Cas9 and reverse transcriptase mutants with improved activity in prime editing applications

Abstract

This invention pertains to fusion protein mutants comprising a Prime Editing enzyme having a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence comprises a SpCas9 H840A nickase mutant protein of SEQ ID NO:152 and the second amino acid sequence comprises a Moloney Murine Leukemia Virus reverse transcriptase protein mutant (MMLV RTase mutant), wherein the fusion protein mutant displays at least the equivalent or greater activity of a reference Prime Editing enzyme in genome editing.

Description

CAS9 AND REVERSE TRANSCRIPTASE MUTANTS WITH IMPROVED ACTIVITY IN PRIME EDITING APPLICATIONS

[0001] This application claim benefit of U.S. Serial No.63 / 668,607 filed July 8, 2024, the entirety of which is incorporated by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing that has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. The XML copy, created on July 8, 2025, is named 6391-0023WO01_Sequence.xml, and is 142 kbytes in size. FIELD OF THE INVENTION

[0003] This invention pertains to the ability of a nickase CRISPR / Cas9 mutant to cleave double-stranded DNA on one strand in a targeted manner in living cells when complexed with sgRNAs. BACKGROUND OF THE INVENTION

[0004] SpCas9 is an RNA guided endonuclease from the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)- Cas (CRISPR-associated) bacterial adaptive immune system of Streptococcus pyogenes[1]. Cas9 is guided to a 23-nt DNA target sequence by a target site-specific 20-nt complementary RNA (part of the 44-nt crRNA) and a universal 89- nt tracrRNA, collectively referred to as the guide RNA (gRNA) complex. The Cas9-gRNA ribonucleotprotein (RNP) complex mediates double-stranded DNA breaks (DSBs) which are then typically repaired by the non-homologous end joining (NHEJ), microhomology mediatedend joining, or homology-directed repair (HDR) system if a suitable template nucleic acid is present.

[0005] S. pyogenes Cas9 protein contains two endonuclease domains that function together to generate a double-strand DNA break by cleaving both the target (guide complementary) and non-target (guide noncomplementary) strands of a double-stranded DNA (dsDNA). These conserved domains are the RuvC and HNH domains. There are two known mutations that can alter Cas9, which produces a double-stranded cut, into a ‘Nickase’ that results in single-stranded cuts. Cas9 D10A variant generates the nick on the targeted strand, while the Cas9 H840A variant generates the nick on the non-targeted strand[1]. The nickase Cas9 variants have been used to facilitate CRISPR-targeted genome editing approaches that do not rely on the introduction of a dsDNA break, examples of which include cytosine / adenine base editors[2, 3] and more recently the Cas9 prime editor[4].

[0006] Prime Editing is a new technology that utilizes a Cas9 nickase fused to an engineered reverse transcriptase. The fusion is coupled with a prime editing guide RNA (pegRNA) that recognizes the target site and contains the desired edit. The prime editor was developed by David Liu and it contains the Cas9 nickase, H840A, and a highly mutagenized reverse transcriptase from Moloney Murine Leukemia Virus (MMLV RTase). The engineered RTase is derived from multiple patents that would require expensive licenses for use and sale of this technology[4].

[0007] There is a long-felt need to improve prime editor capabilities through discovery of novel mutations within the Cas9 nickase and MMLV RTase.BRIEF SUMMARY OF THE INVENTION

[0008] This invention pertains to the ability to create a genomic / DNA change utilizing a a SpCas9 H840A nickase mutant and a Reverse Transcriptase (RT) in a method known as “Prime Editing.”

[0009] In a first aspect, a fusion protein mutant including a Prime Editing enzyme is provided. The Prime Editing enzyme includes a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence comprises a SpCas9 H840A nickase mutant protein of SEQ ID NO:152 and the second amino acid sequence comprises a Moloney Murine Leukemia Virus reverse transcriptase protein mutant (MMLV RTase mutant). The fusion protein mutant displays at least the equivalent or greater activity of a reference Prime Editing enzyme in genome editing.

[0010] In a second aspect, a nucleic acid sequence encoding the fusion protein of claims of the first aspect is provided.

[0011] In a third aspect, an isolated ribonucleoprotein complex is provided. The isolated ribonucleoprotein complex includes the fusion protein mutant of the first aspect and a gRNA. In a first respect, the gRNA includes a pegRNA.

[0012] In a fourth aspect, a CRISPR / Cas endonuclease system including the fusion protein mutant of the first aspect is provided. In a first respect, the CRISPR / Cas endonuclease system is encoded by a DNA expression vector.

[0013] In a fifth aspect, a method of performing gene editing in a eukaryotic cell is provided. The method includes a step of contacting a candidate editing target site locus with an active CRISPR / Cas endonuclease system having the fusion protein mutant of the first aspect

[0014] In a sixth aspect, a kit for performing gene editing in a eukaryotic cell is provided. The kit includes the fusion protein mutant of the first aspect and optionally a gRNA. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention pertains to using methods to select bacterial prime editing variants having novel mutations within the Cas9 nickase and MMLV RTase possessing more potent prime editors. To our knowledge, no comprehensive screen with selection has been performed for MMLV RTase, nor the prime editor in its entirety, and the current amino acid substitutions found in the published prime editor were isolated through rational mutagenesis or random mutagenesis by error prone PCR. Focusing on the complete prime editor construct, we made 25 mutant libraries of every possible amino acid substitution spanning the entire open reading frame of the prime editor (Cas9 and MMLV RTase genes and the amino acid linker between the two proteins). We screened the libraries for substitutions that would enable prime editing in bacteria with the highest overall potency.

[0016] The term “mutant MMLV-II RTase protein” (or “Mutant MMLV-II protein”) refers to a MMLV RTase protein having three amino acid substitutions within the MMLV RTase amino acid sequence relative to the WT MMLV RTase (D524G, E562Q, and D583N; see SEQ ID NO: 154).

[0017] The term “mutant PE2 RTase protein” (or “Mutant PE2 M-MLV RT protein”) refers to a MMLV RTase protein having five amino acid substitutions within the MMLV RTase amino acid sequence (D200N; L603W; T330P; T306K; and W313F; see SEQ ID NO: 156).

[0018] The term “mutant PE2 fusion protein” refers to a Cas9 (H840A)-MMLV RTase fusion protein having five amino acid substitutions within the MMLV RTase amino acid sequence (D200N; L603W; T330P; T306K; and W313F; see SEQ ID NO:155).

[0019] The terms, “Mutant ID,” and “ID,” as used in the disclosure refer to the change in amino acid and codon at a given position relative to a reference protein (e.g., wild-type Cas9 protein). For example, a Mutant ID or ID characterized as “D2P_GAC_CCG” refers to a mutant amino acid at position 2, where Aspartic acid in the reference protein is changed to Proline in the mutant protein, and where the corresponding codon GAC in the open reading frame of the reference protein is changed to the codon CCG in the open reading frame of the mutant protein.

[0020] The term “Cas9 (H840A) protein” encompasses a protein having the identical amino acid sequence of the naturally-occurring Streptococcus pyogenes Cas9 bearing the single amino acid substation at position 840 where an Alanine is substituted for Histidine (e.g., SEQ ID NO:152) and that has biochemical and biological activity when combined with a suitable guide RNA (for example sgRNA, dual crRNA:tracrRNA, or pegRNA compositions) to form an active CRISPR-Cas endonuclease system.

[0021] The term “CRISPR / Cas endonuclease system” refers to a CRISPR / Cas endonuclease system that includes a functional Cas9 protein or mutant thereof and a suitable gRNA.

[0022] The term “isolated nucleic acid” include DNA, RNA, cDNA, and vectors encoding the same, where the DNA, RNA, cDNA and vectors are free of other biological materials from which they may be derived or associated, such as cellular components. Typically, an isolated nucleic acid will be purified from other biological materials from which they may be derived or associated, such as cellular components.

[0023] A competent CRISPR-Cas endonuclease system includes a ribonucleoprotein (RNP) complex formed with a mutant Cas9 protein or a mutant Cas9-MMLV RTase fusion protein and an isolated guide RNA selected from one of a pegRNA, a dual crRNA:tracrRNA combination or a chimeric single-molecule sgRNA. Applications

[0024] In a first aspect, a fusion protein mutant including a Prime Editing enzyme is provided. The Prime Editing enzyme includes a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence comprises a SpCas9 H840A nickase mutant protein of SEQ ID NO:152 and the second amino acid sequence comprises a Moloney Murine Leukemia Virus reverse transcriptase protein mutant (MMLV RTase mutant). The fusion protein mutant displays at least the equivalent or greater activity of a reference Prime Editing enzyme in genome editing. In a first respect, the fusion protein mutant includes a member of the group selected from one of Tables 2, 6, 7, 8, 9, 10, and 11.

[0025] In a second aspect, a nucleic acid sequence encoding the fusion protein of claims of the first aspect is provided.

[0026] In a third aspect, an isolated ribonucleoprotein complex is provided. The isolated ribonucleoprotein complex includes the fusion protein mutant of the first aspect and a gRNA. In a first respect, the gRNA includes a pegRNA.

[0027] In a fourth aspect, a CRISPR / Cas endonuclease system including the fusion protein mutant of the first aspect is provided. In a first respect, the CRISPR / Cas endonuclease system is encoded by a DNA expression vector. In an additional respect, the DNA expression vector is a plasmid-borne vector. In an additional respect, the DNA expression vector is selected from a bacterial expression vector and a eukaryotic expression vector.

[0028] In a fifth aspect, a method of performing gene editing in a eukaryotic cell is provided. The method includes a step of contacting a candidate editing target site locus with an active CRISPR / Cas endonuclease system having the fusion protein mutant of the first aspect

[0029] In a sixth aspect, a kit for performing gene editing in a eukaryotic cell is provided. The kit includes the fusion protein mutant of the first aspect and optionally a gRNA.

[0030] The applications of Cas9-based tools are many and varied. They include, but are not limited to: plant gene editing, yeast gene editing, mammalian gene editing, editing of cells in the organs of live animals, editing of embryos, rapid generation of knockout / knock-in animal lines, generating an animal model of disease state, correcting a disease state, inserting a reporter gene, and whole genome functional screening.EXAMPLE 1

[0031] A bacterial prime editing selection and enrichment strategy reveals Cas9 and RTase mutations that facilitate the most potent prime editor.

[0032] Functional prime editing has recently been described in E. coli using a variety of insertions / deletions and substitutions [5]. We used expression of a prime editor and PEG- RNA that targets a kanamycin resistance gene that contains a 10-base disruption between codons 1 and 2. This prime editing strategy is designed to remove the disrupting 10-base element and restore kanamycin resistance gene expression, and thus confer resistance to the antibiotic kanamycin. The screen is setup such that a library of Cas9 Prime Editor mutants and PEG RNA are expressed from one plasmid, and the target site and non-functional kanamycin resistance gene is present on a second plasmid. Practically, bacterial cells that stably replicate the target site-containing plasmid are made competent, transformed using Cas9 prime editor and PEG RNA plasmid, and selected on Kanamycin-containing solid media.

[0033] Twenty-five saturation mutagenesis libraries (Table 1) spanning the entirety of the prime editor (Cas9 and MMLV RTase genes and the amino acid linker between the two proteins) were generated with nicking mutagenesis and the resulting library complexity was analyzed with tiled-amplicon NGS. These libraries were delivered into E. coli cells as described above to be greater than 99% confident that each possible codon change was observed at least once. The resulting colonies were pooled, plasmids from this pool were purified, and the resulting pool was sequenced with overlapping tiled amplicon NGS. Pre- and post-enrichment pools were sequenced and compared simultaneously with total read count normalization to determine enrichment for each substitution within the pool. Paired endreads were merged by fastp [6], trimmed to keep the desired mutated region by Cutadapt [7], and filtered out undesired reads with wrong length or multiple codon mutation. Codon frequency and enrichment analysis were completed by in-house python script. The results included over 32,000 amino acid substitutions that are at equal or greater frequency than the identical substitution observed in the pre-enriched pool (Table 2). Among these results are substitutions that have issued patents; however, in most cases, these patented positions are far from the most frequently observed mutations indicating the most ideal prime editing mutations are within this pool.

[0034] Table 1. Exemplary primers used for saturation mutagenesis of the entire prime editor protein.

[0035] SEQ ID Sequence (5'-3') SEQ ID NO: 1 CTAAAGAGGAGAAAGGATCTNNKGACAAAAAGTACTCTATTGGC SEQ ID NO: 2 AGAGGAGAAAGGATCTATGNNKAAAAAGTACTCTATTGGCC SEQ ID NO: 3 AGAAAGGATCTATGGACNNKAAGTACTCTATTGGCCTG SEQ ID NO: 4 AAGGATCTATGGACAAANNKTACTCTATTGGCCTGGA SEQ ID NO: 5 GGATCTATGGACAAAAAGNNKTCTATTGGCCTGGATATC SEQ ID NO: 6 CTATGGACAAAAAGTACNNKATTGGCCTGGATATCGG SEQ ID NO: 7 GACAAAAAGTACTCTNNKGGCCTGGATATCGGG SEQ ID NO: 8 GACAAAAAGTACTCTATTNNKCTGGATATCGGGACCAAC SEQ ID NO: 9 AAAGTACTCTATTGGCNNKGATATCGGGACCAACA SEQ ID NO: 10 CTCTATTGGCCTGNNKATCGGGACCAACAG SEQ ID NO: 11 TTGGCCTGGATNNKGGGACCAACAG SEQ ID NO: 12 GGCCTGGATATCNNKACCAACAGCGTC SEQ ID NO: 13 CTGGATATCGGGNNKAACAGCGTCGGG SEQ ID NO: 14 ATATCGGGACCNNKAGCGTCGGGTG SEQ ID NO: 15 TCGGGACCAACNNKGTCGGGTGGGC SEQ ID NO: 16 GGACCAACAGCNNKGGGTGGGCTGT SEQ ID NO: 17 GACCAACAGCGTCNNKTGGGCTGTTATCA SEQ ID NO: 18 ACAGCGTCGGGNNKGCTGTTATCAC SEQ ID NO: 19 GCGTCGGGTGGNNKGTTATCACCGA SEQ ID NO: 20 TCGGGTGGGCTNNKATCACCGACGASEQ ID Sequence (5'-3') SEQ ID NO: 21 GGGTGGGCTGTTNNKACCGACGAGTATA SEQ ID NO: 22 GGGTGGGCTGTTATCNNKGACGAGTATAAAGTAC SEQ ID NO: 23 GTGGGCTGTTATCACCNNKGAGTATAAAGTACCTTC SEQ ID NO: 24 GGCTGTTATCACCGACNNKTATAAAGTACCTTCGAA SEQ ID NO: 25 CTGTTATCACCGACGAGNNKAAAGTACCTTCGAAAAA SEQ ID NO: 26 TTATCACCGACGAGTATNNKGTACCTTCGAAAAAGTTC SEQ ID NO: 27 TCACCGACGAGTATAAANNKCCTTCGAAAAAGTTCAA SEQ ID NO: 28 ACCGACGAGTATAAAGTANNKTCGAAAAAGTTCAAAGTGC SEQ ID NO: 29 CGACGAGTATAAAGTACCTNNKAAAAAGTTCAAAGTGCTGG SEQ ID NO: 30 AGTATAAAGTACCTTCGNNKAAGTTCAAAGTGCTGGG SEQ ID NO: 31 ATAAAGTACCTTCGAAANNKTTCAAAGTGCTGGGCAA SEQ ID NO: 32 GTACCTTCGAAAAAGNNKAAAGTGCTGGGCAAC SEQ ID NO: 33 TTCGAAAAAGTTCNNKGTGCTGGGCAACAC SEQ ID NO: 34 CGAAAAAGTTCAAANNKCTGGGCAACACCGAT SEQ ID NO: 35 AAAAGTTCAAAGTGNNKGGCAACACCGATCG SEQ ID NO: 36 GTTCAAAGTGCTGNNKAACACCGATCGCC SEQ ID NO: 37 AAGTGCTGGGCNNKACCGATCGCCA SEQ ID NO: 38 TGCTGGGCAACNNKGATCGCCATTC SEQ ID NO: 39 CTGGGCAACACCNNKCGCCATTCAATC SEQ ID NO: 40 CTGGGCAACACCGATNNKCATTCAATCAAAAAGA SEQ ID NO: 41 GCAACACCGATCGCNNKTCAATCAAAAAGAAC SEQ ID NO: 42 CAACACCGATCGCCATNNKATCAAAAAGAACTTGAT SEQ ID NO: 43 CACCGATCGCCATTCANNKAAAAAGAACTTGATTG SEQ ID NO: 44 CGATCGCCATTCAATCNNKAAGAACTTGATTGGTG SEQ ID NO: 45 CGCCATTCAATCAAANNKAACTTGATTGGTGCG SEQ ID NO: 46 CATTCAATCAAAAAGNNKTTGATTGGTGCGCTGT SEQ ID NO: 47 ATTCAATCAAAAAGAACNNKATTGGTGCGCTGTTGTT SEQ ID NO: 48 ATCAAAAAGAACTTGNNKGGTGCGCTGTTGTTT SEQ ID NO: 49 TCAAAAAGAACTTGATTNNKGCGCTGTTGTTTGACTC SEQ ID NO: 50 AAAGAACTTGATTGGTNNKCTGTTGTTTGACTCCG SEQ ID NO: 51 CTTGATTGGTGCGNNKTTGTTTGACTCCGG SEQ ID NO: 52 TTGGTGCGCTGNNKTTTGACTCCGG SEQ ID NO: 53 GTGCGCTGTTGNNKGACTCCGGGGA SEQ ID NO: 54 CGCTGTTGTTTNNKTCCGGGGAAACC SEQ ID NO: 55 CTGTTGTTTGACNNKGGGGAAACCGCC SEQ ID NO: 56 TTGTTTGACTCCNNKGAAACCGCCGAG SEQ ID NO: 57 TTGACTCCGGGNNKACCGCCGAGGC SEQ ID NO: 58 ACTCCGGGGAANNKGCCGAGGCGAC SEQ ID NO: 59 CCGGGGAAACCNNKGAGGCGACTCGSEQ ID Sequence (5'-3') SEQ ID NO: 60 GGGAAACCGCCNNKGCGACTCGCCT SEQ ID NO: 61 GAAACCGCCGAGNNKACTCGCCTTAAAC SEQ ID NO: 62 CCGCCGAGGCGNNKCGCCTTAAACG SEQ ID NO: 63 GCCGAGGCGACTNNKCTTAAACGTACAG SEQ ID NO: 64 AGGCGACTCGCNNKAAACGTACAGC SEQ ID NO: 65 CGACTCGCCTTNNKCGTACAGCACG SEQ ID NO: 66 ACTCGCCTTAAANNKACAGCACGTCGC SEQ ID NO: 67 GCCTTAAACGTNNKGCACGTCGCCG SEQ ID NO: 68 CCTTAAACGTACANNKCGTCGCCGGTACA SEQ ID NO: 69 TAAACGTACAGCANNKCGCCGGTACACTC SEQ ID NO: 70 GTACAGCACGTNNKCGGTACACTCGG SEQ ID NO: 71 CAGCACGTCGCNNKTACACTCGGCG SEQ ID NO: 72 CACGTCGCCGGNNKACTCGGCGTAA SEQ ID NO: 73 GTCGCCGGTACNNKCGGCGTAAGAA SEQ ID NO: 74 CGCCGGTACACTNNKCGTAAGAATCGC SEQ ID NO: 75 CCGGTACACTCGGNNKAAGAATCGCATTTG SEQ ID NO: 76 GTACACTCGGCGTNNKAATCGCATTTGCTA SEQ ID NO: 77 CACTCGGCGTAAGNNKCGCATTTGCTATTT SEQ ID NO: 78 CACTCGGCGTAAGAATNNKATTTGCTATTTGCAGG SEQ ID NO: 79 GCGTAAGAATCGCNNKTGCTATTTGCAGGA SEQ ID NO: 80 GGCGTAAGAATCGCATTNNKTATTTGCAGGAAATCTT SEQ ID NO: 81 GTAAGAATCGCATTTGCNNKTTGCAGGAAATCTTTAGC SEQ ID NO: 82 AGAATCGCATTTGCTATNNKCAGGAAATCTTTAGCAAC SEQ ID NO: 83 ATCGCATTTGCTATTTGNNKGAAATCTTTAGCAACGA SEQ ID NO: 84 GCATTTGCTATTTGCAGNNKATCTTTAGCAACGAGAT SEQ ID NO: 85 TTGCTATTTGCAGGAANNKTTTAGCAACGAGATGG SEQ ID NO: 86 ATTTGCAGGAAATCNNKAGCAACGAGATGGC SEQ ID NO: 87 ATTTGCAGGAAATCTTTNNKAACGAGATGGCAAAAGT

[0036] Table 2. Exemplary amino acid changes that show potential benefit in Prime Editing application. NGS reads were analyzed against pre- and post-selection libraries and denoted as fold change.EXAMPLE 2

[0037] Evaluating single mutants for increased prime editing activity.

[0038] The top 23 amino acid substitutions from MMLV RTase Library 1 were introduced by site-directed mutagenesis, using standard PCR conditions and primers (Table 3, SEQ ID NO: 88 - 149). The resulting plasmids were delivered into E. coli cells as described in Example 1. After 18 hours, the individual bacterial cells were counted and compared to the unmodified variant, denoted as PE-M63 (Table 4). Seven of the 23 substitutions resulted in a higher number of colonies, over PE-M63. These 7 substitutions are the following: S1377A, S1393A, E1406R, D1407S, E1408F, V1420F and G1423H.

[0039] The top 8 amino acid substitutions from MMLV RTase Library 1 were introduced by site-directed mutagenesis, using standard PCR conditions and primers (Table 3, SEQ ID NO: 88 - 149). The resulting plasmids were delivered into HEK293 cells and collected after 72 hours. The cells were lysed, the DNA extracted and amplified using primers to detect the HEK3 gene (Table 5) and treated with EcoRI to determine the percentage of prime editing. The resulting DNA was analyzed by a Fragment Analyzer and the data is summarized in Table 6. Seven of the 8 substitutions were successfully delivered into HEK293 cells. All of 7 mutants resulted in increased prime editing activity over PE-M63 and 6 of the 7 mutants resulted in a slight increase in editing over the published prime editor developed by David Liu. These substitutions are the following: S1377A, S1393A, S1397L, E1406R, D1407S, V1420F and G1423H. The only substitution from this library that is currently covered under patent claims is E1406R, and based on the editing in human cells, it is not the top performing mutant from this library. The remaining substitution, E1408F, is still in the cloning process and awaiting delivery into human cells.

[0040] Table 3. Primers used for saturation mutagenesis of the desired single mutation. SEQ ID NO Sequence Name Sequence (5'-3') SEQ ID NO: PE pACYT S1373D GTGGGGATAGCGGTGGTAGCGATGGTGGTTCAAGCGGTAGCGAA 88 TOP SEQ ID NO: PE pACYT S1373D TTCGCTACCGCTTGAACCACCATCGCTACCACCGCTATCCCCAC 89 BTM SEQ ID NO: PE pACYT S1373K GTGGGGATAGCGGTGGTAGCAAAGGTGGTTCAAGCGGTAGCGAA 90 TOP SEQ ID NO: PE pACYT S1373K TTCGCTACCGCTTGAACCACCTTTGCTACCACCGCTATCCCCAC 91 BTM SEQ ID NO: PE pACYT S1377A GTGGTAGCAGCGGTGGTTCAGCGGGTAGCGAAACACCGGGTACAAG 92 TOP SEQ ID NO: PE pACYT S1377A CTTGTACCCGGTGTTTCGCTACCCGCTGAACCACCGCTGCTACCAC 93 BTM SEQ ID NO: PE pACYT S1379I AGCGGTGGTTCAAGCGGTATTGAAACACCGGGTACAAGCGAAAGC 94 TOP SEQ ID NO: PE pACYT S1379I GCTTTCGCTTGTACCCGGTGTTTCAATACCGCTTGAACCACCGCT 95 BTM SEQ ID NO: PE pACYT P1382D GTGGTTCAAGCGGTAGCGAAACAGATGGTACAAGCGAAAGCGCAACAC 96 TOP SEQ ID NO: PE pACYT GTGTTGCGCTTTCGCTTGTACCATCTGTTTCGCTACCGCTTGAACCAC 97 P1382DBTM SEQ ID NO: PE pACYT E1386K AGCGAAACACCGGGTACAAGCAAAAGCGCAACACCGGAAAGC 98 TOP SEQ ID NO: PE pACYT E1386K GCTTTCCGGTGTTGCGCTTTTGCTTGTACCCGGTGTTTCGCT 99 BTM SEQ ID NO: PE pACYT S1393A AGCGCAACACCGGAAAGCGCGGGTGGTAGCTCAGGTGGTAGTAGC 100 TOP SEQ ID NO: PE pACYT S1393A GCTACTACCACCTGAGCTACCACCCGCGCTTTCCGGTGTTGCGCT 101 BTM SEQ ID NO: PE pACYT S1397R CCGGAAAGCAGTGGTGGTAGCCGTGGTGGTAGTAGCACTTTAAATATTGA 102 TOP GGATGAGC SEQ ID NO: PE pACYT S1397R GCTCATCCTCAATATTTAAAGTGCTACTACCACCACGGCTACCACCACTGCT 103 BTM TTCCGGSEQ ID NO Sequence Name Sequence (5'-3') SEQ ID NO: PE pACYT G1399T AGCAGTGGTGGTAGCTCAGGTACCAGTAGCACTTTAAATATTGAGGATGA 104 TOP GCATCG SEQ ID NO: PE pACYT G1399T CGATGCTCATCCTCAATATTTAAAGTGCTACTGGTACCTGAGCTACCACCA 105 BTM CTGCT SEQ ID NO: PE pACYT E1406R CTCAGGTGGTAGTAGCACTTTAAATATTCGTGATGAGCATCGTTTACATGA 106 TOP GACATCAAA SEQ ID NO: PE pACYT E1406R TTTGATGTCTCATGTAAACGATGCTCATCACGAATATTTAAAGTGCTACTA 107 BTM CCACCTGAG SEQ ID NO: PE pACYT E1406S CTCAGGTGGTAGTAGCACTTTAAATATTAGCGATGAGCATCGTTTACATGA 108 TOP GACATCAAA SEQ ID NO: PE pACYT E1406S TTTGATGTCTCATGTAAACGATGCTCATCGCTAATATTTAAAGTGCTACTAC 109 BTM CACCTGAG SEQ ID NO: PE pACYT D1407S TCAGGTGGTAGTAGCACTTTAAATATTGAGAGCGAGCATCGTTTACATGA 110 TOP GACATCAAAA SEQ ID NO: PE pACYT D1407S TTTTGATGTCTCATGTAAACGATGCTCGCTCTCAATATTTAAAGTGCTACTA 111 BTM CCACCTGA SEQ ID NO: PE pACYT E1408F GTGGTAGTAGCACTTTAAATATTGAGGATTTTCATCGTTTACATGAGACAT 112 TOP CAAAAGAAC SEQ ID NO: PE pACYT E1408F GTTCTTTTGATGTCTCATGTAAACGATGAAAATCCTCAATATTTAAAGTGCT 113 BTM ACTACCAC SEQ ID NO: PE pACYT E1408L GTGGTAGTAGCACTTTAAATATTGAGGATCTGCATCGTTTACATGAGACAT 114 TOP CAAAAGAAC SEQ ID NO: PE pACYT E1408L GTTCTTTTGATGTCTCATGTAAACGATGCAGATCCTCAATATTTAAAGTGCT 115 BTM ACTACCAC SEQ ID NO: PE pACYT R1410I AGTAGCACTTTAAATATTGAGGATGAGCATATTTTACATGAGACATCAAAA 116 TOP GAACCCGAC SEQ ID NO: PE pACYT R1410I GTCGGGTTCTTTTGATGTCTCATGTAAAATATGCTCATCCTCAATATTTAAA 117 BTM GTGCTACT SEQ ID NO: PE pACYT T1414Y TTGAGGATGAGCATCGTTTACATGAGTATTCAAAAGAACCCGACGTGAGC 118 TOP TT SEQ ID NO: PE pACYT T1414Y AAGCTCACGTCGGGTTCTTTTGAATACTCATGTAAACGATGCTCATCCTCA 119 BTM A SEQ ID NO: PE pACYT E1417P GGATGAGCATCGTTTACATGAGACATCAAAACCGCCCGACGTGAGCTTAG 120 TOP GGTCSEQ ID NO Sequence Name Sequence (5'-3') SEQ ID NO: PE pACYT E1417P GACCCTAAGCTCACGTCGGGCGGTTTTGATGTCTCATGTAAACGATGCTCA 121 BTM TCC SEQ ID NO: PE pACYT V1420F TCGTTTACATGAGACATCAAAAGAACCCGACTTTAGCTTAGGGTCAACGT 122 TOP GGCTTT SEQ ID NO: PE pACYT V1420F AAAGCCACGTTGACCCTAAGCTAAAGTCGGGTTCTTTTGATGTCTCATGTA 123 BTM AACGA SEQ ID NO: PE pACYT V1420H TCGTTTACATGAGACATCAAAAGAACCCGACCATAGCTTAGGGTCAACGT 124 TOP GGCTTT SEQ ID NO: PE pACYT V1420H AAAGCCACGTTGACCCTAAGCTATGGTCGGGTTCTTTTGATGTCTCATGTA 125 BTM AACGA SEQ ID NO: PE pACYT S1421Y TGAGACATCAAAAGAACCCGACGTGTATTTAGGGTCAACGTGGCTTTCTG 126 TOP AC SEQ ID NO: PE pACYT S1421Y GTCAGAAAGCCACGTTGACCCTAAATACACGTCGGGTTCTTTTGATGTCTC 127 BTM A SEQ ID NO: PE pACYT G1423H ACATCAAAAGAACCCGACGTGAGCTTACATTCAACGTGGCTTTCTGACTTC 128 TOP CCC SEQ ID NO: PE pACYT G1423H GGGGAAGTCAGAAAGCCACGTTGAATGTAAGCTCACGTCGGGTTCTTTTG 129 BTM ATGT SEQ ID NO: PE pACYT G1438R GGCGTGGGCGGAGACTCGTGGAATGGGGTTAGCTGTCCGC 130 TOP SEQ ID NO: PE pACYT G1438R GCGGACAGCTAACCCCATTCCACGAGTCTCCGCCCACGCC 131 BTM SEQ ID NO: PE pACYT P1448T GGGTTAGCTGTCCGCCAAGCAACCTTGATCATCCCGTTAAAGGCAACGTC 132 TOP SEQ ID NO: PE pACYT P1448T GACGTTGCCTTTAACGGGATGATCAAGGTTGCTTGGCGGACAGCTAACCC 133 BTM SEQ ID NO: PE pCMV E1406R 134 TOP GCGGCAGCAGCACCCTAAATATAAGAGATGAGCACCGGCTACATGAGAC SEQ ID NO: PE pCMV E1406R 135 BTM GTCTCATGTAGCCGGTGCTCATCTCTTATATTTAGGGTGCTGCTGCCGC SEQ ID NO: PE pCMV V1420F GCTACATGAGACCTCAAAAGAGCCAGATTTCTCTCTAGGGTCCACATGGCT 136 TOP GTC SEQ ID NO: PE pCMV V1420F GACAGCCATGTGGACCCTAGAGAGAAATCTGGCTCTTTTGAGGTCTCATG 137 BTM TAGCSEQ ID NO Sequence Name Sequence (5'-3') SEQ ID NO: PE pCMV S1377A 138 TOP CTGGAGGATCTAGCGGAGGATCCGCCGGCAGCGAGACACCAGGA SEQ ID NO: PE pCMV S1377A 139 BTM TCCTGGTGTCTCGCTGCCGGCGGATCCTCCGCTAGATCCTCCAG SEQ ID NO: PE pCMV S1397L 140 TOP GAGCAGTGGCGGCAGCCTGGGCGGCAGCAGCACC SEQ ID NO: PE pCMV S1397L 141 BTM GGTGCTGCTGCCGCCCAGGCTGCCGCCACTGCTC SEQ ID NO: PE pCMV D1407S 142 TOP CGGCAGCAGCACCCTAAATATAGAAAGCGAGCACCGGCTACATGAGACCT SEQ ID NO: PE pCMV D1407S 143 BTM AGGTCTCATGTAGCCGGTGCTCGCTTTCTATATTTAGGGTGCTGCTGCCG SEQ ID NO: PE pCMV G1423H TGAGACCTCAAAAGAGCCAGATGTTTCTCTACACTCCACATGGCTGTCTGA 144 TOP TTTTCCTCAG SEQ ID NO: PE pCMV G1423H CTGAGGAAAATCAGACAGCCATGTGGAGTGTAGAGAAACATCTGGCTCTT 145 BTM TTGAGGTCTCA SEQ ID NO: PE pCMV S1393A 146 TOP CGAGTCAGCAACACCAGAGAGCGCCGGCGGCAGCAGCGG SEQ ID NO: PE pCMV S1393A 147 BTM CCGCTGCTGCCGCCGGCGCTCTCTGGTGTTGCTGACTCG SEQ ID NO: PE pCMV E1408F CGGCAGCAGCACCCTAAATATAGAAGATTTCCACCGGCTACATGAGACCT 148 TOP CAAAAGA SEQ ID NO: PE pCMV E1408F TCTTTTGAGGTCTCATGTAGCCGGTGGAAATCTTCTATATTTAGGGTGCTG 149 BTM CTGCCG

[0041] Table 4. Bacterial colony counts of PE-M63 and single mutant prime editors. Mutant Colony Count PE-M63 131 S1373D 112 S1373K 100 S1377A 250S1379I 49 P1382D 65 E1386K 41 S1393A 207 S1397R 71 G1399T 68 E1406R 211 E1406S 115 D1407S 213 E1408F 320 E1408L 103 R1410I 92 T1414Y 21 E1417P 9 V1420H 57 V1420F 151 S1421Y 60 G1423H 222 G1438R 79 P1448T 27

[0042] Table 5. Primers used to amplify HEK3 gene from human cells. SEQ ID NO Sequence Name Sequence (5'-3') SEQ ID NO: 150 HEK3 FWD AGGGACGACTTTAGACCTTAGA SEQ ID NO: 151 HEK3 REV GTCTCTGACCACTGCGATATG

[0043] Table 6. Prime editing efficiency by percentage of EcoRI cleavage by PE- M63 and single mutant prime editors after 72 hours post-delivery into HEK293 cells. PE Variant % EcoRI Cleavage Triplicate Average St Dev PE-M63 21 20.8 19.5 20.43 0.81 David Liu PE2 26.2 25.1 24.6 25.30 0.82 E1406R 27.1 26.5 25.8 26.47 0.65 G1423H 28.2 29.3 30.1 29.20 0.95 D1407S 29.7 29.3 29.4 29.47 0.21 S1397L 29.6 29.6 29.8 29.67 0.12 S1377A 29.2 31.1 28.9 29.73 1.19 S1393A 30.5 29.7 29.3 29.83 0.61 V1420F 32.5 29.7 28.4 30.20 2.10 Cells Alone 0 0 0 0.00 0.00 EXAMPLE 3

[0044] Additional Cas9 and RTase mutations were identified with a revised bacterial prime editing selection and enrichment strategy using a modified strategy as described in Examples 1 and 2.

[0045] Functional prime editing has recently been described in E. coli using a variety of insertions / deletions and substitutions [5]. We used expression of a prime editor and PEG- RNA that targets a kanamycin resistance gene that contains a 10-base disruption between codons 1 and 2. This prime editing strategy is designed to remove the disrupting 10-base element and restore kanamycin resistance gene expression, and thus confer resistance to the antibiotic kanamycin. The screen was modified such that a library of Cas9 Prime Editormutants and PEG RNA are expressed from one plasmid, and the target site and non-functional kanamycin resistance gene is present on the chromosome. Practically, bacterial cells that repair and stably replicate the kanamycin resistance gene are made competent, transformed using Cas9 prime editor and PEG RNA plasmid, and selected on Kanamycin-containing solid media.

[0046] Twenty-five saturation mutagenesis libraries (Table 1) spanning the entirety of the prime editor (Cas9 and MMLV RTase genes and the amino acid linker between the two proteins) were generated with nicking mutagenesis and the resulting library complexity was analyzed with tiled-amplicon NGS. These libraries were delivered into E. coli cells as described above to be greater than 99% confident that each possible codon change was observed at least once. The resulting colonies were pooled, plasmids from this pool were purified, and supplied back into the same E. coli cells for subsequent rounds and pooling, until E. coli cells reached saturation (up to five rounds). The resulting pools from each round were sequenced with overlapping tiled amplicon NGS. Pre- and post-enrichment pools were sequenced and compared simultaneously with total read count normalization to determine enrichment for each substitution within the pool. Paired end reads were merged by fastp [6], trimmed to keep the desired mutated region by Cutadapt [7], and filtered out undesired reads with wrong length or multiple codon mutation. Codon frequency and enrichment analysis were completed by in-house python script. For M63 RTase libraries 1, 2, 4 and 5, the results included over 1,200 amino acid substitutions that are at equal or greater frequency than the identical substitution observed in the pre-enriched pool (Table 7). Among these results are substitutions that have issued patents; however, in most cases, these patented positions are far from the most frequently observed mutations indicating the most ideal prime editing mutationsare within this pool. The top 10 substitutions from each library from Table 2 will be cloned and tested in human cells for prime editing efficiency (denoted in Table 3).

[0047] Table 7. Amino acid changes that show potential benefit in Prime Editing application. NGS reads were analyzed against pre- and post-selection libraries and denoted as fold change. M63 Fold- Fold- Fold- Fold- Fold- Library Position Mutation Change Change Change Change Change Round 1 Round 2 Round 3 Round 4 Round 5 1 1412 H1412A 4.6 36.1 44.0 194.0 434.7 1 1421 S1421P 3.5 57.1 202.3 392.9 337.4 1 1412 H1412I 2.6 42.3 76.9 158.2 144.0 1 1386 E1386L 1.3 18.2 49.2 104.6 108.3 1 1440 M1440G 1.3 6.9 24.3 39.4 74.1 1 1423 G1423Q 3.8 21.5 53.9 75.7 64.3 1 1419 D1419Q 0.8 13.3 55.9 69.5 53.6 1 1406 E1406L 0.8 8.7 13.7 49.7 51.4 1 1400 S1400T 2.9 25.3 22.0 56.6 35.8 1 1381 T1381L 2.0 14.1 21.1 54.2 35.0 2 1521 K1521V 1.0 4.7 38.9 244.6 N / A 2 1480 R1480P 0.3 1.4 17.9 123.1 N / A 2 1507 T1507W 1.2 2.9 12.4 53.1 N / A 2 1500 R1500Y 2.2 10.4 18.8 16.8 N / A 2 1504 K1504P 1.6 11.3 16.0 13.1 N / A 2 1458 T1458R 3.6 16.0 18.1 12.8 N / A 2 1524 E1524P 2.5 4.5 8.1 12.5 N / AM63 Fold- Fold- Fold- Fold- Fold- Library Position Mutation Change Change Change Change Change Round 1 Round 2 Round 3 Round 4 Round 5 2 1491 C1491H 1.6 10.8 17.3 12.0 N / A 2 1470 E1470S 0.5 8.3 15.6 11.7 N / A 2 1505 P1505T 1.5 9.0 12.9 10.9 N / A 2 1458 T1458D 2.6 6.4 11.7 10.7 N / A 4 1646 Q1646R 3.1 12.8 113.4 207.5 92.7 4 1660 A1660S 0.4 1.2 0.3 84.0 87.8 4 1680 W1680Q 1.1 6.4 53.5 59.3 82.3 4 1622 Q1622K 0.9 1.5 18.8 50.1 49.0 4 1638 Q1638S 1.2 3.3 19.9 20.0 37.2 4 1632 T1632A 0.7 2.0 8.2 26.2 30.7 4 1680 W1680N 0.6 1.1 10.9 22.8 30.4 4 1670 L1670L 0.7 0.9 13.5 35.8 29.3 4 1629 L1629L 1.3 2.5 29.8 38.0 27.9 4 1627 L1627I 1.1 1.4 19.9 25.0 23.2 5 1741 Q1741P 1.6 44.9 35.2 51.7 N / A 5 1764 T1764N 2.3 13.7 47.2 51.0 N / A 5 1734 L1734S 1.5 26.5 37.9 37.9 N / A 5 1734 L1734E 2.7 13.1 10.3 25.4 N / A 5 1743 K1743C 2.5 6.2 17.8 25.2 N / A 5 1754 T1754S 2.0 5.7 25.3 25.1 N / A 5 1760 L1760I 1.2 6.5 19.3 23.1 N / A 5 1750 Q1750E 1.2 1.6 16.4 21.2 N / AM63 Fold- Fold- Fold- Fold- Fold- Library Position Mutation Change Change Change Change Change Round 1 Round 2 Round 3 Round 4 Round 5 5 1766 P1766R 1.4 20.1 32.1 17.9 N / A 5 1703 E1703V 0.7 10.2 18.6 17.5 N / A EXAMPLE 4

[0048] Additional Cas9 and RTase mutations were identified with a revised bacterial prime editing selection and enrichment strategy using the modified strategy of Example 3. For M63 RTase libraries 3, 6, 7, 8 and 9, the results included 389 amino acid substitutions that are at equal or greater frequency than the identical substitution observed in the pre-enriched pool (Table 8). Among these results are substitutions that have issued patents; however, in most cases, these patented positions are far from the most frequently observed mutations indicating the most ideal prime editing mutations are within this pool. The top 10 substitutions from each library from Table 8 will be cloned and tested in human cells for prime editing efficiency (denoted in Table 8).

[0049] Table 8. Mutant candidates for increased Prime editing efficiency from M63 Lib 36789 Fold- M63 Fold-Change Fold-Change Fold-Change Position Mutation Change Library Round 2 Round 3 Round 4 Round 1 3 1560 R1560A 4.9 2031.4 3478.2 N / A 3 1560 R1560P 0.6 11.5 19.3 N / A 3 1536 L1536V 3.0 3.3 2.7 N / AFold- M63 Fold-Change Fold-Change Fold-Change Position Mutation Change Library Round 2 Round 3 Round 4 Round 1 3 1542 P1542K 2.0 0.7 1.1 N / A 3 1556 F1556A 0.5 4.7 1.0 N / A 3 1560 R1560G 1.6 0.7 0.9 N / A 3 1580 I1580L 2.9 1.6 0.8 N / A 3 1573 W1573S 0.6 0.7 0.8 N / A 3 1560 R1560S 0.8 0.6 0.8 N / A 3 1580 I1580T 1.6 0.7 0.8 N / A 6 1829 M1829A 0.7 0.2 7.0 N / A 6 1786 L1786P 0.5 0.4 6.6 N / A 6 1830 G1830L 4.0 7.1 5.8 N / A 6 1841 V1841P 1.4 3.8 4.2 N / A 6 1836 L1836H 10.9 5.7 3.5 N / A 6 1806 G1806T 1.3 2.4 2.9 N / A 6 1771 V1771T 2.6 2.9 2.8 N / A 6 1783 T1783H 1.0 2.7 2.7 N / A 6 1828 T1828G 1.2 2.9 2.5 N / A 6 1825 G1825Y 0.2 1.4 2.3 N / A 7 1879 L1879A 8.7 6274.0 8364.0 N / A 7 1879 L1879D 0.6 8.1 10.7 N / A 7 1875 P1875N 7.1 12.6 8.8 N / A 7 1925 G1925P 6.7 5.3 3.4 N / A 7 1891 G1891T 14.3 10.6 3.2 N / AFold- M63 Fold-Change Fold-Change Fold-Change Position Mutation Change Library Round 2 Round 3 Round 4 Round 1 7 1883 T1883R 3.7 3.4 2.8 N / A 7 1891 G1891A 0.3 3.4 2.8 N / A 7 1879 L1879Y 1.4 2.7 2.5 N / A 7 1879 L1879S 0.0 2.6 2.4 N / A 7 1891 G1891N 11.9 5.6 2.0 N / A 7 1879 L1879F 1.4 2.4 1.8 N / A 8 1971 L1971R 1.2 1.4 1.6 N / A 8 1952 A1952P 1.2 1.5 1.6 N / A 8 1980 N1980H 1.2 1.4 1.6 N / A 8 1988 A1988G 1.7 0.4 1.4 N / A 8 2002 R2002S 1.1 1.2 1.3 N / A 8 1963 Q1963P 1.1 1.2 1.2 N / A 8 1992 A1992P 0.8 0.8 1.1 N / A 8 1963 Q1963L 0.8 1.0 1.1 N / A 8 1993 H1993P 1.2 1.4 1.1 N / A 8 1972 K1972M 0.6 0.8 1.1 N / A 9 2014 N2014C 0.5 34.7 140.8 120.5 9 2062 I2062P 1.0 5.6 4.8 99.9 9 2047 A2047V 0.9 3.6 4.1 74.4 9 2037 P2037T 3.0 23.4 91.6 50.3 9 2029 K2029Q 5.1 45.5 43.4 43.2 9 2043 H2043P 3.2 42.7 38.1 34.1Fold- M63 Fold-Change Fold-Change Fold-Change Position Mutation Change Library Round 2 Round 3 Round 4 Round 1 9 2025 L2025W 0.9 7.2 19.5 32.1 9 2044 S2044R 1.1 25.7 33.7 30.1 9 2030 R2030M 0.3 29.7 28.1 28.5 9 2042 G2042R 1.6 6.1 30.0 28.0 EXAMPLE 5

[0050] Additional Cas9 and RTase mutations were identified with a revised bacterial prime editing selection and enrichment strategy using the modified strategy of Example 3. For Cas9 libraries 1-8, the results included 262 amino acid substitutions that are at equal or greater frequency than the identical substitution observed in the pre-enriched pool (Table 9). Among these results are substitutions that have issued patents; however, in most cases, these patented positions are far from the most frequently observed mutations indicating the most ideal prime editing mutations are within this pool. The top 5 substitutions from each library from Table 2 will be cloned and tested in human cells for prime editing efficiency (denoted in Table 9).

[0051] Table 9. Mutant candidates for increased Prime editing efficiency for Cas9 Lib1-8 Cas9 Library Position Mutation Fold-Change Rd 1 Fold-Change Rd 2 Fold-Change Rd 3 1 53 F53C 0.63 48.26 16.28 1 31 K31Q 2.01 1.06 2.41 1 79 I79T 0.71 1.59 1.57 1 50 A50P 0.91 1.54 1.53Cas9 Library Position Mutation Fold-Change Rd 1 Fold-Change Rd 2 Fold-Change Rd 3 1 97 F97Y 0.70 1.57 1.49 2 175 N175A 13.16 20.98 3.11 2 197 E197R 8.72 3.89 2.52 2 185 F185STOP 19.05 9.56 2.27 2 104 S104STOP 7.11 7.94 2.09 2 195 L195P 5.57 7.01 1.79 3 271 Y271G 5.43 2347.92 2361.72 3 275 L275A 4.32 47.49 39.25 3 239 G239L 16.68 20.01 14.76 3 264 L264T 22.93 7.40 5.82 3 271 Y271R 0.04 6.00 4.37 4 385 G385S 19.00 7.08 3.11 4 346 K346N 1.64 2.35 2.38 4 325 Y325F 1.32 1.25 1.30 4 308 V308D 1.08 1.10 1.28 4 326 D326E 1.26 1.38 1.26 5 446 F446L 11.32 3.81 1.44 5 441 E441A 1.26 0.99 1.43 5 442 K442STOP 1.15 1.23 1.39 5 437 R437Q 1.00 1.30 1.35 5 471 E471D 1.21 1.08 1.31 6 581 S581STOP 10.76 2355.42 2045.97 6 518 F518I 1.03 1.03 1.61Cas9 Library Position Mutation Fold-Change Rd 1 Fold-Change Rd 2 Fold-Change Rd 3 6 502 L502M 1.04 1.16 1.37 6 517 Y517N 0.98 1.00 1.28 6 516 E516Q 48.69 7.34 1.18 7 674 Q674H 1.54 1346.54 1471.33 7 654 R654D 17.73 6.70 7.14 7 603 D603V 28.78 15.15 4.70 7 674 Q674L 0.02 3.55 3.23 7 616 L616R 9.90 7.16 2.85 8 741 V741G 0.80 317.22 349.07 8 776 N776A 8.98 5.69 1.69 8 775 K775S 1.65 9.06 1.44 8 771 N776Q 15.07 5.16 0.88 8 775 H799Y 12.61 5.33 0.71 EXAMPLE 6

[0052] Additional Cas9 and RTase mutations were identified with a revised bacterial prime editing selection and enrichment strategy using the modified strategy of Example 3. For Cas9 libraries 9-15, the results included over 2,900 amino acid substitutions that are at equal or greater frequency than the identical substitution observed in the pre-enriched pool (Table 10). Among these results are substitutions that have issued patents; however, in most cases, these patented positions are far from the most frequently observed mutations indicating the most ideal prime editing mutations are within this pool. The top substitutions from each library from Table 10 will be cloned and tested in human cells for prime editing efficiency.

[0053] Table 10. Mutant candidates for increased Prime editing efficiency for Cas9 Lib9-15. Cas9 Fold-Change Rd Fold-Change Fold-Change Rd Library Position Mutation 1 Rd 2 3 9 888 N888L 30.0 24.0 14.1 9 874 E874T 19.7 3.5 3.6 9 863 N863A 30.1 10.2 2.6 9 881 N881H 35.4 4.3 1.7 10 939 M939V 7.9 2244.1 2586.4 10 905 R905N 80.5 0.0 3.2 10 991 A991STOP 13.4 2.4 1.4 10 915 G915P 41.0 3.4 1.1 11 1074 W1074S 4.0 1681.8 886.9 11 1046 F1046S 7.9 3.3 3.4 11 1024 K1024A 15.4 0.0 2.1 12 1105 F1105G 5.8 142.3 96.4 12 1125 D1125E 0.2 0.2 4.1 13 1278 K1278I 127.6 10233.8 11443.4 13 1273 I1273Y 11.1 478.7 199.1 13 1261 Q1261S 126.4 11.1 4.6 13 1217 A1217F 95.5 7.7 1.5 13 1244 K1244L 51.4 9.2 1.4 13 1219 E1219M 40.9 3.9 1.3 13 1260 E1260S 45.6 6.0 0.7 14 1329 T1329A 3.3 505.1 619.4Cas9 Fold-Change Rd Fold-Change Fold-Change Rd Library Position Mutation 1 Rd 2 3 14 1396 S1396C 4.4 204.8 219.0 14 1380 E1380T 145.5 10.4 2.3 14 1354 G1354N 29.4 10.2 1.1 15 487 S487P 22.4 1984.8 1142.2 15 608 D608L 28.4 1552.9 982.9 15 1043 M1043A 12.4 44.6 33.6 EXAMPLE 7

[0054] Additional Cas9 and RTase mutations were identified with a revised bacterial prime editing selection and enrichment strategy using the modified strategy of Example 3. For Cas9 and M63 libraries, additional Mutant ID’s having increased Prime editing efficiency were identified. EXAMPLE 8

[0055] Amino acid and nucleic acid sequences

[0056] The following amino acid and nucleic acid sequences support this disclosure. SEQ ID NO: 152 SpyCas9 (H840A) protein AA sequence MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTR RKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNL SDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQE EFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKIL TFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLG TYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQT VKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLY YLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLN AKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKY FFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESI LPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA KGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SEQ ID NO: 153 WT MMLV protein AA sequence TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGI KPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTV LDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQY VDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFV DEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALV KQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDAD HTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAH IHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPD TSTLLIENSSP SEQ ID NO: 154 Mutant MMLV-II protein AA sequence TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGI KPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTV LDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQY VDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFV DEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALV KQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDAD HTWYTGGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNVYTNSRYAFATAH IHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPD TSTLLIENSSP SEQ ID NO: 155 Mutant PE2 MMLV RT protein AA sequence TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGI KPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTV LDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQY VDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT PRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFV DEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALV KQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDAD HTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPD TSTLLIENSSP SEQ ID NO: 156 Mutant M63 protein AA sequence TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSREARLGI KPHIRRLYDQGILVPCQSPWNTPLRPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTV LDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQY VDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWITDARKETVMGQPTPKT PRELREFLGKAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFV DEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLNILAPHAVEALV KQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDAD HTWYTGGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAQLIALTQALKMAEGKKLNVYTNSRYAFATAH WHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPD TSTLLIENSSP

[0057] References 1. Jinek, M., et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012.337(6096): p.816-21. 2. Komor, A.C., et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 2016.533(7603): p.420-4. 3. Gaudelli, N.M., et al., Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature, 2017.551(7681): p.464-471. 4. Anzalone, A.V., et al., Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 2019.576(7785): p.149-157. 5. Tong, Y., et al., A versatile genetic engineering toolkit for E. coli based on CRISPR- prime editing. Nat Commun, 2021.12(1): p.5206. 6. Chen, S., et al., fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 2018.34(17): p.884-890. 7. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. journal.2011.17(1): p.10-2.

[0058] All references, including publications, patent applications, and patents cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0059] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS:

1. A fusion protein mutant comprising a Prime Editing enzyme having a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence comprises a SpCas9 H840A nickase mutant protein of SEQ ID NO:152 and the second amino acid sequence comprises a Moloney Murine Leukemia Virus reverse transcriptase protein mutant (MMLV RTase mutant), wherein the fusion protein mutant displays at least the equivalent or greater activity of a reference Prime Editing enzyme in genome editing.

2. The fusion protein mutant of claim 1, comprising a member of the group selected from one of Tables 2, 6, 7, 8, 9, 10, and 11.

3. A nucleic acid sequence encoding the fusion protein of claims 1 or 2.

4. An isolated ribonucleoprotein complex, wherein the isolated ribonucleoprotein complex comprises the fusion protein of claim 1 or 2 and a gRNA.

5. The isolated ribonucleoprotein complex of claim 5, wherein the gRNA comprises a pegRNA.

6. A CRISPR / Cas endonuclease system comprising the fusion protein of claims 1 or 2.

7. The CRISPR / Cas endonuclease system of claim 7, wherein the CRISPR / Cas endonuclease system is encoded by a DNA expression vector.

8. The CRISPR / Cas endonuclease system of claim 8, the DNA expression vector is a plasmid-borne vector.

9. The CRISPR / Cas endonuclease system of claim 9, wherein the DNA expression vector is selected from a bacterial expression vector and a eukaryotic expression vector.

10. A method of performing gene editing in a eukaryotic cell, comprising a step of contacting a candidate editing target site locus with an active CRISPR / Cas endonuclease system having the fusion protein of claim 1 or 2.

11. A kit for performing gene editing in a eukaryotic cell, wherein the kit comprises the fusion protein of claim 1 or 2 and optionally a gRNA.