n, which contain five nucleotide changes on the AAVS1 target, as well as untethered (UT) MG160-4 and MG160-4(H230R) to MG71-2n. Various mismatches in the pegRNA across the PBS region were tested to determine whether improved editing could be achieved. In Figures 63B, 63D, 63F, and 63H, PBS lengths of 10, 13, 16, and 20 nucleotides were achieved with perfect complementarity of 8 nucleotides in the region flanking the reverse transcription template (RTT) and various mismatches (mm), resulting in PBS lengths of 10 (2 mismatches), 13 (5 mismatches), 16 (8 mismatches), and 20 (12 mismatches) nucleotides. The bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while the bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 64A]Figure 64A shows guide RNA modifications of MG3-6 to improve editing levels in mammalian cells. MG3-6 wild-type mRNA was used to determine the modification rate (including SNPs and indels) of the target amplicon AAVS1 in NGS samples. The guide RNA consists of a target scaffold and spacer, and the pegRNA contains a guide RNA with a PBS and RTT sequence. Modifications modL1 to modL4 increase the GC content of hairpins 1 to 3 (modL1 to modL3) of the scaffold, and modL4 combines modifications of all hairpins in the scaffold. Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold integration. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 64B]Guide RNA modification of MG3-6 to improve editing levels in mammalian cells. Figures 64B-64C show editing rates for two nucleotide changes in the AAVS1 target measured over PBS lengths of 10 and 13 nucleotides for wild-type and modified scaffolds modL1-modL4 using MMLV2 tethered to the C-terminus of MG3-6(H586A). As controls, "Untreated" represents cells untreated during transfection, and MG3-6(H586A) represents cells transfected without reverse transcriptase, nickase, and pegRNA. Bars labeled "Correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "Incorrect edit" represent intended edits that were incorporated and contained errors in the NGS amplicon due to misintegration by RT and / or pegRNA scaffold integration. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 64C]Guide RNA modification of MG3-6 to improve editing levels in mammalian cells. Figures 64B-64C show editing rates for two nucleotide changes in the AAVS1 target measured over PBS lengths of 10 and 13 nucleotides for wild-type and modified scaffolds modL1-modL4 using MMLV2 tethered to the C-terminus of MG3-6(H586A). As controls, "Untreated" represents cells untreated during transfection, and MG3-6(H586A) represents cells transfected without reverse transcriptase, nickase, and pegRNA. Bars labeled "Correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "Incorrect edit" represent intended edits that were incorporated and contained errors in the NGS amplicon due to misintegration by RT and / or pegRNA scaffold integration. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 64D]Figures 64D-64E show guide RNA modifications of MG3-6 to improve editing levels in mammalian cells. Figures 64D-64E show editing rates for two nucleotide changes in the AAVS1 target measured over PBS lengths of 8, 10, 13, and 16 nucleotides with perfect complementarity to the target or PBS lengths of 10 (2 mismatches), 13 (5 mismatches), 16 (8 mismatches), and 20 (12 mismatches) using nickase MG3-6 (H586A) and untethered MMLV1, MMLV2, MG151-98 (D166AA, H171N), and MG151-98 (D166AA, K297P). The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to mis-incorporation by RT and / or pegRNA scaffold incorporation, the bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents the intended edit and scaffold incorporation of pegRNA. [Figure 64E]Figures 64D-64E show guide RNA modifications of MG3-6 to improve editing levels in mammalian cells. Figures 64D-64E show editing rates for two nucleotide changes in the AAVS1 target measured over PBS lengths of 8, 10, 13, and 16 nucleotides with perfect complementarity to the target or PBS lengths of 10 (2 mismatches), 13 (5 mismatches), 16 (8 mismatches), and 20 (12 mismatches) using nickase MG3-6 (H586A) and untethered MMLV1, MMLV2, MG151-98 (D166AA, H171N), and MG151-98 (D166AA, K297P). The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to mis-incorporation by RT and / or pegRNA scaffold incorporation, the bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents the intended edit and scaffold incorporation of pegRNA. [Figure 65A] Figure 65 shows a comparison of MG3-6 and MG3-6 / 3-8 target recognition using guide RNAs with various PBS lengths. Using MG3-6 wild-type and MG3-6 / 3-8 mRNA, the modification rates (including SNPs and indels) of the target amplicons AAVS1 (Figure 65A) and B2M (Figure 65B) were determined for guide RNAs or pegRNAs with PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides. The guide RNAs consist of a target scaffold and spacer, and the pegRNAs contain guide RNAs with PBS and RTT sequences. MG3-6 / 3-8 showed higher levels of target modification (including indels) compared to MG3-6. The control "untreated" represents cells that were not treated during transfection. [Figure 65B]Figure 65 shows a comparison of MG3-6 and MG3-6 / 3-8 target recognition using guide RNAs with various PBS lengths. Using MG3-6 wild-type and MG3-6 / 3-8 mRNA, the modification rates (including SNPs and indels) of the target amplicons AAVS1 (Figure 65A) and B2M (Figure 65B) were determined for guide RNAs or pegRNAs with PBS lengths of 2, 4, 6, 8, 10, 13, 16, and 20 nucleotides. The guide RNAs consist of a target scaffold and spacer, and the pegRNAs contain guide RNAs with PBS and RTT sequences. MG3-6 / 3-8 showed higher levels of target modification (including indels) compared to MG3-6. The control "untreated" represents cells that were not treated during transfection. [Figure 66A] Figure 66A shows the identification of MG14-241 targets for compatibility with the prime editing system. Wild-type MG14-241 mRNA or plasmid was used to determine the modification rate (including SNPs and indels) levels of various targets. Guide RNAs for various targets (G1, H1, B2, E2, F2, and G2) resulted in varying levels of modification rates at target E2 (a region of AAVS1), with the highest level of indels (approximately 60%). Bars labeled "correct edits" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 66B]Figure 66B shows the identification of the MG14-241 target for compatibility with the prime editing system. MG14-241 mRNA was used to determine the modification rate (including SNPs and indels) of the target amplicon AAVS1 in NGS samples. The guide RNA consists of a target scaffold and spacer, and the pegRNA contains a guide RNA with a PBS and RTT sequence. As the length of the PBS increased, the modification rate decreased. The control "untreated" represents cells that were not treated during transfection. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, while the bar labeled "incorrect edit" indicates that the intended edit was incorporated and contained errors in the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold integration. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 66C]Figures 66C-66D show the identification of the MG14-241 target for compatibility with the primed editing system. Figures 66C-66D show the editing rates of five nucleotide changes on the AAVS1 target across eight different PBS lengths (2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) using the untethered reverse transcriptases MMLV1, MMLV2, MG151-98 (D166AA, H171N), and MG151-98 (D166AA, K297P) and the nickase MG14-241n. MG14-241n (no RT) represents the nickase and pegRNA without reverse transcriptase in cell transfection. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to mis-incorporation by RT and / or pegRNA scaffold incorporation, the bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents the intended edit and scaffold incorporation of pegRNA. [Figure 66D]Figures 66C-66D show the identification of the MG14-241 target for compatibility with the primed editing system. Figures 66C-66D show the editing rates of five nucleotide changes on the AAVS1 target across eight different PBS lengths (2, 4, 6, 8, 10, 13, 16, and 20 nucleotides) using the untethered reverse transcriptases MMLV1, MMLV2, MG151-98 (D166AA, H171N), and MG151-98 (D166AA, K297P) and the nickase MG14-241n. MG14-241n (no RT) represents the nickase and pegRNA without reverse transcriptase in cell transfection. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to mis-incorporation by RT and / or pegRNA scaffold incorporation, the bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents the intended edit and scaffold incorporation of pegRNA. [Figure 67A] Figure 67A shows the design of engineered cell lines, RT-Cas chimeric proteins, and RNA cargo templates to evaluate integration by TPRT. Figure 67B shows a schematic diagram showing artificial sequences integrated into HEK293 cells via lentivirus to generate engineered cell lines with target sites for integration. [Figure 67B] Figure 67B shows the design of engineered cell lines, RT-Cas chimeric proteins, and RNA cargo templates to assess TPRT-mediated integration. Figure 67B shows the percentage of indels generated by five different sgRNAs targeting the engineered landing pad. Figure 67C shows a schematic diagram illustrating the four different conformations of each RT-Cas9WT / nickase fusion generated for testing. [Figure 67C]Figure 67B shows the design of engineered cell lines, RT-Cas chimeric proteins, and RNA cargo templates to assess TPRT-mediated integration. Figure 67B shows the percentage of indels generated by five different sgRNAs targeting the engineered landing pad. Figure 67C shows a schematic diagram illustrating the four different conformations of each RT-Cas9WT / nickase fusion generated for testing. [Figure 67D] Figure 67D shows the designs of engineered cell lines, RT-Cas chimeric proteins, and RNA cargo templates to assess TPRT-mediated integration. Figure 67D shows six cargo designs generated to test TPRT-mediated integration. [Figure 68] A schematic diagram of the primers used for left and right edge PCR to detect integration is shown. [Figure 69A] Detection of cargo integration using Cas9 WT-MG140-3 and sg4 is shown using Tapestation on LE (box indicates band of interest; Figure 69A), Sanger sequencing on LE PCR (sequences matching the landing pad and cargo are shown; Figure 69B), and Sanger sequencing on RE PCR (sequences matching the cargo are shown, but also show insertion of another product (Cas9); Figure 69C). [Figure 69B] Detection of cargo integration using Cas9 WT-MG140-3 and sg4 is shown using Tapestation on LE (box indicates band of interest; Figure 69A), Sanger sequencing on LE PCR (sequences matching the landing pad and cargo are shown; Figure 69B), and Sanger sequencing on RE PCR (sequences matching the cargo are shown, but also show insertion of another product (Cas9); Figure 69C). [Figure 69C]Detection of cargo integration using Cas9 WT-MG140-3 and sg4 is shown using Tapestation on LE (box indicates band of interest; Figure 69A), Sanger sequencing on LE PCR (sequences matching the landing pad and cargo are shown; Figure 69B), and Sanger sequencing on RE PCR (sequences matching the cargo are shown, but also show insertion of another product (Cas9); Figure 69C). [Figure 70A] Detection of cargo integration using MG140-3-Cas9 WT and sg4. Tape station on LE (Figure 70A) and Sanger sequencing on LE PCR (Figure 70B) show a match to the landing pad and mCherry cargo. [Figure 70B] Detection of cargo integration using MG140-3-Cas9 WT and sg4. Tape station on LE (Figure 70A) and Sanger sequencing on LE PCR (Figure 70B) show a match to the landing pad and mCherry cargo. [Figure 71] 1 shows detection of cargo integration using Cas9 WT-MG140-8 and sg4 by Sanger sequencing in LE. [Figure 72A] Detection of cargo integration using MG153-18-CAs9 WT and sg4 by tape station at LE (Figure 72A) and Sanger sequencing at LE (Figure 72B). [Figure 72B] Detection of cargo integration using MG153-18-CAs9 WT and sg4 by tape station at LE (Figure 72A) and Sanger sequencing at LE (Figure 72B). [Figure 73A]Retron RT activity against a cognate ncRNA loaded with a 2.2 kb cargo is shown. Figure 73A shows a schematic of the substrate design for testing retron RT activity and processivity. A universal template was used to test retron nonspecific activity, primed with an ssDNA priming oligo annealed to the 3' end of the RNA. Retron ncRNAs are primed at 5' and 3' inverted repeats (IRs), facilitated by the presence of terminal 5' and 3' retron ncRNA elements. For both substrates, the cargo sequence was flanked by the reverse complement (rc) of the LE and RE recognition motifs of MG92-4 TnpA. This sequence was then flanked by approximately 100 nt of RNA sequence, which, when converted to cDNA, could be quantified by multiplexed TaqMan qPCR to assess how much of the 5' (FAM) and 3' (HEX) ends of the cDNA molecule was synthesized by the RT. For retron ncRNA substrates, sequences were inserted within previously identified substitutable regions of the ncRNA msd. [Figure 73B] Figure 73B shows retron RT activity against a cognate ncRNA loaded with a 2.2 kb cargo. Figure 73B shows the amount of ssDNA detected by FAM and HEX using multiplexed TaqMan qPCR. A no-RT control was generated by not adding any RT-expressing template to the cell-free expression system. The dashed line indicates 10-fold above the highest background of no RT signal. TGIRT is the GII intron control RT, MMLV is the retrovirus control RT, and Ec86 is the retron control RT. The label "gen" indicates that the RT was tested using the universal template, and "ncRNA" indicates that the RT was tested using its cognate ncRNA loaded with cargo. [Figure 73C]Figure 73C shows retron RT activity against cognate ncRNAs carrying a 2.2 kb cargo. Figure 73C shows confirmation of 2.2 kb ssDNA generated by RT using the TapeStation D5000. Lane numbers correspond to the following: Lane 1: ladder; Lane 2: no RT gene; Lane 3: TGI RT gene; Lane 4: MG154-1 nRNA; Lane 5: MG157-1 ncRNA; Lane 6: MG157-3 ncRNA; Lane 7: MG157-4 ncRNA; Lane 8: MG158-1 ncRNA; Lane 9: MG159-3 ncRNA; Lane 10: MG173-1 ncRNA. [Figure 74A] Screening of the ability of retron RT MG173-1 to synthesize cDNA in mammalian cells is shown. Figure 74A shows a schematic diagram illustrating the methodology used to detect cDNA synthesis in mammalian cells. The first (FAM) and last (HEX) 100 bp of a 4.1 kb RNA template were detected using TaqMan-based qPCR. [Figure 74B] Screening of the ability of retron RT MG173-1 to synthesize cDNA in mammalian cells is shown. Figure 74A shows a schematic diagram illustrating the methodology used to detect cDNA synthesis in mammalian cells. The first (FAM) and last (HEX) 100 bp of a 4.1 kb RNA template were detected using TaqMan-based qPCR. [Figure 75A] Figure 75A shows PCR insertion reactions and Sanger sequencing of TnpA92-4 using a 2.2 kb retron-generated cDNA cargo. Figure 75A: Lane 1: PCR of a no-template control (NTC) insertion reaction using ssDNA ultramer target and MG173-1-generated cDNA cargo. Lane 2: PCR of a TnpA92-4 insertion reaction using ssDNA ultramer target and MG173-1-generated cDNA cargo. [Figure 75B]Figure 75B shows the PCR insertion reaction and Sanger sequencing of TnpA92-4 with a 2.2 kb retron-generated cDNA cargo. Figure 75B: Sanger sequencing of the chimeric insertion product generated by TnpA92-4-mediated insertion of the MG173-1-generated cargo into a ssDNA ultramer target. Figure 75B discloses SEQ ID NO: 2579. [Figure 76A] Targeting therapeutic sites with MG71-2 is shown. Figure 76A: WT mRNA of MG71-2 with indels at therapeutically relevant sites (hPDK1, G6PC1 Q347*, and PAH R408W) using various guide RNAs. The highest indels were found in guide 1 of the hPDK1 gene and guide 2 of the PAH gene, which targets the R408W mutation. Other guides tested for G6PC1 did not result in indel detection in these guides. The positive control contained a guide RNA targeting AAVS1. Bars labeled "correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edit" represent intended edits that were incorporated and contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. The bar labeled "Edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. * indicates that fewer than 1000 reads were acquired for that NGS sample, and error bars represent the standard deviation of two biological replicates. [Figure 76B]Figure 76B shows targeting of the therapeutic site using MG71-2. Figure 76B: Targeting of the HBB gene mutation E7V using guide RNA and pegRNA with various PBS lengths of 8, 10, and 13 nucleotides. Indels are slightly reduced with pegRNA compared to guide RNA. Editing levels across eight different PBS lengths are shown. The bar labeled "correct edit" represents intended edits without errors within the NGS amplicon, while the bar labeled "incorrect edit" represents intended edits that were incorporated and contained errors within the NGS amplicon due to RT misintegration and / or pegRNA scaffold integration. The bar labeled "edit" represents intended edits with errors within the NGS amplicon (excluding pegRNA scaffold integration), while the bar labeled "scaffold integration" represents intended edits and pegRNA scaffold integration. * indicates that fewer than 1000 reads were obtained for that NGS sample, and error bars represent the standard deviation of two biological replicates. [Figure 76C]Targeting of therapeutic sites using MG71-2 is shown. Figures 76C-76H: Prime editing experiments were then performed using pegRNAs with the spacers from Figures 76A-76B. The prime editing systems were MG160-4(H230R) tethered to the N-terminus of MG71-2n (MG160-4(H230R)-MG71-2n) and MMLV2 tethered to the N-terminus of MG71-2n (MMLV2-MG71-2n). Figures 76C-76D: MG160-4(H230R)-MG71-2n and MMLV2-MG71-2n were targeted for disruption of microRNA recognition sites by using pegRNAs containing 3- or 5-nucleotide (nt) mismatches incorporated into the RT template (RTT) of the pegRNA. The highest level of editing was observed with PBS10 for the 3-nt mismatch incorporation into the hPDK1 microRNA recognition site. Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample, and error bars represent the standard deviation of two biological replicates. [Figure 76D]Targeting of therapeutic sites using MG71-2 is shown. Figures 76C-76H: Prime editing experiments were then performed using pegRNAs with the spacers from Figures 76A-76B. The prime editing systems were MG160-4(H230R) tethered to the N-terminus of MG71-2n (MG160-4(H230R)-MG71-2n) and MMLV2 tethered to the N-terminus of MG71-2n (MMLV2-MG71-2n). Figures 76C-76D: MG160-4(H230R)-MG71-2n and MMLV2-MG71-2n were targeted for disruption of microRNA recognition sites by using pegRNAs containing 3- or 5-nucleotide (nt) mismatches incorporated into the RT template (RTT) of the pegRNA. The highest level of editing was observed with PBS10 for the 3-nt mismatch incorporation into the hPDK1 microRNA recognition site. Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample, and error bars represent the standard deviation of two biological replicates. [Figure 76E]Targeting of the therapeutic site using MG71-2 is shown. Figures 76C-76H: Prime editing experiments were then performed using pegRNA with the spacer from Figures 76A-76B. The prime editing systems were MG160-4(H230R) tethered to the N-terminus of MG71-2n (MG160-4(H230R)-MG71-2n) and MMLV2 tethered to the N-terminus of MG71-2n (MMLV2-MG71-2n). Figures 76E-76F: Prime editing systems targeting PAH R408W over PBS lengths of 8, 10, and 13 nt with RTT lengths varying from 29 nt and 32 nt did not show detectable levels of editing. Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample, and error bars represent the standard deviation of two biological replicates. [Figure 76F]Targeting of the therapeutic site using MG71-2 is shown. Figures 76E-76F: A primed editing system targeting PAH R408W over 8, 10, and 13 nt PBS lengths with RTTs varying in length from 29 nt to 32 nt showed no detectable levels of editing. Bars labeled "correct edits" represent intended edits without errors within the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors within the NGS amplicon due to RT misintegration and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors within the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample, and error bars represent the standard deviation of two biological replicates. [Figure 76G] Targeting of therapeutic sites using MG71-2 is shown. Figures 76G-76H: MG160-4(H230R)-MG71-2n and MMLV2-MG71-2n targeted HBB E7V mutations across multiple PBS lengths and achieved editing above background levels. The bar labeled "correct editing" represents intended editing without errors within the NGS amplicon. The bar labeled "incorrect editing" represents intended editing with errors within the NGS amplicon due to misintegration by RT and / or PEG-RNA scaffold integration. The bar labeled "editing" represents intended editing with errors within the NGS amplicon (excluding PEG-RNA scaffold integration), and the bar labeled "scaffold integration" represents intended editing and PEG-RNA scaffold integration. * indicates that fewer than 1000 reads were obtained for that NGS sample, and error bars represent the standard deviation of two biological replicates. [Figure 76H]Targeting of therapeutic sites using MG71-2 is shown. Figures 76G-76H: MG160-4(H230R)-MG71-2n and MMLV2-MG71-2n targeted HBB E7V mutations across multiple PBS lengths and achieved editing above background levels. The bar labeled "correct editing" represents intended editing without errors within the NGS amplicon. The bar labeled "incorrect editing" represents intended editing with errors within the NGS amplicon due to misintegration by RT and / or PEG-RNA scaffold integration. The bar labeled "editing" represents intended editing with errors within the NGS amplicon (excluding PEG-RNA scaffold integration), and the bar labeled "scaffold integration" represents intended editing and PEG-RNA scaffold integration. * indicates that fewer than 1000 reads were obtained for that NGS sample, and error bars represent the standard deviation of two biological replicates. [Figure 77A] Figure 77A shows data demonstrating that MG71-2 recognizes multiple guide RNAs across a variety of targets, enabling it to integrate larger genome changes. Figure 77A: WT mRNA of MG71-2 with indels on two targets (TRAC and AAVS1) using a variety of guide RNAs. Target sites D3 and D4 on AAVS1 showed some of the highest levels of editing and were 69 nt apart on the AAVS1 target. The spacers for D3 and D4 were oriented in the correct orientation to be compatible with TWIN, PASTE, and template jumping (Tj) prime editing methods. [Figure 77B]Figure 77B shows data demonstrating that MG71-2 recognizes multiple guide RNAs across various targets, enabling it to integrate larger genome changes. Figure 77B: Tape station gel image confirming the replacement of a 69-nt sequence in the AAVS1 target with a 38-nt Bxb1 sequence using Bxb1-specific primers. Lanes G3 and H3 represent two replicates of MMLV2-MG71-2n using pegRNA containing the Bxb1 sequence and a nicking guide (PASTE method). Lanes A4 and B4 represent two replicates of MMLV2-MG71-2n using pegRNA containing the Bxb1 sequence but without a nicking guide. Lanes C4 and D4 represent samples from MG151-98 (H171N, K297P, 166AA)-MG71-2n using pegRNA containing the Bxb1 sequence but without a nicking guide. Lanes E4 and D4 represent pegRNA containing the Bxb1 sequence and a nicking guide (PASTE method). [Figure 77C] Figures 77C-77D: Tapestation fragment analysis for lanes G3, H3, E4, and F4 confirming amplicons containing Bxb1 sequences. [Figure 77D] Figures 77C-77D: Tapestation fragment analysis for lanes G3, H3, E4, and F4 confirming amplicons containing Bxb1 sequences. [Figure 78A]Optimization of the MG71-2n system using selected reverse transcriptases is shown in Figures 78A-78D. MG160-4(H230R) was cloned at either the N- or C-terminus of MG71-2n using a 33-amino acid linker. Furthermore, MG160-4(H230R) and MG71-2n were introduced into five different insertion sites (S311, S355, T396, I822, and V1176). The introduced constructs had 33-amino acid linkers at the 5' and 3' ends of MG160-4(H230R) at the insertion site. The introduced constructs were tested for 5-nt changes and 24-nt insertions into the AAVS1 target across four different PBS lengths. MG160-4(H230R) on the N-terminus of MG71-2n showed the highest level of editing. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to mis-incorporation by RT and / or pegRNA scaffold incorporation, the bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents the intended edit and scaffold incorporation of pegRNA. [Figure 78B]Optimization of the MG71-2n system using selected reverse transcriptases is shown in Figures 78A-78D. MG160-4(H230R) was cloned at either the N- or C-terminus of MG71-2n using a 33-amino acid linker. Furthermore, MG160-4(H230R) and MG71-2n were introduced into five different insertion sites (S311, S355, T396, I822, and V1176). The introduced constructs had 33-amino acid linkers at the 5' and 3' ends of MG160-4(H230R) at the insertion site. The introduced constructs were tested for 5-nt changes and 24-nt insertions into the AAVS1 target across four different PBS lengths. MG160-4(H230R) on the N-terminus of MG71-2n showed the highest level of editing. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to mis-incorporation by RT and / or pegRNA scaffold incorporation, the bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents the intended edit and scaffold incorporation of pegRNA. [Figure 78C]Optimization of the MG71-2n system using selected reverse transcriptases is shown in Figures 78A-78D. MG160-4(H230R) was cloned at either the N- or C-terminus of MG71-2n using a 33-amino acid linker. Furthermore, MG160-4(H230R) and MG71-2n were introduced into five different insertion sites (S311, S355, T396, I822, and V1176). The introduced constructs had 33-amino acid linkers at the 5' and 3' ends of MG160-4(H230R) at the insertion site. The introduced constructs were tested for 5-nt changes and 24-nt insertions into the AAVS1 target across four different PBS lengths. MG160-4(H230R) on the N-terminus of MG71-2n showed the highest level of editing. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to mis-incorporation by RT and / or pegRNA scaffold incorporation, the bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents the intended edit and scaffold incorporation of pegRNA. [Figure 78D]Optimization of the MG71-2n system using selected reverse transcriptases is shown in Figures 78A-78D. MG160-4(H230R) was cloned at either the N- or C-terminus of MG71-2n using a 33-amino acid linker. Furthermore, MG160-4(H230R) and MG71-2n were introduced into five different insertion sites (S311, S355, T396, I822, and V1176). The introduced constructs had 33-amino acid linkers at the 5' and 3' ends of MG160-4(H230R) at the insertion site. The introduced constructs were tested for 5-nt changes and 24-nt insertions into the AAVS1 target across four different PBS lengths. MG160-4(H230R) on the N-terminus of MG71-2n showed the highest level of editing. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to mis-incorporation by RT and / or pegRNA scaffold incorporation, the bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents the intended edit and scaffold incorporation of pegRNA. [Figure 78E]Optimization of the MG71-2n system using selected reverse transcriptases is shown in Figures 78E-78H. Various linker lengths (14AA, 15AA, 26AA, and 32AA) fusing MG160-4 to the N-terminus of MG71-2 were tested along with the original 33AA linker. The 32AA and 33AA linkers had similar levels of editing for both the 5-nt change and the 24-nt insertion on the AAVS1 target. Bars labeled "correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edit" represent intended edits that were incorporated but contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 78F] Optimization of the MG71-2n system using selected reverse transcriptases is shown in Figures 78E-78H. Various linker lengths (14AA, 15AA, 26AA, and 32AA) fusing MG160-4 to the N-terminus of MG71-2 were tested along with the original 33AA linker. The 32AA and 33AA linkers had similar levels of editing for both the 5-nt change and the 24-nt insertion on the AAVS1 target. Bars labeled "correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edit" represent intended edits that were incorporated but contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 78G]Optimization of the MG71-2n system using selected reverse transcriptases is shown in Figures 78E-78H. Various linker lengths (14AA, 15AA, 26AA, and 32AA) fusing MG160-4 to the N-terminus of MG71-2 were tested along with the original 33AA linker. The 32AA and 33AA linkers had similar levels of editing for both the 5-nt change and the 24-nt insertion on the AAVS1 target. Bars labeled "correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edit" represent intended edits that were incorporated but contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 78H] Optimization of the MG71-2n system using selected reverse transcriptases is shown in Figures 78E-78H. Various linker lengths (14AA, 15AA, 26AA, and 32AA) fusing MG160-4 to the N-terminus of MG71-2 were tested along with the original 33AA linker. The 32AA and 33AA linkers had similar levels of editing for both the 5-nt change and the 24-nt insertion on the AAVS1 target. Bars labeled "correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edit" represent intended edits that were incorporated but contained errors in the NGS amplicon due to misincorporation by RT and / or pegRNA scaffold incorporation. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 78I]Optimization of the MG71-2n system using selected reverse transcriptases is shown in Figures 78I-78L. Various linker lengths (7AA, 14AA, 15AA, 16AA, 26AA, 32AA, 44AA, and 58AA) fusing RT MG160-473 or MG151-98 (H171N, Δ166AA) to the N-terminus of MG71-2 were tested along with the original 33AA linker and screened for incorporation of a 5-nt change and a 24-nt insertion on the AAVS1 target. Bars labeled "correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edit" represent intended edits that were incorporated but contained errors in the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 78J] Optimization of the MG71-2n system using selected reverse transcriptases is shown in Figures 78I-78L. Various linker lengths (7AA, 14AA, 15AA, 16AA, 26AA, 32AA, 44AA, and 58AA) fusing RT MG160-473 or MG151-98 (H171N, Δ166AA) to the N-terminus of MG71-2 were tested along with the original 33AA linker and screened for incorporation of a 5-nt change and a 24-nt insertion on the AAVS1 target. Bars labeled "correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edit" represent intended edits that were incorporated but contained errors in the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 78K]Optimization of the MG71-2n system using selected reverse transcriptases is shown in Figures 78I-78L. Various linker lengths (7AA, 14AA, 15AA, 16AA, 26AA, 32AA, 44AA, and 58AA) fusing RT MG160-473 or MG151-98 (H171N, Δ166AA) to the N-terminus of MG71-2 were tested along with the original 33AA linker and screened for incorporation of a 5-nt change and a 24-nt insertion on the AAVS1 target. Bars labeled "correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edit" represent intended edits that were incorporated but contained errors in the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 78L] Optimization of the MG71-2n system using selected reverse transcriptases is shown in Figures 78I-78L. Various linker lengths (7AA, 14AA, 15AA, 16AA, 26AA, 32AA, 44AA, and 58AA) fusing RT MG160-473 or MG151-98 (H171N, Δ166AA) to the N-terminus of MG71-2 were tested along with the original 33AA linker and screened for incorporation of a 5-nt change and a 24-nt insertion on the AAVS1 target. Bars labeled "correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edit" represent intended edits that were incorporated but contained errors in the NGS amplicon due to misincorporation by the RT and / or pegRNA scaffold incorporation. The bar labeled "edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. [Figure 79A]Targeting of therapeutic sites using MG3-6-3-8 and MG3-6 is shown. Figure 79A: WT mRNA of MG3-6 / 3-8 with indels at therapeutically relevant sites (A1AT, PAH R408W, G6PC1 Q347*, G6PC1 R83C, and hPDK1) using various guide RNAs. The guide RNAs, represented by dark gray bars, indicate the spacer sequences selected for designing pegRNAs. Bars labeled "correct edits" represent intended edits without errors within the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated but contained errors within the NGS amplicon due to RT misintegration and / or pegRNA scaffold integration. Bars labeled "edits" represent intended edits with errors within the NGS amplicon (excluding pegRNA scaffold integration), while bars labeled "scaffold integration" represent intended edits and pegRNA scaffold integration. * indicates that less than 1000 reads were obtained for that NGS sample. [Figure 79B]Targeting of therapeutic sites using MG3-6-3-8 and MG3-6 is shown. Figures 79B-79K: Prime editing systems MG3-6-3-8n (MG160-4(H230R) tethered to the C-terminus of MG3-6-3-8n-MG160-4(H230R) and MMLV2 tethered to the C-terminus of MG3-6-3-8n (MG3-6-3-8n-MMLV2-) were tested for prime editing at therapeutically relevant sites. No editing was detected at sites PAH R408W, G6PC1:R83C, and hPDK1. A1AT and G6PC1 For Q347*, some detectable levels of editing were observed. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, while the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. The bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while the bar labeled "scaffold incorporation" represents the intended edit and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 79C]Targeting of therapeutic sites using MG3-6-3-8 and MG3-6 is shown. Figures 79B-79K: Prime editing systems MG3-6-3-8n (MG160-4(H230R) tethered to the C-terminus of MG3-6-3-8n-MG160-4(H230R) and MMLV2 tethered to the C-terminus of MG3-6-3-8n (MG3-6-3-8n-MMLV2-) were tested for prime editing at therapeutically relevant sites. No editing was detected at sites PAH R408W, G6PC1:R83C, and hPDK1. A1AT and G6PC1 For Q347*, some detectable levels of editing were observed. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, while the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. The bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while the bar labeled "scaffold incorporation" represents the intended edit and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 79D]Targeting of therapeutic sites using MG3-6-3-8 and MG3-6 is shown. Figures 79B-79K: Prime editing systems MG3-6-3-8n (MG160-4(H230R) tethered to the C-terminus of MG3-6-3-8n-MG160-4(H230R) and MMLV2 tethered to the C-terminus of MG3-6-3-8n (MG3-6-3-8n-MMLV2-) were tested for prime editing at therapeutically relevant sites. No editing was detected at sites PAH R408W, G6PC1:R83C, and hPDK1. A1AT and G6PC1 For Q347*, some detectable levels of editing were observed. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, while the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. The bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while the bar labeled "scaffold incorporation" represents the intended edit and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 79E]Targeting of therapeutic sites using MG3-6-3-8 and MG3-6 is shown. Figures 79B-79K: Prime editing systems MG3-6-3-8n (MG160-4(H230R) tethered to the C-terminus of MG3-6-3-8n-MG160-4(H230R) and MMLV2 tethered to the C-terminus of MG3-6-3-8n (MG3-6-3-8n-MMLV2-) were tested for prime editing at therapeutically relevant sites. No editing was detected at sites PAH R408W, G6PC1:R83C, and hPDK1. A1AT and G6PC1 For Q347*, some detectable levels of editing were observed. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, while the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. The bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while the bar labeled "scaffold incorporation" represents the intended edit and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 79F]Targeting of therapeutic sites using MG3-6-3-8 and MG3-6 is shown. Figures 79B-79K: Prime editing systems MG3-6-3-8n (MG160-4(H230R) tethered to the C-terminus of MG3-6-3-8n-MG160-4(H230R) and MMLV2 tethered to the C-terminus of MG3-6-3-8n (MG3-6-3-8n-MMLV2-) were tested for prime editing at therapeutically relevant sites. No editing was detected at sites PAH R408W, G6PC1:R83C, and hPDK1. A1AT and G6PC1 For Q347*, some detectable levels of editing were observed. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, while the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. The bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while the bar labeled "scaffold incorporation" represents the intended edit and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 79G]Targeting of therapeutic sites using MG3-6-3-8 and MG3-6 is shown. Figures 79B-79K: Prime editing systems MG3-6-3-8n (MG160-4(H230R) tethered to the C-terminus of MG3-6-3-8n-MG160-4(H230R) and MMLV2 tethered to the C-terminus of MG3-6-3-8n (MG3-6-3-8n-MMLV2-) were tested for prime editing at therapeutically relevant sites. No editing was detected at sites PAH R408W, G6PC1:R83C, and hPDK1. A1AT and G6PC1 For Q347*, some detectable levels of editing were observed. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, while the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. The bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while the bar labeled "scaffold incorporation" represents the intended edit and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 79H]Targeting of therapeutic sites using MG3-6-3-8 and MG3-6 is shown. Figures 79B-79K: Prime editing systems MG3-6-3-8n (MG160-4(H230R) tethered to the C-terminus of MG3-6-3-8n-MG160-4(H230R) and MMLV2 tethered to the C-terminus of MG3-6-3-8n (MG3-6-3-8n-MMLV2-) were tested for prime editing at therapeutically relevant sites. No editing was detected at sites PAH R408W, G6PC1:R83C, and hPDK1. A1AT and G6PC1 For Q347*, some detectable levels of editing were observed. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, while the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. The bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while the bar labeled "scaffold incorporation" represents the intended edit and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 79I]Targeting of therapeutic sites using MG3-6-3-8 and MG3-6 is shown. Figures 79B-79K: Prime editing systems MG3-6-3-8n (MG160-4(H230R) tethered to the C-terminus of MG3-6-3-8n-MG160-4(H230R) and MMLV2 tethered to the C-terminus of MG3-6-3-8n (MG3-6-3-8n-MMLV2-) were tested for prime editing at therapeutically relevant sites. No editing was detected at sites PAH R408W, G6PC1:R83C, and hPDK1. A1AT and G6PC1 For Q347*, some detectable levels of editing were observed. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, while the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. The bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while the bar labeled "scaffold incorporation" represents the intended edit and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 79J]Targeting of therapeutic sites using MG3-6-3-8 and MG3-6 is shown. Figures 79B-79K: Prime editing systems MG3-6-3-8n (MG160-4(H230R) tethered to the C-terminus of MG3-6-3-8n-MG160-4(H230R) and MMLV2 tethered to the C-terminus of MG3-6-3-8n (MG3-6-3-8n-MMLV2-) were tested for prime editing at therapeutically relevant sites. No editing was detected at sites PAH R408W, G6PC1:R83C, and hPDK1. A1AT and G6PC1 For Q347*, some detectable levels of editing were observed. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, while the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. The bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while the bar labeled "scaffold incorporation" represents the intended edit and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 79K]Targeting of therapeutic sites using MG3-6-3-8 and MG3-6 is shown. Figures 79B-79K: Prime editing systems MG3-6-3-8n (MG160-4(H230R) tethered to the C-terminus of MG3-6-3-8n-MG160-4(H230R) and MMLV2 tethered to the C-terminus of MG3-6-3-8n (MG3-6-3-8n-MMLV2-) were tested for prime editing at therapeutically relevant sites. No editing was detected at sites PAH R408W, G6PC1:R83C, and hPDK1. A1AT and G6PC1 For Q347*, some detectable levels of editing were observed. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, while the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. The bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while the bar labeled "scaffold incorporation" represents the intended edit and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 79L]Targeting of therapeutic sites with MG3-6-3-8 and MG3-6 is shown. Figures 79L-79O: MG160-4(H230R) tethered to the N-terminus of MG3-6n or MG3-6-3-8n was compared to editing by MMLV2 tethered to the C-terminus of MG3-6n or MG3-6-3-8n. These constructs targeted the four therapeutic sites, A1A and hPDK1. Bars labeled "correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edit" represent intended edits that were incorporated but contained errors in the NGS amplicon due to misintegration by RT and / or pegRNA scaffold integration. The bar labeled "Edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 79M] Targeting of therapeutic sites with MG3-6-3-8 and MG3-6 is shown. Figures 79L-79O: MG160-4(H230R) tethered to the N-terminus of MG3-6n or MG3-6-3-8n was compared to editing by MMLV2 tethered to the C-terminus of MG3-6n or MG3-6-3-8n. These constructs targeted the four therapeutic sites, A1A and hPDK1. Bars labeled "correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edit" represent intended edits that were incorporated but contained errors in the NGS amplicon due to misintegration by RT and / or pegRNA scaffold integration. The bar labeled "Edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 79N]Targeting of therapeutic sites with MG3-6-3-8 and MG3-6 is shown. Figures 79L-79O: MG160-4(H230R) tethered to the N-terminus of MG3-6n or MG3-6-3-8n was compared to editing by MMLV2 tethered to the C-terminus of MG3-6n or MG3-6-3-8n. These constructs targeted the four therapeutic sites, A1A and hPDK1. Bars labeled "correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edit" represent intended edits that were incorporated but contained errors in the NGS amplicon due to misintegration by RT and / or pegRNA scaffold integration. The bar labeled "Edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 79O] Targeting of therapeutic sites with MG3-6-3-8 and MG3-6 is shown. Figures 79L-79O: MG160-4(H230R) tethered to the N-terminus of MG3-6n or MG3-6-3-8n was compared to editing by MMLV2 tethered to the C-terminus of MG3-6n or MG3-6-3-8n. These constructs targeted the four therapeutic sites, A1A and hPDK1. Bars labeled "correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edit" represent intended edits that were incorporated but contained errors in the NGS amplicon due to misintegration by RT and / or pegRNA scaffold integration. The bar labeled "Edit" represents intended edits including errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "Scaffold incorporation" represents intended edits and scaffold incorporation of pegRNA. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 80A]Optimization of the MG3-6n system using MG160-4 and MG160-4(H230R) is shown. Figures 80A-80B: MG160-4 was cloned into the N-terminus of MG3-6n with various linker lengths, including 33 AA (the original linker length) and 32 AA, 44 AA, and 58 AA. These prime editing systems were then tested to correct the two stop codons in the linker between hygromycin and the BFP-engineered cell line. PEG RNAs with PBS lengths of 8, 10, and 13 nucleotides were tested. Using PEG RNAs with a PBS length of 8 nt, the highest level of editing was observed using a fusion construct with 58 AA. As the PBS length increased, the differences between linker systems showed less variability in editing levels when using prime editing systems with different linker lengths. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to mis-incorporation by RT and / or pegRNA scaffold incorporation, the bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents the intended edit and scaffold incorporation of pegRNA. [Figure 80B]Optimization of the MG3-6n system using MG160-4 and MG160-4(H230R) is shown. Figures 80A-80B: MG160-4 was cloned into the N-terminus of MG3-6n with various linker lengths, including 33 AA (the original linker length) and 32 AA, 44 AA, and 58 AA. These prime editing systems were then tested to correct the two stop codons in the linker between hygromycin and the BFP-engineered cell line. PEG RNAs with PBS lengths of 8, 10, and 13 nucleotides were tested. Using PEG RNAs with a PBS length of 8 nt, the highest level of editing was observed using a fusion construct with 58 AA. As the PBS length increased, the differences between linker systems showed less variability in editing levels when using prime editing systems with different linker lengths. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to mis-incorporation by RT and / or pegRNA scaffold incorporation, the bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents the intended edit and scaffold incorporation of pegRNA. [Figure 80C]Optimization of the MG3-6n system using MG160-4 and MG160-4(H230R) is shown. Figures 80C-80D: MG160-4(H230R) and MG3-6n were further introduced into five different insertion sites (K115, V208, K368, D550, and L881). The introduced constructs had 33 amino acid linkers at the 5' and 3' ends of MG160-4(H230R) at the insertion site. The introduced constructs were tested for correction of two stop codons in the linker between hygromycin and BFP-engineered cell lines across three different PBS lengths. The highest level of editing was observed when MG160-4(H230R) was tethered to the N-terminus of MG3-6n. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to mis-incorporation by RT and / or pegRNA scaffold incorporation, the bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents the intended edit and scaffold incorporation of pegRNA. [Figure 80D]Optimization of the MG3-6n system using MG160-4 and MG160-4(H230R) is shown. Figures 80C-80D: MG160-4(H230R) and MG3-6n were further introduced into five different insertion sites (K115, V208, K368, D550, and L881). The introduced constructs had 33 amino acid linkers at the 5' and 3' ends of MG160-4(H230R) at the insertion site. The introduced constructs were tested for correction of two stop codons in the linker between hygromycin and BFP-engineered cell lines across three different PBS lengths. The highest level of editing was observed when MG160-4(H230R) was tethered to the N-terminus of MG3-6n. The bar labeled "correct edit" represents the intended edit without any errors in the NGS amplicon, the bar labeled "incorrect edit" represents the intended edit with errors in the NGS amplicon due to mis-incorporation by RT and / or pegRNA scaffold incorporation, the bar labeled "edit" represents the intended edit with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), and the bar labeled "scaffold incorporation" represents the intended edit and scaffold incorporation of pegRNA. [Figure 81A]Screening of native reverse transcriptase tethered to the N-terminus of MG71-2n targeting AAVS1 is shown. Figure 81A: Overview of candidate MG198 tethered to the N-terminus of MG71-2n targeting a 5-nt change in AAVS1 using pegRNA with various PBS lengths (8, 10, 13, and 16 nt). Editing levels above background were observed for candidates MG198-6 and MG198-7. Bars labeled "correct edit" represent intended edits with no errors in the NGS amplicon, while bars labeled "incorrect edit" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT misincorporation and / or pegRNA scaffold incorporation. Bars labeled "edit" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and scaffold incorporation of pegRNA. * indicates that less than 1000 reads were obtained for that NGS sample. [Figure 81B]Screening of native reverse transcriptase tethered to the N-terminus of MG71-2n, which targets AAVS1, is shown in Figures 81B-81C. MG160 candidates MG160-45, MG160-121, MG160-136, and MG160-232 were tethered to the N-terminus of MG71-2n, which targets a 5-nt change in AAVS1, using pegRNA with various PBS lengths (8, 10, and 13 nt). All MG160 candidates showed insufficient activity, although slightly above background levels, compared to MMLV2 tethered to the N-terminus of MG160-4 (H230R) and MG71-2n. Bars labeled "correct edits" represent intended edits without errors in the NGS amplicon, while bars labeled "incorrect edits" represent intended edits that were incorporated and contained errors in the NGS amplicon due to RT mis-incorporation and / or pegRNA scaffold incorporation. Bars labeled "edits" represent intended edits with errors in the NGS amplicon (excluding pegRNA scaffold incorporation), while bars labeled "scaffold incorporation" represent intended edits and pegRNA scaffold incorporation. * indicates that fewer than 1000 reads were acquired for that NGS sample. [Figure 81C]Screening ...

Claims

1. A fusion protein comprising a nickase linked to a reverse transcriptase using a linker, wherein the reverse transcriptase comprises at least about 80% sequence identity with any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.

2. A fusion protein comprising a nuclease linked to a reverse transcriptase using a linker, wherein the reverse transcriptase comprises at least about 80% sequence identity with any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.

3. A fusion protein comprising a catalytically deficient nuclease linked to a reverse transcriptase using a linker, wherein the reverse transcriptase comprises at least about 80% sequence identity with any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.

4. 1. A gene editing system comprising: a) nickase; b) a guide nucleic acid configured to form a complex with the nickase and hybridize with a target nucleic acid sequence; c) a reverse transcriptase having at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585, and configured to form a complex with the nickase.

5. The gene editing system of claim 4, wherein the nickase is a modifying endonuclease.

6. The gene editing system of claim 5, wherein the modified endonuclease is a type II CRISPR endonuclease.

7. The gene editing system of claim 5, wherein the modified endonuclease is a V-type CRISPR endonuclease.

8. The gene editing system of claim 6 or 7, wherein the type II CRISPR endonuclease or the type V CRISPR endonuclease has nickase activity.

9. 6. The gene editing system of claim 5, wherein the modified endonuclease is selected from the group consisting of spCas9(H840A), spCas9(D10A), nMG3-6(D13A), nMG3-6(H586A), nMG3-6(N609A), Cas12a, and MG29-1.

10. 6. The gene editing system of claim 5, wherein the modified endonuclease comprises at least about 80% sequence identity to any one of SEQ ID NOs: 152-154.

11. The gene editing system of any one of claims 4 to 10, wherein the nickase and the reverse transcriptase are fused together.

12. The gene editing system of any one of claims 4 to 10, wherein the nickase and the reverse transcriptase are linked by a linker.

13. The gene editing system of claim 12, wherein the linker comprises at least 10, 20, or 30 amino acids.

14. The gene editing system of claim 12, wherein the linker comprises about 30 to 35 amino acids.

15. The gene editing system of claim 12, wherein the linker comprises about 30 amino acids.

16. 13. The gene editing system of claim 12, wherein the linker comprises at least 80% sequence identity to SEQ ID NO:

103.

17. The gene editing system of claim 12, wherein the linker comprises at least 80% sequence identity to any one of SEQ ID NOs: 155-160.

18. The gene editing system of any one of claims 4 to 10, wherein the nickase and the reverse transcriptase are unlinked.

19. The gene editing system of any one of claims 4 to 18, wherein the guide nucleic acid comprises a spacer sequence and crRNA.

20. The gene editing system of any one of claims 4 to 19, wherein the guide nucleic acid further comprises a reverse transcriptase template (RTT).

21. 21. The gene editing system of claim 20, wherein the bases in the RTT comprise bulk modifications selected from the group of complex sugars, or complex amino groups, and / or other modifications that are compatible with RNA.

22. The gene editing system of any one of claims 4 to 21, wherein the guide nucleic acid further comprises a primer binding site.

23. 23. The gene editing system of Claim 22, wherein the primer binding site is at the 3' end of the guide nucleic acid.

24. 24. The gene editing system of Claim 22 or 23, wherein the primer binding site comprises at least 2, 4, 6, 8, 10, 13, 16, 20, 24, 28, 32, 36, 40, 45, 50, 55, 60, or 65 nucleotides.

25. The gene editing system of any one of claims 4 to 24, wherein the nuclease is non-covalently linked to the guide nucleic acid.

26. The gene editing system of any one of claims 4 to 24, wherein the nuclease is covalently linked to the guide nucleic acid.

27. The gene editing system of any one of claims 4 to 24, wherein the nuclease is fused to the guide nucleic acid.

28. The gene editing system of any one of claims 4 to 24, further comprising a transposase, an integrase, or a homing endonuclease.

29. The gene editing system of any one of claims 4 to 28, further comprising a retrotransposon.

30. 30. The gene editing system of any one of claims 4 to 29, wherein the reverse transcriptase comprises at least about two-fold higher processivity than Moloney murine leukemia virus (MMLV) reverse transcriptase.

31. 30. The gene editing system of any one of claims 4 to 29, wherein the reverse transcriptase comprises a processivity that is at least about 2-fold lower than Moloney murine leukemia virus (MMLV) reverse transcriptase.

32. 32. The gene editing system of any one of claims 4-31, wherein the reverse transcriptase comprises an error rate of less than about 2.5%, 2.0%, 1.5%, 1%, 0.5%, 0.25%, 0.10%, or 0.05%.

33. 33. The gene editing system of any one of claims 4-32, wherein the reverse transcriptase comprises an error rate of less than about 2.5%, 2.0%, 1.5%, 1%, 0.5%, 0.25%, 0.10%, or 0.05% compared to Moloney murine leukemia virus (MMLV) reverse transcriptase.

34. 1. A gene editing system comprising: a) a nuclease; b) a guide nucleic acid configured to form a complex with the nuclease and hybridize with a target nucleic acid sequence; c) a reverse transcriptase having at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585, and configured to form a complex with the nuclease.

35. The gene editing system of Claim 34, wherein the nuclease is a double-stranded nuclease.

36. 36. The gene editing system of claim 34 or 35, wherein the nuclease is a type II CRISPR endonuclease.

37. 37. The gene editing system of claim 36, wherein the CRISPR endonuclease is Cas9.

38. 38. The gene editing system of claim 37, wherein the Cas9 is catalytically deficient Cas9 (dCas9).

39. The gene editing system of any one of claims 34 to 38, wherein the nuclease and the reverse transcriptase are fused together.

40. The gene editing system of any one of claims 34 to 38, wherein the nuclease and the reverse transcriptase are linked by a linker.

41. 41. The gene editing system of Claim 40, wherein the linker comprises at least 10, 20, or 30 amino acids.

42. The gene editing system of claim 40, wherein the linker comprises about 30 to 35 amino acids.

43. 41. The gene editing system of Claim 40, wherein the linker comprises about 30 amino acids.

44. 41. The gene editing system of Claim 40, wherein the linker comprises at least 80% sequence identity to SEQ ID NO:

103.

45. The gene editing system of claim 40, wherein the linker comprises at least 80% sequence identity to any one of SEQ ID NOs: 155-160.

46. The gene editing system of any one of claims 34 to 38, wherein the nuclease and the reverse transcriptase are unlinked.

47. The gene editing system of any one of claims 34 to 46, wherein the guide nucleic acid further comprises a primer binding site.

48. 48. The gene editing system of Claim 47, wherein the primer binding site is at the 3' end of the guide nucleic acid.

49. 49. The gene editing system of Claim 47 or 48, wherein the primer binding site comprises at least 2, 4, 6, 8, 10, 13, 16, 20, 24, 28, 32, 36, 40, 45, 50, 55, 60, or 65 nucleotides.

50. 50. The gene editing system of any one of claims 34 to 49, wherein the nuclease is non-covalently linked to the guide nucleic acid.

51. 50. The gene editing system of any one of claims 34 to 49, wherein the nuclease is covalently linked to the guide nucleic acid.

52. 50. The gene editing system of any one of claims 34 to 49, wherein the nuclease is fused to the guide nucleic acid.

53. 53. The gene editing system of any one of claims 34 to 52, further comprising a transposase, integrase, or homing endonuclease.

54. A gene editing system described in any one of claims 34 to 53, further comprising a retrotransposon.

55. 55. The gene editing system of any one of claims 34 to 54, wherein the reverse transcriptase comprises at least about two-fold higher processivity than Moloney murine leukemia virus (MMLV) reverse transcriptase.

56. 55. The gene editing system of any one of claims 34 to 54, wherein the reverse transcriptase comprises a processivity that is at least about 2-fold lower than Moloney murine leukemia virus (MMLV) reverse transcriptase.

57. 57. The gene editing system of any one of Claims 34-56, wherein the reverse transcriptase comprises an error rate of less than about 2.5%, 2.0%, 1.5%, 1%, 0.5%, 0.25%, 0.10%, or 0.05%.

58. 57. The gene editing system of any one of claims 34-56, wherein the reverse transcriptase comprises an error rate of less than about 2.5%, 2.0%, 1.5%, 1%, 0.5%, 0.25%, 0.10%, or 0.05% compared to Moloney murine leukemia virus (MMLV) reverse transcriptase.

59. 1. A gene editing system comprising: a) nickase; b) a guide nucleic acid configured to form a complex with the nickase and hybridize with a target nucleic acid sequence; c) a reverse transcriptase configured to form a complex with said nickase, said reverse transcriptase comprising: X １ X ２ DD motif, X １ is F or Y, and X １ If Y, then X ２ is A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, V, W, or Y.

60. The X ２ is A or I.

61. The X １ X ２ The gene editing system of claim 59, wherein the DD motif is YADD (sequence number 2572) or YIDD (sequence number 2573).

62. The X １ X ２ 60. The gene editing system of claim 59, wherein the DD motif is FADD (SEQ ID NO: 2574), FVDD (SEQ ID NO: 2575), FIDD (SEQ ID NO: 2576), or FLDD (SEQ ID NO: 2577).

63. 63. The gene editing system of any one of claims 59-62, wherein the reverse transcriptase has at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.

64. 1. A gene editing system comprising: a) a nuclease; b) a guide nucleic acid configured to form a complex with the nuclease and hybridize with a target nucleic acid sequence; c) a reverse transcriptase configured to form a complex with said nuclease, said reverse transcriptase comprising X １ X ２ DD motif, X １ is F or Y, and X １ If Y, then X ２ is A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, V, W, or Y.

65. The X ２ is A or I. The gene editing system of claim 64.

66. The X １ X ２ The gene editing system of claim 64, wherein the DD motif is YADD (sequence number 2572) or YIDD (sequence number 2573).

67. The X １ X ２ 65. The gene editing system of claim 64, wherein the DD motif is FADD (SEQ ID NO: 2574), FVDD (SEQ ID NO: 2575), FIDD (SEQ ID NO: 2576), or FLDD (SEQ ID NO: 2577).

68. 68. The gene editing system of any one of claims 64-67, wherein the reverse transcriptase has at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.

69. An isolated reverse transcriptase having at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.

70. A nucleic acid encoding the fusion protein of any one of claims 1 to 3 or the gene editing system of any one of claims 4 to 68.

71. 71. The nucleic acid of claim 70, wherein the nucleic acid is DNA or RNA.

72. 72. The nucleic acid of claim 71, wherein the RNA is mRNA.

73. A vector comprising the nucleic acid of any one of claims 70 to 72.

74. An adeno-associated virus or lipid nanoparticle comprising a nucleic acid according to any one of claims 70 to 72 or a vector according to claim 73.

75. A cell comprising the nucleic acid of any one of claims 70 to 72 or the vector of claim 73.

76. 76. The cell of claim 75, wherein the cell is a human cell.

77. 76. The cell of claim 75, wherein the cell is a eukaryotic cell.

78. 76. The cell of claim 75, wherein the cell is a mammalian cell.

79. 76. The cell of claim 75, wherein the cell is an immortalized cell.

80. 76. The cell of claim 75, wherein the cell is an insect cell.

81. 76. The cell of claim 75, wherein the cell is a yeast cell.

82. 76. The cell of claim 75, wherein the cell is a plant cell.

83. 76. The cell of claim 75, wherein the cell is a fungal cell.

84. 76. The cell of claim 75, wherein the cell is a prokaryotic cell.

85. 76. The cell of claim 75, wherein the cell is A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC1, BSC40, BMT10, WI38, HeLa, Saos, C2C12, L cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof.

86. 76. The cell of claim 75, wherein the cell is an engineered cell.

87. 76. The cell of claim 75, wherein the cell is a stable cell.

88. 69. A method for modifying double-stranded and / or single-stranded nucleic acid, the method comprising contacting a cell with a fusion protein of any one of claims 1 to 3 or a gene editing system of any one of claims 4 to 68.

89. 1. A method for modifying double-stranded and / or single-stranded nucleic acids, comprising: a) providing a cell with a guide nucleic acid that binds to a target strand of said nucleic acid; b) providing the cell with a nuclease or nickase to cleave the nucleic acid at the binding site of the guide nucleic acid; c) providing said cell with a reverse transcriptase to synthesize a modification on said target strand of said nucleic acid at the site of cleavage by said nickase and / or nuclease.

90. 90. The method of claim 89, wherein the reverse transcriptase has at least about 80% sequence identity to any one of SEQ ID NOs: 161-629, 767-1220, 1959-2522, and 2582-2585.

91. 90. The method of claim 89, wherein the modification is an insertion, deletion, or mutation.

92. 90. The method of claim 89, further comprising providing an RNA or DNA template.

93. 90. The method of claim 89, wherein the nucleic acid is a genome or a vector.

94. 90. The method of claim 89, further comprising providing said cell with a transposase, integrase, or homing endonuclease.

95. 90. The method of claim 89, further comprising providing said cell with a retrotransposon.

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