RNA-guided kilobase-scale genome recombination engineering

Abstract

The invention provides a recombinant engineering editing system using CRISPR (clustered regularly interspaced short palindromic repeats) and recombinase as well as a method, a vector, a nucleic acid composition and a kit thereof. The methods and systems provide means for altering target DNA, including genomic DNA, in host cells. In particular, the present invention provides systems and compositions that utilize aptamers to bind to a component of a recombinant-editing system, such as a CAS protein or guide RNA, to enhance targeting of genomic DNA in a cell.

Description

Related Applications and Incorporation by Reference

[0001] This application claims priority under 35 USC §119(e) to U.S. patent application serial number 63 / 308,830, U.S. patent application serial number 63 / 308,834, and U.S. patent application serial number 63 / 308,837 (each filed on February 10, 2022), and PCT / US2022 / 075850 (filed on September 1, 2022), which are incorporated herein by reference in their entirety.

[0002] Reference is made to U.S. patent application serial number 62 / 984,618 filed on March 3, 2020, U.S. patent application serial number 63 / 146,447 filed on February 5, 2021, and PCT / US2021 / 020513 filed on March 2, 2021.

[0003] The above-mentioned application, and all documents cited therein or during the prosecution of the same ("documents cited in the application") and all documents cited or referenced in documents cited in the application, and all documents cited or referenced herein ("documents cited herein"), and all documents cited or referenced in documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated by reference and may be employed in the practice of the present invention. More specifically, all cited documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Field of the Invention

[0004] The present invention relates to an RNA-guided recombineering editing system using bacteriophage recombinase and a method, vector, nucleic acid composition and kit thereof. Sequence Listing Statement

[0005] The contents of the electronic sequence listing entitled STDU2-41722_601_SQL.xml (size: 758,685 bytes; and creation date: February 10, 2023) are incorporated herein by reference in its entirety. Background of the Invention

[0006] The clustered regularly interspaced short palindromic repeat (CRISPR) system, originally discovered in bacteria and archaea as part of the immune system to defend against invading viruses, forms the basis of genome editing technologies that can be programmed to target specific segments of the genome or other DNA for editing at precise locations. Although a variety of CRISPR-based tools are available, most are suitable for editing short sequences. Long sequence editing is highly sought after in the engineering of model systems, therapeutic cell generation, and gene therapy. Previous studies have developed technologies to improve Cas9-mediated homology-5-directed repair (HDR) (KS Pawelczak, et al., ACS Chem. Biol. 13, 389–396 (2018)), as well as tools that utilize nucleic acid-modifying enzymes carrying Cas9, such as prime-editing (AV Anzalone, et al., Nature. 576, 149–157 (2019)), demonstrating editing lengths up to 80 base pairs (bp). Despite these advances, there is a continuing need for large-scale mammalian genome engineering with high efficiency and fidelity.

[0007] Citation or identification of any document in this application does not constitute an admission that the document is available as prior art to the present invention. Brief description of the invention

[0008] Provided herein are systems and methods for facilitating nucleic acid editing in a manner that allows large-scale nucleic acid editing with high accuracy and low off-target errors. These systems and methods employ recombinant protein components and optionally CRISPR components.

[0009] For example, disclosed herein is a system comprising a binding protein, a nucleic acid molecule comprising a guide RNA sequence complementary to a target DNA sequence, and a recombinant protein. The recombinant protein can be a single-stranded DNA annealing protein (SSAP), including but not limited to a microbial recombinant protein, for example, RecE, RecT, lambda exonuclease (Exo), Bet protein (betA, redB), exonuclease gp6, single-stranded DNA binding protein gp2.5, or a derivative or variant thereof. In some embodiments, the system further comprises a donor DNA. In some embodiments, the target DNA sequence is a genomic DNA sequence in a host cell. In certain embodiments, there is no CRISPR component. In certain embodiments, the system comprises a recruitment system for recruiting recombinant proteins and a nucleic acid for guiding recombinant proteins to a target. In certain embodiments, the recruitment system recruits recombinant proteins, nucleic acids guiding recombinant proteins, and CRISPR components.

[0010] In one aspect, the present invention provides a recombination system comprising an SSAP and lacking a CRISPR component. In certain embodiments, the present invention provides a system or composition comprising: (i) a nucleic acid molecule comprising a guide RNA sequence complementary to a target DNA sequence; and (ii) a recombinant protein, wherein the recombinant protein comprises an exonuclease, a single-stranded DNA annealing protein (SSAP) or a single-stranded DNA binding protein (SSB), or a combination of two or more thereof; or, (iii) one or more nucleic acid molecules encoding or delivering (i) and / or (ii) for in vivo expression in a cell; or, (iv) one or more vectors containing one or more nucleic acid molecules of (iii) for in vivo expression in a cell. In certain embodiments, the system or composition does not comprise a CRISPR protein, or does not comprise a Cas protein, or does not comprise a Cas9 protein, or does not comprise a Cas12a protein

[0011] In certain embodiments, the system or composition comprises a recruitment system for recruiting guide nucleic acids and recombinant proteins. In certain embodiments, the recruitment system comprises at least one aptamer sequence and an aptamer binding protein, which is functionally connected to the recombinant protein as a part of the fusion protein. In certain embodiments, at least one aptamer sequence is an RNA aptamer sequence or a peptide aptamer sequence. In certain embodiments, the one nucleic acid molecule or a plurality of nucleic acid molecules additionally comprises at least one RNA aptamer sequence or comprises one, two, three or more RNA aptamer sequences. In certain embodiments, two aptamer sequences comprise the same sequence or comprise a sequence bound to the same aptamer binding protein.

[0012] In certain embodiments, the aptamer binding protein comprises an MS2 coat protein, or a functional derivative or variant thereof. In certain embodiments, the aptamer binding protein comprises a bacteriophage N peptide, or a functional derivative or variant thereof. In certain embodiments, at least one peptide aptamer sequence is conjugated to a guide RNA. In certain embodiments, at least one peptide aptamer sequence comprises 1 to 24 peptide aptamer sequences. In certain embodiments, two or more aptamer sequences comprise the same sequence. In certain embodiments, the aptamer sequence comprises a GCN4 peptide sequence.

[0013] In certain embodiments, the recombinant protein N-terminus is linked to the aptamer binding protein C-terminus. In certain embodiments, the recombinant protein and the aptamer binding protein are operably linked via a linker.

[0014] In certain embodiments, the recombinant system or composition comprises at least one nuclear localization sequence (NLS), optionally wherein the NLS is linked to the recombinant protein. In certain embodiments, the NLS is located at the C-terminus of the recombinant protein or the N-terminus of the recombinant protein.

[0015] In certain embodiments, the recombinant protein comprises a microbial recombinant protein or an active portion thereof, a mitochondrial recombinant protein or an active portion thereof, a viral recombinant protein or an active portion thereof, or a eukaryotic recombinant protein or an active portion thereof, including but not limited to the recombinant proteins or derivatives or variants or functional portions thereof listed in Table 12. In certain embodiments, the recombinant protein comprises an amino acid sequence having at least 70% identity, or at least 75% identity, or at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 92% identity, or at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity with the recombinant proteins or derivatives or variants or functional portions thereof listed in Table 12.

[0016] In certain embodiments, the system or composition comprises a donor nucleic acid.In certain embodiments, the donor nucleic acid comprises a homology arm.

[0017] In certain embodiments, the recombination system is contained in a cell (eg, a eukaryotic cell, a mammalian cell, an animal cell, a human cell, or a plant cell).

[0018] The recruitment system is applicable to a variety of combinations and configurations of recombinant proteins. For example, by selecting and incorporating multiple nucleic acid aptamers, the system can include multiple recombinant proteins, which can be the same or different and exist in various proportions. In certain embodiments, the system includes an exonuclease. In certain embodiments, the system includes an SSAP. In certain embodiments, the system includes an SSB. In certain embodiments, the system includes an exonuclease and an SSAP. In certain embodiments, the system includes an exonuclease and an SSB. In certain embodiments, the system includes an SSAP and an SSB. In certain embodiments, the system includes an exonuclease and an SSAP and does not include an SSB. In certain embodiments, the system includes an exonuclease and an SSB and does not include an SSAP. In certain embodiments, the system includes an SSAP and an SSB and does not include an exonuclease. In certain embodiments, the system includes an exonuclease, an SSAP, and an SSB.

[0019] In one aspect, the invention provides a recombination system comprising SSAP and reverse transcriptase (RT). In certain embodiments, the invention provides a system or composition comprising: (i) reverse transcriptase (RT); (ii) a nucleic acid molecule comprising a guide RNA sequence complementary to a target DNA sequence and an RNA for reverse transcription, or a nucleic acid molecule comprising a guide RNA sequence complementary to a target DNA sequence and an RNA for reverse transcription; and (iii) a recombinant protein, wherein the recombinant protein comprises an exonuclease, a single-stranded DNA annealing protein (SSAP) or a single-stranded DNA binding protein (SSB), or a combination of two or more thereof; or, (iv) a nucleic acid molecule encoding or delivering (i) and / or (ii) and / or (iii) for in vivo expression in a cell; or, (v) a vector containing a nucleic acid molecule of (iv) for in vivo expression in a cell.

[0020] In certain embodiments, the system or composition further comprises a Cas protein; or (iv) comprises one or more nucleic acid molecules encoding or delivering (i), and / or (ii) and / or (iii) and / or Cas proteins for in vivo expression in cells; or (v) one or more vectors additionally contain one or more nucleic acid molecules encoding Cas proteins.

[0021] In certain embodiments, one or more components are provided in the form of a complex. For example, a protein or fusion protein and a nucleic acid are provided in the form of a ribonucleoprotein (RNP). Non-limiting examples of RNP include CRISPR-guide RNA complexes and SSAP-guide RNA complexes. In certain embodiments, the fusion protein comprises one or more components. Non-limiting examples include Cas9-SSAP fusion, Cas9-RT fusion, and SSAP-RT fusion.

[0022] In certain embodiments, the system or composition comprises a recruitment system for recruiting guide nucleic acids and recombinant proteins. In certain embodiments, the recruitment system comprises at least one aptamer sequence and an aptamer binding protein, which is functionally connected to the recombinant protein as a part of the fusion protein. In certain embodiments, at least one aptamer sequence is an RNA aptamer sequence or a peptide aptamer sequence. In certain embodiments, the one nucleic acid molecule or a plurality of nucleic acid molecules additionally comprises at least one RNA aptamer sequence or comprises one, two, three or more RNA aptamer sequences. In certain embodiments, two aptamer sequences comprise the same sequence or comprise a sequence bound to the same aptamer binding protein.

[0023] In certain embodiments, the aptamer binding protein comprises an MS2 coat protein, or a functional derivative or variant thereof. In certain embodiments, the aptamer binding protein comprises a bacteriophage N peptide, or a functional derivative or variant thereof. In certain embodiments, at least one peptide aptamer sequence is conjugated to a guide RNA. In certain embodiments, at least one peptide aptamer sequence comprises 1 to 24 peptide aptamer sequences. In certain embodiments, two or more aptamer sequences comprise the same sequence. In certain embodiments, the aptamer sequence comprises a GCN4 peptide sequence.

[0024] In certain embodiments, the recombinant protein N-terminus is linked to the aptamer binding protein C-terminus. In certain embodiments, the recombinant protein and the aptamer binding protein are operably linked via a linker.

[0025] In certain embodiments, the recombinant system or composition comprises at least one nuclear localization sequence (NLS), optionally wherein the NLS is linked to the recombinant protein. In certain embodiments, the NLS is located at the C-terminus of the recombinant protein or the N-terminus of the recombinant protein.

[0026] In certain embodiments, the recombinant protein comprises a microbial recombinant protein or an active portion thereof, a mitochondrial recombinant protein or an active portion thereof, a viral recombinant protein or an active portion thereof, or a eukaryotic recombinant protein or an active portion thereof, including but not limited to the recombinant proteins or derivatives or variants or functional portions thereof listed in Table 12. In certain embodiments, the recombinant protein comprises an amino acid sequence having at least 70% identity, or at least 75% identity, or at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 92% identity, or at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity with the recombinant proteins or derivatives or variants or functional portions thereof listed in Table 12.

[0027] In certain embodiments, the system or composition comprises a donor nucleic acid.In certain embodiments, the donor nucleic acid comprises homology arms.

[0028] In certain embodiments, the recombination system is contained in a cell (eg, a eukaryotic cell, a mammalian cell, an animal cell, a human cell, or a plant cell).

[0029] In one aspect, the present invention provides a recombinant method, which includes providing in a cell, system or composition, (i) a nucleic acid molecule comprising a guide RNA sequence that is complementary to a target DNA sequence; wherein the target DNA sequence comprises a genomic DNA sequence in the cell, and (ii) a recombinant protein, wherein the recombinant protein comprises an exonuclease, a single-stranded DNA annealing protein (SSAP) or a single-stranded DNA binding protein (SSB), or a combination of two or more thereof; or, (iii) one or more nucleic acid molecules encoding or delivering (i) and / or (ii) for in vivo expression in the cell; or, (iv) one or more vectors containing one or more nucleic acid molecules of (iii) for in vivo expression in the cell.

[0030] In certain embodiments, (i) and (ii) further comprise a Cas protein or a nucleic acid polymerase, including but not limited to a natural or engineered polymerase with reverse transcriptase activity, such as a reverse transcriptase (RT) or a Cas protein and RT; or (iii) comprises one or more nucleic acid molecules encoding or delivering (i) and / or (ii) and / or Cas protein and / or RT for in vivo expression in a cell; or (iv) one or more vectors additionally contain one or more nucleic acid molecules encoding a Cas protein and / or RT.

[0031] In certain embodiments, one or more components are provided in the form of a complex. For example, a protein or fusion protein and a nucleic acid are provided in the form of a ribonucleoprotein (RNP). Non-limiting examples of RNP include CRISPR-guide RNA complexes and SSAP-guide RNA complexes. In certain embodiments, the fusion protein comprises one or more components. Non-limiting examples include Cas9-SSAP fusion, Cas9-RT fusion, and SSAP-RT fusion.

[0032] In certain embodiments, the target DNA sequence comprises the genomic sequence of albumin (ALB), AAVS1, HSP90AA1, DYNLT1, ACTB, BCAP31, HIST1H2BK, CLTA, or RAB11A.

[0033] In certain embodiments, the system or composition comprises a recruitment system for recruiting guide nucleic acids and recombinant proteins. In certain embodiments, the recruitment system comprises at least one aptamer sequence and an aptamer binding protein, which is functionally connected to the recombinant protein as a part of the fusion protein. In certain embodiments, at least one aptamer sequence is an RNA aptamer sequence or a peptide aptamer sequence. In certain embodiments, a nucleic acid molecule or a plurality of nucleic acid molecules additionally comprises at least one RNA aptamer sequence or comprises one, two, three or more RNA aptamer sequences. In certain embodiments, two aptamer sequences comprise the same sequence or comprise a sequence bound to the same aptamer binding protein.

[0034] In certain embodiments, the aptamer binding protein comprises an MS2 coat protein, or a functional derivative or variant thereof. In certain embodiments, the aptamer binding protein comprises a bacteriophage N peptide, or a functional derivative or variant thereof. In certain embodiments, at least one peptide aptamer sequence is conjugated to a guide RNA. In certain embodiments, at least one peptide aptamer sequence comprises 1 to 24 peptide aptamer sequences. In certain embodiments, two or more aptamer sequences comprise the same sequence. In certain embodiments, the aptamer sequence comprises a GCN4 peptide sequence.

[0035] In certain embodiments, the recombinant protein N-terminus is connected to the aptamer binding protein C-terminus. In certain embodiments, the recombinant protein and the aptamer binding protein are operably connected via a linker. In certain embodiments, the linker comprises 39115.

[0036] In certain embodiments, the recombinant system or composition comprises at least one nuclear localization sequence (NLS), optionally wherein the NLS is connected to the recombinant protein. In certain embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the NLS is located at the recombinant protein C-terminus or the recombinant protein N-terminus.

[0037] In certain embodiments, the recombinant protein comprises a microbial recombinant protein or an active portion thereof, a mitochondrial recombinant protein or an active portion thereof, a viral recombinant protein or an active portion thereof, or a eukaryotic recombinant protein or an active portion thereof, including but not limited to the recombinant proteins or derivatives or variants or functional portions thereof listed in Table 12. In certain embodiments, the recombinant protein comprises an amino acid sequence having at least 70% identity, or at least 75% identity, or at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 92% identity, or at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity with the recombinant proteins or derivatives or variants or functional portions thereof listed in Table 12.

[0038] In certain embodiments, the system or composition comprises a donor nucleic acid.In certain embodiments, the donor nucleic acid comprises homology arms.

[0039] In certain embodiments, the recombination system is contained in a cell (eg, a eukaryotic cell, a mammalian cell, an animal cell, a human cell, or a plant cell).

[0040] In some embodiments, the Cas protein is Cas9 or Cas12a. In some embodiments, the Cas protein is catalytically dead. In some embodiments, the Cas9 protein is wild-type Streptococcus pyogenes Cas9 or wild-type Staphylococcus aureus Cas9. In some embodiments, the Cas9 protein is a Cas9 nickase (e.g., wild-type Streptococcus pyogenes Cas9 with an amino acid substitution D10A at position 10).

[0041] Also disclosed are eukaryotic cells comprising the system or vector disclosed herein.

[0042] Further disclosed herein are methods for altering a target genomic DNA sequence in a host cell. The method includes contacting a system, composition, or vector as described herein with a target DNA sequence (e.g., introducing a system, composition, or vector as described herein into a host cell comprising a target genomic DNA sequence). Also disclosed herein are kits containing one or more reagents or other components useful, necessary, or sufficient for practicing any of these methods.

[0043] In some embodiments, the invention provides a system or composition comprising: (i) a nucleic acid polymerase, such as one or more reverse transcriptases (RT); (ii) a nucleic acid molecule comprising a guide RNA sequence complementary to a target DNA sequence and an RNA for reverse transcription, or a plurality of nucleic acid molecules comprising a guide RNA sequence complementary to a target DNA sequence and an RNA for reverse transcription; and (iii) a recombinant protein, wherein the recombinant protein comprises an exonuclease, a single-stranded DNA annealing protein (SSAP) or a single-stranded DNA binding protein (SSB), or a combination of two or more thereof; or, (iv) one or more nucleic acid molecules encoding or delivering (i), (ii) and (iii) for in vivo expression in a cell; or, (v) one or more vectors containing one or more nucleic acid molecules of (iv) for in vivo expression in a cell. In the system or composition involving RT ("RT system or composition"), (iv) may involve (i) being an enzyme, (ii) being one or more nucleic acid molecules, and (iii) being multiple nucleic acid molecules; or (i) being one or more nucleic acid molecules encoding one or more enzymes, (ii) being one or more nucleic acid molecules, and (iii) being a protein, or (i), (ii) and (iii) all being nucleic acid molecules. In some embodiments, the RT system or composition may include more than one reverse transcriptase. When more than one reverse transcriptase is present, multiple RNAs may be used for reverse transcription. In some embodiments, in the RT system or composition, (i), (ii) and (iii) further comprise a Cas protein; or (iv) further comprises one or more nucleic acid molecules encoding a Cas protein, for example, (iv) comprises one or more nucleic acid molecules encoding or delivering (i), and / or (ii) and / or (iii) and / or Cas proteins for in vivo expression in cells; or (v) one or more vectors additionally contain one or more nucleic acid molecules encoding a Cas protein.

[0044] Reverse transcriptases that can be used according to the present invention include, but are not limited to, reverse transcriptase, retrotransposon reverse transcriptase, retron reverse transcriptase, LINE-1 reverse transcriptase, Ec86 reverse transcriptase, human immunodeficiency virus (HIV) RT, avian myoblastosis virus (AMV) RT, Moloney murine leukemia virus (M-MLV) RT, group II intron RT, group II intron-like RT, chimeric RT, MaLuoni murine leukemia virus (M-MLV) transcriptase, Rous sarcoma virus (RSV), avian myeloblastosis virus (AMV) reverse transcriptase, Lao Sishi-associated virus (RAV) reverse transcriptase, and myeloblastosis-associated virus (MAV) reverse transcriptase or other avian sarcoma leukosis virus (ASLV) reverse transcriptases as well as other naturally occurring and engineered nucleic acid polymerases. Such engineered polymerases include, but are not limited to, human DNA polymerase η having reverse transcriptase activity in a cellular environment (Su et al. 2019, J. Biol. Chem. 294(15):6073-81), and Taq DNA polymerase engineered to enhance reverse transcription and strand replacement (Barnes et el., Front. Bioeng. Biotechnol., 14 January 2021, doi.org / 10.3389 / fbioe.2020.553474).

[0045] In some embodiments, the RT system or composition further comprises a recruitment system containing at least one aptamer sequence; and an aptamer binding protein, which is functionally connected to the recombinant protein as part of a fusion protein. In some embodiments, in the RT system or composition or a composition with a recruitment system, at least one aptamer sequence is an RNA aptamer sequence or a peptide aptamer sequence. In some embodiments, the RT system or composition or a composition with a recruitment system has a nucleic acid molecule or a plurality of nucleic acid molecules that additionally contain at least one RNA aptamer sequence, such as a nucleic acid molecule or a plurality of nucleic acid molecules that contain two RNA aptamer sequences; for example, wherein the two RNA aptamer sequences contain the same sequence. In some embodiments, the RT system or composition or a composition with a recruitment system has an aptamer binding protein that contains an MS2 coat protein, or a functional derivative or variant thereof; and / or the aptamer binding protein contains a bacteriophage N peptide, or a functional derivative or variant thereof; and / or at least one peptide aptamer sequence is conjugated to a Cas protein; and / or at least one peptide aptamer sequence contains 1 to 24 peptide aptamer sequences; and / or the aptamer sequences contain the same sequence. In some embodiments, the RT system or composition or composition with a recruitment system has an aptamer sequence comprising a GCN4 peptide sequence.

[0046] In some embodiments of the RT system or composition, the N-terminus of the recombinant protein is connected to the C-terminus of the aptamer binding protein; and in some embodiments, the RT system or composition further comprises a linker between the recombinant protein and the aptamer binding protein; for example, in some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:15.

[0047] In some embodiments of the RT system or composition, the system or composition includes at least one nuclear localization sequence (NLS), optionally wherein the NLS is linked to the recombinant protein or Cas protein or reverse transcriptase, or at least one NLS is on each of at least two or three of the recombinant protein, reverse transcriptase or Cas protein; for example, in some embodiments, the nuclear localization sequence comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments of the RT system or composition, the nuclear localization sequence is located at the C-terminus of the recombinant protein on the recombinant protein or Cas protein.

[0048] In some embodiments of the RT system or composition, the recombinant protein comprises a recombinant protein or an active portion thereof. In some embodiments of the RT system or composition, the recombinant protein comprises a mitochondrial recombinant protein or an active portion thereof. In some embodiments of the RT system or composition, the recombinant protein comprises a viral recombinant protein or an active portion thereof. In some embodiments of the RT system or composition, the recombinant protein comprises a eukaryotic recombinant protein or an active portion thereof. In some embodiments of the RT system or composition, the recombinant protein comprises RecE or RecT or RecE and RecT or a derivative or variant or a functional portion thereof. In some embodiments of the RT system or composition, RecE or its derivative or variant comprises an amino acid sequence having at least 70% (or any integer from 70% to 100%, e.g., at least 71%, 72%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) similarity or identity or homology to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8. In some embodiments of the RT system or composition, the fusion protein comprises RecT, or its derivative or variant. In some embodiments of the RT system or composition, RecT, or a derivative or variant thereof, comprises an amino acid sequence having at least 70% (or any integer from 70% to 100%, e.g., at least 71%, 72%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) similarity or identity or homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-14.

[0049] In some embodiments of the RT system or composition, the Cas protein is catalytically inactive (less than 5% nuclease activity compared to a wild-type or non-mutated Cas protein) or catalytically inactive. In some embodiments of the RT system or composition, the Cas protein comprises Cas9 or Cas12a. In some embodiments of the RT system or composition, the Cas9 protein comprises wild-type Streptococcus pyogenes Cas9 or wild-type Staphylococcus aureus Cas9. In some embodiments of the RT system or composition, the Cas protein comprises a nickase. In some embodiments of the RT system or composition, the nickase comprises a wild-type Streptococcus pyogenes Cas9 having an amino acid substitution D10A at position 10.

[0050] In some embodiments, the RT system or composition further comprises a donor nucleic acid.In some embodiments of the RT system or composition, the target DNA sequence is a genomic DNA sequence in a host cell.

[0051] In some embodiments of the RT system or composition, RT and recombinant protein are functionally connected to each other and constitute a fusion protein. In some embodiments of the RT system or composition, the aptamer binding protein and the recombinant protein are functionally connected to each other and constitute a fusion protein. In some embodiments of the RT system or composition, RT and Cas protein are functionally connected to each other and constitute a fusion protein. In some embodiments of the RT system or composition, the recombinant protein and Cas protein are functionally connected to each other and constitute a fusion protein. In some embodiments of the RT system or composition. In some embodiments of the RT system or composition, RT, Cas protein and recombinant protein are functionally connected to each other and constitute a fusion protein.

[0052] With regard to RT, and to linkers or ways of functionally connecting components of embodiments of RT systems or compositions (as well as with regard to linkers or ways of functionally connecting components of systems or compositions discussed herein that do not involve RT), reference is made to WO2020 / 191241, WO2020 / 191153, WO2020 / 191245, WO2020 / 191243, WO2020 / 191233, WO2020 / 191246, WO2020 / 191249, WO2020 / 191239, WO2020 / 191234, WO2020 / 191242, WO2020 / 191248, WO2020191171 and WO2021 / 226558, which relate to so-called prime editing and twin prime editing. Each of WO2020 / 191241, WO2020 / 191153, WO2020 / 191245, WO2020 / 191243, WO2020 / 191233, WO2020 / 191246, WO2020 / 191249, WO2020 / 191239, WO2020 / 191234, WO2020 / 191242, WO2020 / 191248, WO2020191171 and WO2021 / 226558 are hereby incorporated by reference. The RTs of WO2020 / 191241, WO2020 / 191153, WO2020 / 191245, WO2020 / 191243, WO2020 / 191233, WO2020 / 191246, WO2020 / 191249, WO2020 / 191239, WO2020 / 191234, WO2020 / 191242, WO2020 / 191248, WO2020191171 and WO2021 / 226558 can be used in the practice of the present invention. The linkers or functional connection methods of WO2020 / 191241, WO2020 / 191153, WO2020 / 191245, WO2020 / 191243, WO2020 / 191233, WO2020 / 191246, WO2020 / 191249, WO2020 / 191239, WO2020 / 191234, WO2020 / 191242, WO2020 / 191248, WO2020191171 and WO2021 / 226558 can be used in the practice of the present invention.

[0053] In some embodiments, the present invention relates to a cell or eukaryotic cell comprising any RT system or composition described or discussed herein. In some embodiments, the present invention relates to a method for changing a target genomic DNA sequence in a cell comprising a target genomic DNA sequence, comprising introducing any RT system or composition discussed or described herein. In some embodiments, the cell or eukaryotic cell is a mammalian cell, or in the method, the cell or eukaryotic cell is a mammalian cell; for example, a human cell; for example, a stem cell. In some embodiments, the method relates to a target genomic DNA sequence encoding a gene product. In some embodiments, the method includes introducing a cell, including administering to a subject. In some embodiments, the subject involved in the method is a mammalian non-human animal (e.g., a laboratory animal, such as a rodent, a rat, a mouse, a rabbit, or a domestic animal, such as a horse, a dog or a canine, or a cat or a feline, or a zoo animal (a non-domestic animal under human care and guardianship) or a production animal, such as a cow or a pig), or a human. In some embodiments, in the method, administration includes in vivo administration. In some embodiments, in the method, the cell or eukaryotic cell or mammalian cell is an ex vivo or in vitro cell. In some embodiments, the method comprises administering the cells ex vivo or in vitro to a subject after the introducing step; and in such embodiments, the subject is a mammalian non-human animal or a human.

[0054] In some embodiments, the invention relates to the use of a RT system or composition for altering a target DNA sequence in a cell.

[0055] In systems or compositions discussed herein that do not involve RT, aspects of the RT system that are not part of the RT or RT system (eg, linkers) can be applied to systems or compositions discussed herein that do not include RT.

[0056] The linker can be a peptide of 5-30, 10-30, 10-20 or 15 amino acid residues. The linker can be -(Gly-Gly-Gly-Gly-Ser) 2 -(SEQ ID NO:560), -(Gly-Gly-Gly-Gly-Ser) 3 -(SEQ ID NO:561) or -(Gly-Gly-Gly-Gly-Ser) 4 -(SEQ ID NO:562). In certain embodiments, the linker is -(Gly-Gly-Gly-Gly-Ser) 3 -(SEQ ID NO:561). The amino acid sequence of SEQ ID NO:561 may be encoded by the nucleic acid sequence of SEQ ID NO:563.

[0057] In certain embodiments, the linker consists primarily of unhindered amino acids, such as glycine and alanine. Exemplary linkers are polyglycine (particularly poly(Gly-Ser), poly(Gly-Ala) and polyalanine). An exemplary suitable linker as shown in the Examples below is (Gly-Ser), e.g. -(Gly-Gly-Gly-Gly-Ser) 2 -(SEQ IDNO:560)、-(Gly-Gly-Gly-Gly-Ser) 3 -(SEQ ID NO:561) or -(Gly-Gly-Gly-Gly-Ser) 4 -(SEQ ID NO:562).

[0058] The linker can also be a non-peptide linker. For example, an alkyl linker such as -NH-, -(CH2)sC(O)-, where s = 2-20, can be used. These alkyl linkers can be further substituted with any non-sterically hindering group, such as a lower alkyl group (e.g., C 1-4 ), lower acyl, halogen (e.g., Cl, Br), CN, NH 2 , phenyl, etc.

[0059] Accordingly, the goal of the present invention is not to cover any previously known product, method of preparing the product, or method of using the product within the present invention, so the applicant reserves its rights and hereby publicly abandons any previously known product, process, or method. It should also be noted that the present invention is not intended to cover any product, process, or method of preparing the product or using the product that does not meet the written description and implementability requirements of the USPTO (35 U.SC § 112, first paragraph) or the EPO (EPC Article 83) within the scope of the present invention, so the applicant reserves the right and hereby publicly abandons any previously described product, process of preparing the product, or method of using the product. In the practice of the present invention, it may be advantageous to comply with Article 53 (c) of the EPC and Articles 28 (b) and (c) of the EPC. All rights to expressly abandon any embodiment of the subject matter of any granted patent of the applicant in any prior filed application as a family of the present application or any other family or any third party are expressly reserved. Nothing herein shall be construed as a promise.

[0060] It should be noted that in this disclosure, and particularly in the claims and / or paragraphs, terms such as “comprises,” “comprised,” “comprising,” and the like may have the meanings ascribed to them in U.S. patent law; for example, they may mean “includes,” “included,” “including,” and the like; and terms such as “consisting essentially of” and “consists essentially of” have the meanings ascribed to them in U.S. patent law, for example, they allow for elements not expressly recited, but exclude elements found in the prior art or that affect the basic or novel characteristics of the invention.

[0061] These and other embodiments are disclosed or are obvious from and encompassed by the following Detailed Description.

[0062] Other aspects and embodiments of the invention will be apparent from the following detailed description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1A and Figure 1B It was reconstructed using eukaryotic recombinases from yeast and humans ( Figure 1A ) and RecT( Figure 1B )Phylogenetic tree.

[0064] Figure 2A Phylogenetic tree and length distribution of RecE / RecT homologs. Figure 2B is the metagenomic distribution of RecE / T. Figure 2C is a schematic diagram showing the central model disclosed herein. Figure 2D is a graph of the genomic knock-in efficiency of RecE / T homologs.

[0065] Figure 3A and Figure 3B It is EMX1( Figure 3A ) locus and VEGFA( Figure 3B ) plot of high-throughput sequencing (HTS) reads for homology-directed repair (HDR) at the locus. Figure 3C-Figure 3E is HSP90AA1 in HEK293T cells ( Figure 3C )、DYNLT1( Figure 3D ) and AAVS1( Figure 3E ) Plot of mKate knock-in efficiency at the locus. Figure 3F is an image of mKate knock-in efficiency in HEK293T cells using RecT. Figure 3Gis a schematic diagram of an exemplary AAVS1 knock-in strategy and chromatographic traces from the RecT knock-in panel. Figure 3H Schematic diagram and graph of recruitment control experiment and corresponding knock-in efficiency. All results are normalized to NR. (NC, no cleavage; NR, no recombinator).

[0066] Figure 4A-4C is HSP90AA1 in HEK293T cells ( Figure 4A )、DYNLT1( Figure 4B ) and AAVS1( Figure 4C ) Plot of relative mKate knock-in efficiency at the locus relative to the NE group. (NC, no cleavage control group. NR, no recombinant control group.) Figure 4D is an image of an exemplary agarose gel of junction PCR validating mKate knock-in at the AAVS1 locus. Figure 4E and Figure 4F is the absolute LOV knock-in efficiency at the AAVS1 locus ( Figure 4E ) and relative LOV knock-in efficiency ( Figure 4F 4G is the Sanger sequencing result of the connected PCR product of the exemplary mKate knock-in at the AAVS1 locus.

[0067] Figure 5A-5D The cell line is A549 ( Figure 5A )、HepG2( Figure 5B )、HeLa( Figure 5C ) and hESC(H9)( Figure 5D ) plots the genomic knock-in efficiency at different loci. Figure 5E is an image of mKate knock-in in hESC. Fig. 5F and Figure 5G is the genome-wide off-target site (OTS) count of the REDITv1 tool ( Fig. 5F ) and OTS chromosome distribution ( Figure 5G ).

[0068] Figure 6A-6D The AAVS1 locus and the DYNT1 locus in the A549 cell line ( Fig. 6A ), DYNLT1 locus and HSP90AA1 locus in HepG2 cell line ( Figure 6B ), DYNLT1 locus and HSP90AA1 locus in Hela cell line ( Figure 6C ) and the HSP90AA1 locus and OCT4 locus in the hES-H9 cell line ( Fig.6D) relative mKate knock-in efficiency at . (NC, no cleavage control group. NR, no recombinant control group. All data are normalized to the NR group.) Fig. 6E Representative FACS results of HSP90AA1 mKate knock-in in hES-H9 cells.

[0069] Figure 7A-7D is DYNLT1( Fig. 7A ) and HSP90AA1( Figure 7B ) locus and the different homology arm lengths at DYNLT1( Figure 7C ) and HSP90AA1( Fig.7D ) is a graph of the absolute mKate knock-in efficiency of the no recombinant control.

[0070] Figure 8A-8E It is the same as sgEMX1 in REDITv1 system ( Figure 8A-8C ) or sgVEGFA( Figure 8D-Figure 8E )A plot of the insertion / deletion (indel) rates of the top 3 predicted off-target loci associated with .

[0071] Fig. 9A Schematic diagram of a preferred embodiment of REDITv2N and the corresponding knock-in efficiency in HEK293T cells. Fig. 9B and Fig. 9C Comparison of genome-wide off-target site (OTS) counts between REDITv2N and REDITv1 ( Fig. 9B ) and OTS chromosome distribution ( Fig. 9C )’s picture. Fig.9D Schematic diagram of a preferred embodiment of REDITv2D and the corresponding knock-in efficiency. Fig.9E is a graph of the editing efficiency of REDITv1, REDITv2N, and REDITv2D under serum starvation conditions. Fig.9F is the knock-in efficiency of REDITv3 in hESCs. Figure 9G Here is an image of mKate knock-in in hESCs using REDITv3.

[0072] Fig. 10A and Fig. 10B is REDITv2N( Fig. 10A ) and REDITv2D( Fig. 10B ) schematic diagram and graph of the relative mKate knock-in efficiency at the DYNLT1 locus and the HSP90AA1 locus of a preferred embodiment.

[0073] Figures 11A-11Dis an image of an agarose gel showing ligation PCR of the preferred REDITv2N system for mKate knock-in at the DYNLT1 locus and the HSP90AA1 locus. Figure 1 IE is a chromatogram sequence of ligation PCR products at the DYNLT1 locus.

[0074] Fig. 12A and Fig. 12B is REDITv2( Fig. 12A ) and REDITv2N( Fig. 12B ) of the preferred embodiment of the present invention. Stacking (pileup) includes alignments with two or more reads overlapping each other. Flanking pairs (flanking pair) include alignments that appear on opposite strands within 200bp upstream of each other. Target matching (targetmatched) includes alignments matched to the target processed in the upstream sequence (up to 6 mismatches are allowed in the target sequence, including 1 mismatch in the PAM). Fig. 12C is a graph of HTS HDR and indel reads at the EMX1 locus of the REDITv2N system.

[0075] Fig.13A is an image of an agarose gel showing ligation PCR of mKate knock-in at the DYNLT1 locus by the REDITv2D system. Fig. 13B is the chromatogram sequence of the ligated PCR products at the DYNLT1 locus.

[0076] Figure 14A-Figure 14C When treated with different FBS concentrations, REDITv2( Fig.14A )、REDITv2N( Fig. 14B ) and REVITv2D( Fig. 14C ) A graph of mKate knock-in efficiency at the HSP90AA1 locus. Figure 14A-Figure 14C When treated with different serum FBS concentrations, REDITv2( Fig.14D )、REDITv2N( Fig.14E ) and REVITv2D( Fig.14F ) A graph of mKate knock-in efficiency at the HSP90AA1 locus.

[0077] Fig.15 This is an image of nuclear localization of RecE_587 and RecT after EGFP was fused to the REDITv1 system. The nuclei were stained with NucBlue Live Ready Probes Reagent.

[0078] Fig.16A and Fig. 16Bis the relative mKate knock-in efficiency at the HSP90AA1 and DYNLT1 loci after fusion of different nuclear localization sequences to the N-terminus or C-terminus of RecT and RecE_587. Fig. 16C and Fig.16D Is from Fig.16A and Fig. 16B The constructs for DYNLT1 locus ( Fig. 16C ) and HSP90AA1 locus ( Fig.16D ) plot of the absolute mKate knock-in efficiency of the samples.

[0079] Figures 17A-17D The new NLS sequence and the optimal linker were fused to the REDITv2 and REDITv3 variants to the DYNLT1 locus ( Fig.17A and Fig. 17C ) and HSP90AA1 locus ( Fig. 17B and Fig.17D )'s relative ( Fig.17A and Fig. 17B ) and absolute ( Fig. 17C and Fig.17D )A plot of mKate knock-in efficiency. The REDITv2 versions using REDITv2N (D10A or H840A) and REDITv2D (dCas9) are shown on the horizontal axis, along with the number of guides used. Different colors represent different controls and REDIT versions.

[0080] Fig.18 is a graph of the relative editing efficiency of the REDITv3N system at the HSP90AA1 locus in hES-H9 cells.

[0081] Fig.19A is a schematic representation of an exemplary saCas9 expression vector. Figure 19B-Figure 19E The different effectors in the saCas9 system are at the AAVS1 locus ( Fig.19B ) and HSP90AA1 locus ( Fig.19D ) and their absolute efficiencies (respectively Fig.19D and Fig.19E ). (NC, non-cleavage control group. NR, no recombinant control group.

[0082] Fig. 20A is a schematic diagram of RecT truncation. Fig. 20B and Fig. 20C is a graph of the relative mKate knock-in efficiency of wild-type S. pyogenes Cas9 and S. pyogenes Cas9n(D10A) with single and double nicking at the DYNLT1 locus.

[0083] Fig.21AIt is a schematic diagram of the truncation of RecE_587. Fig. 21B and Fig. 21C is a graph of the relative mKate knock-in efficiency of wild-type S. pyogenes Cas9 and S. pyogenes Cas9n(D10A) with single and double nicking at the DYNLT1 locus.

[0084] Fig.22A and Fig. 22B is a combination of various exonucleases from naturally occurring recombineering systems ( Fig.22A ) and single-stranded DNA annealing protein (SSAP) ( Fig. 22B ) Comparison of the efficiency of recombineering-based editing, including NR (no recombinant) as a negative control. Gene editing activity was measured using mKate knock-in assays at genomic loci (DYNLT1 and HSP90AA1). Data shown are the percentages of successful mKate knock-ins using human HEK293 cells, and each experiment was performed in triplicate (n=3).

[0085] Figure 23A-23E A compact recruitment system using boxB and N22 is shown. The REDIT recombinase protein is fused to the N22 peptide, and within the sgRNA is a short recognition sequence for the N22 peptide, boxB ( Fig.23A ). Figure 23B-Figure 23E Graph of gene editing efficiency of the mKate knock-in assay using wild-type SpCas9 and side-by-side comparison with the MS2-MCP recruitment system. Fig. 23B and Fig.23D is the absolute mKate knock-in efficiency at the DYNLT1, HSP90AA1 loci, and Fig.23C and Fig.23E is the relative efficiency. Data shown are the percentages of successful mKate knock-in using HEK293 human cells, and each experiment was performed in triplicate (n=3).

[0086] Figure 24A-24C The SunTag recruitment system is shown. The REDIT recombinant protein is fused to the scFV antibody, and the GCN4 peptide is fused to the Cas9 protein in a tandem manner (10 copies of the GCN4 peptide separated by a linker) ( Fig.24A ). Using mKate knock-in experiments at the DYNLT1 locus ( Fig. 24B ) to measure gene editing knock-in efficiency ( Fig.24C). All data are measured using wild-type SpCas9 mKate knock-in assay to measure gene editing efficiency. The absolute mKate knock-in efficiency at DYNLT1 is shown in the lower right corner of each flow cytometry graph, where the control is no recombinant (NR), which includes scFV fused to GFP protein as a negative control, and all experiments were performed in HEK293 human cells.

[0087] Fig.25A and Fig.25B REDIT with Cas12A system is illustrated. For two different Cpf1 / Cas12a proteins, the Cpf1 / Cas12a-based REDIT system was created by SunTag recruitment design ( Fig.25A Using the mKate knock-in assay, the efficiency at two endogenous loci (DYNLT1 and AAS1) was measured ( Fig.25B ). Shown are the absolute mKate knock-in efficiencies measured by the percentage of mKate+ cells using HEK293 human cells, with each experiment performed in triplicate (n=3) with no recombinant (NR) as the negative control.

[0088] Fig.26A and Fig.26B is to use RecE and RecT homologs at the DYNLT1 locus ( Fig.26A ) and HSP90AA1 locus ( Fig.26B ) was measured by mKate knock-in gene editing assay to measure precise recombineering activity. Absolute mKate knock-in efficiency measured by mKate+ cell percentage using HEK293 human cells is shown, and each experiment was repeated three times (n=3), with negative controls of no recombinant (NR) and no cleavage (NC). Original RecE and RecT from E. coli were also included as positive controls.

[0089] Fig.27A and Fig.27B is a schematic diagram showing SunTag-based recruitment of SSAP RecT to the Cas9-gRNA complex for gene editing ( Fig.27A ) and a graph quantifying SunTag editing efficiency compared to MS2-based strategies ( Fig.27B ).

[0090] Figure 28A-28C A comparison of REDIT with an alternative HDR-enhanced gene editing approach is shown. Fig.28A Schematic diagram showing alternative HDR enhancement methods by fusing the functional domains CtIP or Geminin (Gem) to the Cas9 protein (left) and in combination with REDIT (right). Fig.28BAn alternative small molecule HDR enhancement method is through cell cycle control. Nocodazole was used to synchronize cells at the G2 / M boundary (left) according to the timeline shown (right). Fig.28C Comparison of gene editing efficiency using REDIT and the optional HDR enhancement tools Cas9-HE (CtIP fusion), Cas9-Gem (synaptin fusion) and nocodazole (noc), and REDIT in combination with these methods (Cas9-HE / Cas9-Gem / noc+REDIT). Donor DNA had HA of 200+400bp (DYNLT1) or 200+200bp (HSP90AA1). All assays were performed with no donor, NTC and Cas9 (no enhancement) as controls. #P<0.05 vs. REDIT; ##P<0.01 vs. REDIT.

[0091] Figure 29A-Figure 29D The template design guidance, ligation precision, and capacity of the REDIT gene editing method are demonstrated. Fig.29A Figure 1 is a graph of homology arm (HA) length testing of different template designs using REDIT and Cas9 reference comparison HDR donors (longer HA) or NHEJ / MMEJ donors (zero / shorter HA). The top and bottom graphs are two genomic loci tested using the mKate knock-in assay. Fig.29B is the design of an exemplary junction analysis assay, by isolating knock-in clones followed by genomic PCR using primers (fwd, rev) that bind outside the donor to avoid template amplification. Paired Sanger sequencing of PCR products revealed homologous and nonhomologous edits at 5'-junctions and 3'-junctions. Fig.29C Yes Fig.29B Graph showing the percentage of clones with the indicated junction profiles after Sanger sequencing of knock-in clones in. Editing methods and donor DNA (HA length indicated in parentheses) are listed below. Fig.29D Figure 2 is a graph showing the insertion of a dual GFP / mKate tag using a 2-kb cassette to verify the knock-in efficiency of the REDIT method using Cas9. The HA length of the donor DNA is shown below.

[0092] Figure 30A-Figure 30C The GISseq results are shown ( Figure 6C-6E ), indicating that REDIT is an effective method capable of inserting kilobase-length sequences with few undesired editing events. Fig. 30A Schematic diagram showing the design, procedure and analysis steps for GIS-seq to measure genome-wide insertion sites of knock-in cassettes. High molecular weight (HMW) genomic DNA purification is required to remove potential contamination from donor DNA. Donor DNA has 200 bp HA on each side. Fig. 30B is a representative GIS-seq result showing positive / negative reads at the on-target locus DYNLT1. The expected 2A-mKate knock-in site before the stop codon of the last exon is the center of the trimmed reads (the reads were trimmed to remove the 2A-mKate box). Template mutations that are helpful for avoiding gRNA targeting and distinguishing genomic and edited reads are marked. Fig. 30C Summary of the top GIS-seq insertion sites, comparing the Cas9dn and REDITdn groups, showing the expected on-target insertion sites (highlighted) and the reduced number of off-target insertion sites identified when using REDITdn. MLEs were calculated based on the distribution of filtered and trimmed GIS-seq reads for the (left) DYNLT1 and (right) ACTB loci.

[0093] Figure 31A-Figure 31F We show the dependence of REDIT gene editing on endogenous DNA repair and apply the REDIT approach to human stem cell engineering. Fig.31A Model showing the editing process and major repair pathways involved in gene editing using REDIT or Cas9, highlighting the HDR pathway under chemical interference (inhibition of RAD51). Donor DNA with 200+200 bp HA was used for all inhibitor experiments. Fig.31B and Fig.31C It is shown that for REDIT and Cas9 based on wtCas9 ( Fig.31B ) and for REDITdn and Cas9dn based on Cas9 nickase ( Fig.31C ), Plot of the relative knock-in efficiency of the REDIT tool compared to Cas9 reference treated with RAD51 inhibitor B02 and RI-1 or vehicle treated. All conditions were measured at two genomic loci (DYNLT1 and HSP90AA1) using a 1-kb knock-in assay. Fig.31D is a graph of knock-in efficiency in hESCs (H9) using REDIT and REDITdn tested at three genomic loci compared to the corresponding Cas9 and Cas9dn references. Fig.31E and 31F Figure 2 is a flow cytometry plot of mKate knock-in results in hESCs using REDIT, REDITdn with Cas9, Cas9dn, and NTC controls. Donor DNA in the hESC experiments had 200+200 bp HA at all loci tested.

[0094] Figure 32A-32B Chemical interference with dCas9 REDIT is shown. Fig.32A ) and no ( Fig.32B) The gene editing efficiency was determined when the cells were treated with mammalian DNA repair pathway inhibitors (Mirin, RI-1 and B02) under the condition of cell cycle inhibitor (Thy, double thymidine) blocking. Statistical analysis was performed by t-test results using 1% FDR by two-stage step-up method.

[0095] Fig.33A and Fig.33B Schematic diagrams of DNA components (gene editing vector and template DNA) and mouse tail vein injection.

[0096] Figure 34A-Figure 34C It is the result of tail vein injection of gene editing vector into mice. Fig.34A Schematic diagram and gel electrophoresis of PCR analysis of liver hepatocytes from injected mice. Fig.34B is the result of Sanger sequencing of PCR amplicons. Fig.34C It is a schematic diagram of the second-generation sequencing and a quantitative graph of knock-in ligation errors.

[0097] Fig.35A and Fig.35B Schematic diagram of DNA components (gene editing and control vectors) and adeno-associated virus (AAV) processing, respectively. Fig.35C Figure 2 is a fluorescent image of the lungs of AAV-treated mice and the corresponding quantitative graphs of tumor numbers.

[0098] 36A-36C show the predicted structures of E. coli RecT (EcRecT) alone ( FIG. 36A ) and with bound single-stranded DNA ( FIG. 36B , 36C ).

[0099] Figure 37A-Figure 37B The predicted interactions of EcRecT SSAP amino acids with ssDNA are shown.

[0100] Figure 38A- Fig.38F The development of dCas9 gene editors by mining microbial SSAPs is shown. (Figure 38A) Schematic diagram of dCas9 editors with single-stranded annealing proteins (SSAPs). (Figure 38B) Design of genomic knock-in assays for measuring gene editing efficiency (left); Workflow of SSAP screening experiments (right). ( Fig.38C ) Construct design for screening gene editing efficiency of SSAP with 800 bp transgene using 2A-mKate knock-in assay. Fig.38D ) Initial screening results of three SSAPs: Bet protein from λ phage (LBet), RecT protein from Rac prophage (RacRecT), and gp2.5 from T7 phage (T7gp2.5). ( Fig.38E ) RecT-like SSAP candidates were screened by metagenomic homolog mining and knock-in assays. The most active candidates were labeled as dCas9-SSAP. NTC: non-target control. In addition to the no-donor control, donor templates were added to all groups, and their homology arm (HA) lengths were: DYNLT1, 200+200bp; HSP90AA1, 200+400bp; ACTB, 200+400bp. ( Fig.38F ) Using three types of donor designs with different HA lengths, gene editing efficiency was measured at the DYNLT1 (left) and HSP90AA1 (right) loci in HEK293T cells. Unless otherwise stated, all results in this figure and the following figure are from repeated experiments, error bars represent standard error of the mean (SEM), n = 3.

[0101] Figures 39A-39H show on-target and off-target editing errors of dCas9-SSAP. (Figure 39A) Deep sequencing to measure the level of indel formation at the endogenous target when using dCas9-SSAP and Cas9 reference. The donor template used was the 200bp-HA HDR template. The assay details are described in the methods. (Figure 39B) Clonal Sanger sequencing to analyze the accuracy of knock-in editing with different HDR and MMEJ donors using dCas9-SSAP and Cas9 reference. The donor templates used were the 200bp-HAHDR template and the 25bp-HAMMEJ template (Methods and Supplementary Notes). ( Fig.39C - Figure 39E) Genome-wide detection of the insertion site of the knock-in cassette using unbiased sequencing shows ( Fig.39C ) workflow, (Figure 39D) representative reads aligned at the knock-in genomic site, and (e) summary of detected on-target and off-target insertion sites. (Figure 39F-Figure 39G) Workflow and results for measuring cell fitness effects, which are defined by the percentage of live cells after editing (normalized to mock control). (Figure 39H) Summary analysis of knock-in accuracy of dCas9-SSAP editors compared to Cas9 HDR and Cas9 MMEJ methods. Accuracy is defined as the total yield (%) of correct knock-ins within all edited results (correct knock-ins, knock-ins with indels, and NHEJ indels).

[0102] Fig.40 A- Figure 40G Validation of the dCas9-SSAP editor and comparison with the Cas9 reference and other HDR enhancement methods are shown. Fig.40A) Comparison of efficiency using dCas9-SSAP and other alternative Cas9, nCas9, and HDR enhancement tools. Cas9-HE (CtIP-fused Cas9), Cas9-Gem (synaptin-fused Cas9), nCas9 (Cas9-D10A nickase reference), and nCas9-hRAD51 (improved Cas9 nickase editor). The donor template is the same as in Figure 1. ( Fig.40B ) Imaging validation of mKate knock-in at an endogenous genomic locus using the dCas9-SSAP editor. Fig.40 C) Design of knock-in donors with different lengths of transgenes. Fig.40D ) Knock-in efficiency of different transgene lengths using dCas9-SSAP editor. The donor HA length of DYNLT1 is 200bp+200bp, and the donor HA length of HSP90AA1 is 200bp+400bp. ( Fig.40E ) Performance of dCas9-SSAP editors at seven endogenous loci in HEK293T cells compared to Cas9 reference. ND, no donor control; NT, non-target control. ( Figure 40F-40G ) Gene knock-in editing in human embryonic stem cells (hESC, H9) using the dCas9-SSAP editor, including quantification of HDR efficiency ( Fig.40F ) and flow cytometry analysis ( Figure 40G All statistical analyses used multiple t-tests to compare all genomic targets, performed by the two-stage step-up method of Benjamini, Krieger, and Yekutieli, at a false discovery rate (FDR) of 1%.

[0103] Figure 41A-Figure 41D shows chemical interference for detecting the editing mechanism of dCas9-SSAP editor. When treated with DNA repair pathway inhibitors (Mirin, RI1 and B02), the gene editing efficiency of dCas9-SSAP editor was not used (Figure 41A, Figure 41B) or treated with (Figure 41C, Figure 41D) cell cycle synchronization (DTB, double thymidine blocking). All donor templates are the same as those in Figure 38. Statistical analysis is from the t-test results performed using 1% FDR by the two-stage ascending method of Benjamini, Krieger and Yekutieli.

[0104] Fig.42A - Figure 42D shows the minimization of the dCas9-SSAP editor as a compact CRISPR knock-in tool for easy delivery. ( Fig.42A ) Schematic diagram showing the predicted secondary structure and priming site of EcRecT used to construct a truncated EcRecT protein based on structure prediction. ( Fig.42B) Relative knock-in efficiency of various truncation designs. All groups were normalized to the Cas9 reference (performed separately for each target). (Figure 42C) Schematic diagram of the dSaCas9-mSSAP system in an AAV construct using compact SaCas9 (left, element sizes not shown to scale), and (Figure 42D) Knock-in efficiency at AAVS1 and HSP90AA1 endogenous targets in HEK293T cells by in vitro delivery of AAV2 vectors carrying the original and minimized dSaCas9-SSAP editors.

[0105] Figure 43A-Figure 43E shows gel electrophoresis and sequencing verification of knock-in specific PCR products using dCas9-SSAP. (Figure 43A) Agarose gel results of knock-in specific connection PCR at DYNLT1 locus. (Figure 43B-Figure 43E) Sanger sequencing chromatograms of genome connections from knock-in experiments at DYNLT1 locus. For all samples, the applicant uses the 5' end (Figure 43B, Figure 43C) and 3' end (Figure 43D, Figure 43E) of the cross-connection (junction-spanning) primer amplification genomic DNA outside the donor DNA to confirm the knock-in.

[0106] Fig.44 Phylogenetic trees and amino acid alignments of representative RecT homologs are shown along with annotated conserved domains of the proteins.

[0107] Fig.45 A- Fig.45 B shows deep sequencing comparing short sequence editing by dCas9-SSAP and Cas9 editors. ( Fig.45 A) Donor design for a 16-bp substitution at EMX1. Fig.45 B) Analysis of accurate HDR and indel editing results at the EMX1 genomic locus using deep sequencing. The first round of PCR used sequencing primers completely outside the donor to ensure that the sequencing results were not contaminated by the donor template, which was verified by a non-target control in which donor DNA was delivered to the cells.

[0108] Figures 46A-46B are schematic diagrams showing the workflow used in Sanger sequencing of knock-in products (Figure 46A) and the sequencing method used in the deep on-target indel assay (Figure 46B). The assay described here corresponds to Figure 41. gPCR, genomic PCR. Seq-F / seq-R are primers used for Sanger sequencing, binding upstream / downstream of the knock-in template.

[0109] Figure 47A-47BShown is a Sanger sequencing chromatogram of a genomic junction from a dCas9-SSAP experiment at the DYNLT1 locus. Sequences in the red box were not accurately repaired. For all samples, the 5' end of the genomic DNA was amplified using a cross-junction primer ( Fig.47A ) and the 3' end ( Fig.47B ) to confirm knock-in accuracy. The genomic binding primers used were completely outside the donor DNA to avoid contamination.

[0110] Figure 48A-Figure 48B Shown is a Sanger sequencing chromatogram of a genomic junction from a dCas9-SSAP experiment at the HSP90AA1 locus. For all samples, the 5' end of the genomic DNA was amplified using a cross-junction primer ( Fig.48A ) and the 3' end ( Fig.48B ) to confirm knock-in accuracy. The genome binding primers used were completely outside the donor DNA to avoid contamination.

[0111] Figure 49A-Figure 49B shows genome-wide insertion site mapping and quantification. (Figure 49A) Overall workflow of the unbiased genome-wide insertion site mapping process. On-target and off-target insertion sites are recovered from reads aligned with the reference genome (hg38). The complete protocol and data analysis process are detailed in the method. (Figure 49B) Quantification of genome-wide insertion sites counting all aligned reads (with valid UMIs) shows that the abundance of insertion sites on two genomic loci (DYNLT1 and HSP90AA1) using Cas9-SSAP is reduced compared to Cas9HDR. The abundance of insertion sites is measured in RPKU or reads per thousand UMIs.

[0112] Figures 50A-50B show testing of the dCas9-SSAP Editor tool using single guide (Figure 50A) and dual guide (Figure 50B) designs at three genomic targets (shown in the upper panel). The donor DNA used was the same as that shown in Figure 3a, using an 800-bp knock-in design.

[0113] Figures 51A-51C show the validation of dCas9-SSAP knock-in efficiency in three additional cell lines, HepG2 (Figure 51A), HeLa (Figure 51B), and U2OS (Figure 51C). For all cell lines tested, the knock-in experiments used similar donor DNA with an 800-bp cassette encoding the 2A-mKate transgene.

[0114] Figure 52A-Figure 52C shows a full set of flow cytometry analysis data for human stem cell engineering using the dCas9-SSAP editor. Flow cytometry analysis of knock-in gene editing at the endogenous locus of HSP90AA1 (Figure 52A), ACTB (Figure 52B), OCT4 (Figure 52C) in human embryonic stem cells (hESC, H9) using dCas9-SSAP compared to non-target controls and Cas9 (Cas9 HDR) references.

[0115] Fig.53 is a schematic diagram showing the secondary structure of the RecT protein predicted using an online tool (CFSSP, see Methods). The prediction results (upper panel showing secondary structure, lower panel showing alignment) formed the basis for the development of truncated functional RecT variants.

[0116] Figure 54A- Fig.54C Optimization of dCas9-SSAP is shown for efficient and durable gene editing. (FIG. 54A) Knock-in efficiency of SSAP dose optimization. Donor HA length was ~200bp for DYNLT1 and ~300bp for HSP90AA1. n=3 biologically independent experiments. (FIG. 54B) Performance of dCas9-SSAP editors in 7 endogenous loci in HEK293T cells compared to Cas9 reference after SSAP dose optimization and donor HA extension. For DYNLT1, the donor HA length was ~200bp, for HSP90AA1, the donor HA length was 673bp+750bp, for ACTB, the donor HA length was 500bp+800bp, for BCAP31, the donor HA length was 608bp+740bp, for HIST1H2BK, the donor HA length was 212bp+413bp, for CLTA, the donor HA length was 705bp+602bp, and for RAB11A, the donor HA length was 464bp+440bp. All knock-in donors targeted the C-terminus of the endogenous protein, except for the CLTA / RAB11A donors that targeted the N-terminus. n=3 biologically independent experiments. ( Fig.54C ) Stability of transgene expression at HSP90AA1 (left) and ACTB (right) after sorting on day 3 after dCas9-SSAP knock-in. Variable sorting efficiency leads to different starting mKate+ rates (Figure 57). Error bars in a and b represent standard error of the mean (SEM).

[0117] Fig.55A - Figure 55C shows the optimization of donor dose and donor DNA homology arms. ( Fig.55A) When using the dCas9-SSAP editor, the genomic mKate knock-in efficiency at the DYNLT1, HSP90AA1, and ACTB loci was quantified for donor dose optimization. Non-target, non-target control. For DYNLT1, the donor HA length was 200bp+200bp, for HSP90AA1, the donor HA length was 200bp+400bp, and for ACTB, the donor HA length was 200bp+400bp. When using the dCas9-SSAP editor, the mKate knock-in efficiency at the HSP90AA1 (Figure 55B) and ACTB (Figure 55C) loci was quantified for donor homology arm (HA) optimization. Non-target, non-target control. For HSP90AA1, the donor HA length was 200bp+200bp or 673bp+750bp, and for ACTB, the donor HA length was 200bp+400bp or 500bp+800bp. n=3 biologically independent experiments. All results in this figure are from repeated experiments, and the error bars represent the standard error of the mean (SEM).

[0118] Fig.56A - Figure 56D shows validation of the dCas9-SSAP editor using a protein functional assay. ( Fig.56A ) Design of genomic puromycin / blasticidin resistance cassette knock-in assay to validate functional on-target editing of dCas9-SSAP. Fig.56B ) Immunoblotting confirmed the presence and size of the on-target dCas9-SSAP knock-in products at the HSP90AA1 and ACTB loci, using an anti-V5 antibody that recognizes in-frame fusions with endogenous proteins. The data shown represent 3 biologically independent experiments. (Figure 56C-Figure 56D) dCas9-SSAP was used to verify and quantify the on-target knock-in by colony formation assay. Cells were selected by knock-in resistance box and then quantified using Crystal Violet staining. Scale bar = 500um. n = 4 biologically independent experiments. Error bars represent standard error of the mean (SEM).

[0119] Fig.57A - Figure 57E shows the validation of on-target editing stability. ( Fig.57A )Workflow for long-term time-course experiments to evaluate the stability of editing outputs using the dCas9-SSAP editor. Fig.57B) At different time points after delivery of dCas9-SSAP and donor DNA, flow cytometry analysis was performed on knock-in gene editing at the endogenous loci of HSP90AA1 and ACTB. (Figure 57C-Figure 57D) Representative crystal violet staining images of on-target puromycin knock-in at HSP90AA1 and ACTB loci. Scale bar = 500um. This assay has been performed 3 times with similar results. (Figure 57E) Quantification of HSP90AA1 and ACTB gene expression levels in HEK293T cells by bulk RNA-seq analysis showed that HSP90AA1 expression levels were significantly higher. This resulted in better cell survival in the HSP90AA1 group compared to the ACTB group. Data from 2 biological independent experiments are shown.

[0120] Fig.58 SSAP+Cas9-mediated knock-in editing using deactivated guide RNA (dgRNA) is shown. SSAP+Cas9 contains RecT and wtCas9. mKate knock-in at DYNLT1, HSP90AA1, and ACTB is depicted.

[0121] Fig.59 Shown is a dCas9-SSAP-mediated knock-in of a 600-bp transgene expressing luciferase or mKate at the human albumin (ALB) locus (upper panel) or AAVS1 locus (lower panel) in human HEK293T cells or human hepatocytes. The transgenic knock-in at the albumin locus is highly expressed in hepatocytes but not in HEK293T (upper panel). The transgenic knock-in at the AAVS1 locus is highly expressed in HEK293T but not in hepatocytes (lower panel).

[0122] Fig.60 Shown is the knock-in of a 800-bp transgene expressing luciferase or mKate mediated by dCas9-SSAP at the human albumin (ALB) locus (upper panel) or AAVS1 locus (lower panel) in human HEK293T cells or human hepatocytes. The transgenic knock-in at the albumin locus is highly expressed in hepatocytes but not in HEK293T (upper panel). The transgenic knock-in at the AAVS1 locus is highly expressed in HEK293T but not in hepatocytes (lower panel).

[0123] Fig.61Electroporation of RNPs containing 800 bp mKate encoding transgene in K562 cells is shown. Cells were electroporated with RNPs containing purified Cas9 or dCas9 protein complexed with guide RNA (double-stranded 800 bp mKate transgene) (with or without RecT). Knock-ins were located at the HSP90AA1 (left) or HIST1H2BK (right) loci.

[0124] Fig.62 Shown is the delivery of RNPs containing Cas9 or dCAS9 (with or without SSAP) to mouse primary hematopoietic stem cells (HSCs) and delivery of AAV6 to knock in a transgene expressing GFP.

[0125] Fig.63 Transgene expression is shown.

[0126] Figures 64A-64B depict SSAP-mediated transgene knock-in using R-loop-forming guides without CRISPR elements. (Figure 64A) Model of guide RNA-SSAP-mediated gene editing showing MCP-MS2 aptamer paired with SSAP and R-loop-gRNA. (Figure 64B) Vector / plasmid design for expression of guide RNA, dsDNA donor, and SSAP. ( Fig.64C ) Original FACS plot showing identification of subset expressing mCherry. Fig.64D ) Transgene knock-in at the ACTB locus using different guide lengths (18nt, 20nt, 25nt) in the presence or absence of RecT.

[0127] Figure 65A-Figure 65B shows R-loop-guide RNA design. R-loop-guide RNA comprises two components, guide and backbone, represented by guide-backbone and backbone-guide configurations (e.g., guide at the 5' or 3' end of the backbone). The guide sequence is designed to match the target DNA. One or more aptamers may be incorporated, including but not limited to MS2, PP7, and BoxB. MS2 is depicted.

[0128] Fig.66 Chimeric guide RNAs containing the MS2 / PP7-aptamer are shown.

[0129] Fig.67 The effect of varying guide length on knock-in efficiency at the ACTB locus is shown, comparing R-loop-SSAP (no CRISPR), Cas9 HDR, and dCas9-SSAP. Donor-only includes only the mKate knock-in donor.

[0130] Fig.68The effect of varying guide length on knock-in efficiency at the HIST locus is shown, comparing R-loop-SSAP (no CRISPR), Cas9 HDR, and dCas9-SSAP. Donors only include only mKate knock-in donors.

[0131] Fig.69 The effect of varying guide length on knock-in efficiency at the HSP90AA1 locus is shown, comparing R-loop-SSAP (no CRISPR), Cas9 HDR, and dCas9-SSAP. Donors only include only mKate knock-in donors.

[0132] Fig.70 Shown is the knock-in of a 600-bp transgene expressing luciferase or mKate mediated by R-loop-SSAP at the human albumin (ALB) locus (upper panel) or AAVS1 locus (lower panel) in human HEK293T cells or human hepatocytes. The transgenic knock-in at the albumin locus is highly expressed in hepatocytes but not in HEK293T (upper panel). The transgenic knock-in at the AAVS1 locus is highly expressed in HEK293T but not in hepatocytes (lower panel).

[0133] Fig.71 Shown is the knock-in of an 800-bp transgene expressing luciferase or mKate mediated by R-loop-SSAP at the human albumin (ALB) locus (upper panel) or AAVS1 locus (lower panel) in human HEK293T cells or human hepatocytes. The transgenic knock-in at the albumin locus is highly expressed in hepatocytes but not in HEK293T (upper panel). The transgenic knock-in at the AAVS1 locus is highly expressed in HEK293T but not in hepatocytes (lower panel).

[0134] Fig.72 A schematic diagram comparing RNA-mediated SSAP editing without reverse transcriptase (top) and RNA-mediated SSAP editing with reverse transcriptase (bottom) is shown. The RNA template / donor shown includes a homology arm (HA) region with one HA. In some embodiments, the RNA template / donor includes two HA regions, one at each end. The HA matches the genomic region next to the editing site, so SSAP can promote editing.

[0135] Fig.73Insertion rates of 4 bp sequences inserted at the human EMX locus using in vitro transcribed (IVT) RNA templates are shown. System components are 1. gE3 or sg2 or sgHE SpCas9 guide RNAs targeting human EMX1; 2. dg2 of dg3 inactive / inactivated guide RNAs binding to the gE3 / sg2 / sgHE vicinity; and 3. 100 / 200 HA: 100 bp or 200 bp HA regions at both ends next to the 4 bp edit.

[0136] Fig.74 The RNA template expressing U6 in the sense or antisense direction for replacing the 16bp sequence at human EMX1 (introducing 16bp editing) is shown. The system components include 1. SpCas9 guide RNA targeting human EMX1; 2. Inactive / inactivated guide RNA bound to this region. The numbers at the top of each lane of the gel indicate the length of the homology arm (HA) region next to the editing at both ends.

[0137] Fig.75 The dosage relationship of RNA template expressing U6 in sense or antisense direction for replacing a 16 bp sequence at human EMX1 (introducing a 16 bp edit) is shown. The system components include 1. SpCas9 guide RNA targeting human EMX1; 2. Inactive / deactivated guide RNA binding to the region.

[0138] Fig.76 The system of the present invention inserted at the human AAVS1 locus is shown ( Fig.76 A) and repair of the defective Venus (green fluorescent protein) locus ( Fig.76 B).

[0139] Fig.77 Schematic diagram of sgRNA+dgRNA system (top) and example based on TLR locus (bottom) are shown. SpCas9 guide_20bp sgRNA: 20bp guide used in the first guide RNA for sgRNA targeting TLR. SaCas9 guide design is also shown. dg1-dg6: Different 15bp / 16bp inactive / deactivated guides in dgRNA targeting TLR, used in the second guide RNA, together with aptamer for recruitment.

[0140] Fig.78A demonstration of the sgRNA+dgRNA system is shown, including the signal obtained in the GFP (green) channel, indicating repair by the Venus protein. pA19 encodes Cas9 and guide RNAs with sg318 / sg530, which are two guides targeting the TLR gene editing reporter genomic region. BB is the backbone, serving as a negative control. Dg532 / 534 / 536 / 538 are different inactive guide RNAs containing MS2 / PP7 aptamers. The red box indicates the design of the RNA repair template / donor that works best using Venus at the 3' end of the RNA.

[0141] Fig.79 A schematic diagram of a sgRNA+dgRNA system in which the RNA template / donor is directly fused to the dgRNA is shown. In certain embodiments, one or both of the sgRNA and the dgRNA can be a circular RNA (e.g., a structure in which the 5' end and the 3' end of the RNA are covalently linked). Circular RNA can enhance stability and efficiency.

[0142] Fig.80 Demonstration of the system with dgRNA fused to RNA template donor and SSAP is shown. Box highlights that repair by Venus is significantly higher than the control.

[0143] Fig.81 Testing of pol2(CMV) vs pol3(U6) promoter TLR genome editing is shown.

[0144] Fig.82 An example of an sgRNA+dgRNA system based on the EMX1 locus is shown. gE3: 20bp guide for sgRNA targeting EMX1, used in the first guide RNA; dg1-dg8 (15bp / 16bp inactive / inactivated guides in dgRNA targeting EMX1, used in the second guide RNA with fusion RNA template / donor.

[0145] Fig.83A dgRNA with a fused RNA template donor and SSAP targeting the human EMX site is shown. pA19 has Cas9 and a guide RNA with sg334 / sg516, which are two guides targeting the EMX1 gene editing reporter genomic region. BB is the backbone and serves as a negative control. dg518 / 520 / 522 / 526 are different inactive guide RNAs that have MS2 / PP7 aptamers and bind to a position near sg334 / sg516 and are fused to the RNA template / donor via a 36bp linker (36L). All designs are antisense with a 300bp homology arm region (a300+300). Red box: Designs with optimal dg guide RNA positions support higher editing efficiency.

[0146] Fig.84 A schematic diagram of a system incorporating SSAP and prime editing is shown. The MS2 aptamer recruits SSAP-MCP to the Cas9-RT complex. The system provides 1) a local reverse transcription template donor for SSAP; 2) bypasses the endogenous HDR mechanism that is limited to dividing cells; and 3) benefits from the use of homology arm regions of the template / donor and allows SSAP editing using Cas / nCas / dCas.

[0147] Fig.85 SSAP+ prime editing mediated editing at the HEK3 locus (top) and RFN2 locus (bottom) are shown. 293T cells were transfected with Cas9n-RT construct, pegRNA construct, nicking / recruiting sgRNA-MS2 construct, and SSAP-MCP construct.

[0148] Fig.86 Different length edits mediated by SSAP+prime editing at the HEK3 locus (upper panel) and RFN2 locus (lower panel) are shown. 293T cells were transfected with Cas9n-RT construct, pegRNA construct, nicking / recruiting sgRNA-MS2 construct, and SSAP-MCP construct.

[0149] Fig.87A schematic diagram of an editing system incorporating SSAP and retron is shown. The retron-SSAP editor has three components: (1) The retron-sgRNA can be subdivided into three regions: the RNA region that is reverse transcribed (called "msd"), the region that remains RNA in the final molecule (called "msr"), and the final guide RNA region (the guide RNA has MS2 or other aptamers to recruit SSAP). The gRNA region can be derived from the Cas9 backbone. The msr / msd RNA helps to initiate the RT process, which generates reverse transcribed ssDNA directly connected to the sgRNA. (2) The RT of the retron, which recognizes the retron RNA and completes the reverse transcription of the donor template (the connected RNA-DNA hybrid molecule). The RT can be fused to Cas9 or MCP-SSAP. (3) The SSAP protein fused to MCP, optionally also fused to RT (if RT is not fused to Cas9).

[0150] Figures 88A-88D depict SSAP array screening, showing cell viability vs. editing efficiency (fold relative to negative control (Figure 88A, Figure 88C) or mKate knock-in percentage (Figure 88B, Figure 88D)) for ACTB targets (Figure 88A, Figure 88B) and HSP90AA1 targets (Figure 88C, Figure 88D). The positive control is EcRecT.

[0151] Figure 89A- Fig.89C Normalized ( FIG. 89A ) and absolute ( FIG. 89B ) editing efficiencies are depicted, comparing activity at two targets, HSP90AA and ACTB. Fig.89C Cell viability is shown, comparing SSAP usage between HSP90AA1 knock-in and ACTB knock-in. The positive control is EcRecT.

[0152] Figure 90 depicts a comparison of cell viability vs. normalized (A) or absolute (B) editing efficiency for all combined targets by scatter plots. The bar graphs compare the normalized (C) or absolute (D) editing efficiency for each candidate at two targets (HSP90 and QCTB). The positive control is EcRecT.

[0153] Fig.91 Depicted are trees and sequence alignments of SSAP_16 (1, SEQ ID NO: 185), SSAP_10 (2, SEQ ID NO: 179), SSAP_36 (3, SEQ ID NO: 205), SSAP_152 (4, SEQ ID NO: 321) and SSAP_184 (5, SEQ ID NO: 353) compared to EcRecT (SEQ ID NO: 171). See Table 12.

[0154] Fig.92 Depicted are trees and sequence alignments of SSAP_16 (1, SEQ ID NO: 185), SSAP_10 (2, SEQ ID NO: 179), SSAP_36 (3, SEQ ID NO: 205), SSAP_152 (4, SEQ ID NO: 321), SSAP_184 (5, SEQ ID NO: 353), SSAP_197 (6, SEQ ID NO: 366), SSAP_305 (7, SEQ ID NO: 424), SSAP_210 (8; SEQ ID NO: 379) and SSAP_190 (9, SEQ ID NO: 359) compared to EcRecT (SEQ ID NO: 171). See Table 12.

[0155] Fig.93 Depicted are trees and sequence alignments of SSAP_16 (1, SEQ ID NO: 185), SSAP_10 (2, SEQ ID NO: 179), SSAP_36 (3, SEQ ID NO: 205), SSAP_197 (6, SEQ ID NO: 366) and SSAP_210 (8; SEQ ID NO: 379) compared to EcRecT (SEQ ID NO: 171). See Table 12.

[0156] Fig.94 The evolutionary tree of candidate SSAPs is depicted. 296 candidates were selected by applying a set of filters and maximizing evolutionary distance. SSAPs cover diverse phylogenetic families (clades) within the SSAP family.

[0157] Fig.95 A- Fig.95 C depicts the editing efficiency of the top 10 SSAPs using the dCas9 editing system compared to EcRecT and a negative control. ( Fig.95 A) mKate knock-in at the ACTB locus. Fig.95 B) mKate knock-in at the HSP90 locus. ( Fig.95 C) Scatter plot depicting the editing efficiency of candidate SSAPs at the ACTB target and the HSP90AA1 target.

[0158] Fig.96The editing efficiency of the top 10 SSAPs in the Cas-free system was compared with the negative control (pA25 expressing MCP-EBFP) and pCK914 expressing MCP-EcRecT. The gene editing efficiency is for knocking in 800bp mKate donors with homology arms in HEK293 human cells, and 20 bases of guide RNA to match the target genomic insertion site HSP90AA1 (top) or ACTB (bottom). The constructs used are i) guide RNA with MS aptamers to recruit SSAPs; ii) MCP-SSAP fusion protein; and iii) donor DNA, which is inserted into the mKate cargo (cargo) (no promoter).

[0159] Fig.97 A- Fig.97 C depicts the editing efficiency of the top SSAPs compared to EcRecT using the dCas9 editing system with a transcribed AAV donor in primary hepatocytes (mouse). dCas9 was delivered using adenovirus-Cas9 alone under the control of a strong CMV promoter (Adeno-CMV-Cas9). Fig.97 A) AAV donor design: (Top) Typical AAV donor DNA; (Bottom) AAV vector includes a promoter that transcribes the donor cargo into RNA. The donor RNA is transcribed in the antisense orientation to prevent cargo expression. Fig.97 B) or ACTB locus ( Fig.97 C) A 600 bp luciferase vector was knocked in.

[0160] Figure 98A-Figure 98C depicts AAV donor design and editing efficiency. Adenovirus-Cas9 alone virus delivery dCas9 is used under the control of a strong CMV promoter (Adeno-CMV-Cas9). (Figure 98A) Top: 5' release AAV design. A second guide RNA is provided to bind / cut the 5' end of the carrier (hsgRNA cleavage site adjacent to the left homology arm (left HA)). Middle: 3' release AAV design. A second guide RNA is provided to bind / cut the 3' end of the carrier (hsgRNA cleavage site adjacent to the right homology arm (right HA)). Bottom: Complete AAV design. 600bp luciferase carrier is knocked in at the mouse albumin locus (Figure 98B) or ACTB locus (Figure 98C).

[0161] Fig.99 Depicted is the genome engineering of multiple human targets using SSAPs. The editing system includes dCas9 (dSpCas9), guide RNA with an MS2 aptamer, MCP protein fused to a candidate SSAP, and donor DNA that inserts the mKate fluorescent protein cargo in-frame into the indicated endogenous genomic loci.

[0162] Figures 100A-100C depict comparisons of SSAP clones with RecT from Listeria Innoccua phage A118 (LiRecT) and models of RecT complexes in DNA annealing. (Figure 100A) Phylogenetic tree showing clones SSAP-10, SSAP-16, SSAP-152, SSAP-198, EcRecT, and LiRecT. (Figure 100B) Model of RecT-dsDNA complex. (Figure 100C) Model of RecT complexed with DNA annealing duplex intermediate. The model is based on the deposited structures 7UBB and 7UB2.

[0163] Fig.101 A- Fig.101 B depicts engineering of RecT. (A) Model of RecT in complex with a DNA annealing duplex intermediate. Inset shows RecT and DNA strands with extensive interactions (yellow dashed lines) involving selected protein residues (including the core tyrosine amino acid highlighted in the upper right corner). (B) Model of LiRecT showing interactions with dsDNA (highlighted) consistent with knock-in efficiency of an N-terminally truncated mini-SSAP (mSSAP) (see, e.g., FIG. 42 ). DETAILED DESCRIPTION OF THE INVENTION

[0164] The present invention relates to systems and components for DNA editing. In particular, the disclosed system is based on CRISPR targeting and homology-directed repair of phage recombinases. The system has superior recombination efficiency and accuracy on a kilobase scale.

[0165] To facilitate understanding of the present technology, some terms and phrases are defined below. Other terms are explained throughout the detailed description.

[0166] As used herein, the terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)", and variations thereof are intended as open transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The present invention also contemplates additional embodiments "comprising", "consisting of", and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0167] For the recitation of numerical ranges herein, each intermediate value with the same degree of precision is expressly contemplated. For example, for the range of 6-9, in addition to 6 and 9, the values ​​7 and 8 are also contemplated, and for the range of 6.0-7.0, the values ​​6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are expressly contemplated.

[0168] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. For example, any terminology and technology related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The meaning and scope of these terms should be clear; however, in the presence of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definition. In addition, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms.

[0169] The terms "complementary" and "complementarity" refer to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence through traditional Watson-Crick base pairing or other non-traditional types of pairing. The degree of complementarity between two nucleic acid sequences can be expressed in terms of the percentage of nucleotides in a nucleic acid sequence that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 50%, 60%, 70%, 80%, 90%, and 100% complementary). Two nucleic acid sequences are "fully complementary" if all of the consecutive nucleotides of a nucleic acid sequence will form hydrogen bonds with the same number of consecutive nucleotides in a second nucleic acid sequence. Two nucleic acid sequences are "substantially complementary" if the degree of complementarity between the two nucleic acid sequences is at least 60% (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) over a region of at least 8 nucleotides (e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides), or if the two nucleic acid sequences hybridize under at least moderate stringency, preferably high stringency. Exemplary moderate stringency conditions include incubation overnight at 37°C in a solution comprising 20% ​​formamide, 5X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured sheared salmon sperm DNA, followed by washing the filter in 1X SSC at about 37-50°C, or under substantially similar conditions, e.g., the moderate stringency conditions described in Sambrook et al., see below.High stringency conditions are those using, for example, (1) low ionic strength and high temperature washing, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate (SDS) at 50° C., (2) use of a denaturing agent during hybridization, for example, formamide, for example, 50% (v / v) formamide, and 0.1% bovine serum albumin (BSA) / 0.1% Ficoll / 0.1% polyvinylpyrrolidone (PVP) / 50 mM sodium phosphate buffer (pH 6.5), containing 750 mM sodium chloride and 75 mM sodium citrate at 42° C., or (3) use of 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS and 10% dextran sulfate at 42° C., and washes at (i) 42° C. in 0.2×SSC, (ii) 55° C. in 50% formamide, and (iii) 55° C. in 0.1×SSC (preferably in combination with EDTA). Additional details and explanations of hybridization reaction stringency are provided, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (2001); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York (1994).

[0170] When exogenous DNA (e.g., a recombinant expression vector) is introduced into a cell, the cell has been "genetically modified," "transformed," or "transfected" by this DNA. The presence of exogenous DNA results in permanent or transient genetic changes. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, in prokaryotes, yeast, and mammalian cells, the transforming DNA may be maintained on an episomal element (e.g., a plasmid). With regard to eukaryotic cells, stably transformed cells are cells in which the transforming DNA has been integrated into the chromosome, thereby being inherited by daughter cells through chromosome replication. This stability is manifested in the ability of eukaryotic cells to establish cell lines or clones comprising a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or a common ancestor by mitosis. A "cell line" is a clone of primary cells that can stably grow for many generations in vitro.

[0171] As used herein, "nucleic acid" or "nucleic acid sequence" refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine and uracil, and adenine and guanine, respectively. The present technology covers any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component and any chemical variant thereof, such as methylated, hydroxymethylated or glycosylated forms of these bases, etc. The polymer or oligomer may be heterogeneous or homogeneous in composition and may be isolated from a naturally occurring source or may be artificially or synthetically produced. In addition, the nucleic acid may be DNA or RNA, or a mixture thereof, and may exist permanently or transiently in single-stranded or double-stranded form, including homoduplexes, heteroduplexes and hybrid states. In some embodiments, the nucleic acid or nucleic acid sequence comprises other types of nucleic acid structures, e.g., DNA / RNA helices, peptide nucleic acids (PNAs), morpholino nucleic acids (see, e.g., Braasch and Corey, Biochemistry, 41(14):4503-4510 (2002)) and U.S. Pat. No. 5,034,506, which are incorporated herein by reference), locked nucleic acids (LNAs; see Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 97:5633-5638 (2000), which are incorporated herein by reference), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122:8595-8602 (2000), which are incorporated herein by reference), and / or ribozymes. Therefore, the term "nucleic acid" or "nucleic acid sequence" can also encompass chains containing non-natural nucleotides, modified nucleotides and / or non-nucleotide structural elements (e.g., "nucleotide analogs") that can exhibit the same functions as natural nucleotides; in addition, as used herein, the term "nucleic acid sequence" refers to oligonucleotides, nucleotides or polynucleotides and fragments or portions thereof, as well as DNA or RNA of genomic or synthetic origin, which can be single-stranded or double-stranded, and represent a sense strand or an antisense strand. The terms "nucleic acid", "polynucleotide", "nucleotide sequence" and "oligonucleotide" can be used interchangeably. They refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides, or analogs thereof) of any length.

[0172] A "peptide" or "polypeptide" is a linked sequence of two or more amino acids linked by peptide bonds. Peptides or polypeptides may be natural, synthetic, or a modification or combination of natural and synthetic. Polypeptides include proteins, such as binding proteins, receptors, and antibodies. Proteins may be modified by the addition of sugars, lipids, or other moieties not included in the amino acid chain. The terms "polypeptide" and "protein" are used interchangeably herein.

[0173] As used herein, the term "sequence identity percentage" refers to the percentage of nucleotides or nucleotide analogs in a nucleic acid sequence or amino acids in an amino acid sequence compared to the corresponding nucleotides or amino acids in a reference sequence, and introducing gaps when necessary to achieve the same percentage under the condition of maximum identity percentage. Therefore, in the case where the nucleic acid according to this technology is longer than the reference sequence, other nucleotides in the nucleic acid that are not compared to the reference sequence are not considered when determining sequence identity. Methods and computer programs for comparison are well known in the art, including BLAST, Align 2 and FASTA.

[0174] A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus, or cosmid, into which another DNA segment (eg, an "insert") can be attached or incorporated so as to cause the attached segment to replicate in a cell.

[0175] The term "wild-type" refers to a gene or gene product that has the characteristics of the gene or gene product when isolated from a naturally occurring source. A wild-type gene is the gene most commonly observed in a population and is therefore arbitrarily designated as the "normal" or "wild-type" form of the gene. In contrast, the terms "modified," "mutated," or "polymorphic" refer to a gene or gene product that exhibits modifications (e.g., altered characteristics) in sequence and / or functional properties when compared to a wild-type gene or gene product. It is noteworthy that naturally occurring mutants can be isolated; these mutants are identified by the fact that they have altered characteristics compared to a wild-type gene or gene product.

[0176] RNA-guided CRISPR recombineering system. In bacteria and archaea, the CRISPR / Cas system provides immunity by integrating fragments of invading phage, virus and plasmid DNA into the CRISPR locus, and using the corresponding CRISPR RNA ("crRNA") to guide the degradation of homologous sequences. Each CRISPR locus encodes a "spacer" separated by a repetitive sequence. The transcription of the CRISPR locus produces a "pre-crRNA", which is processed to produce a crRNA containing a spacer repeat fragment, and the crRNA guides the effector nuclease complex to cut the dsDNA sequence complementary to the spacer. Three different types of CRISPR systems are known, type I, type II or type III, and are classified based on the Cas protein type and the prototype spacer adjacent motif (PAM) for selecting the prototype spacer in the invading DNA. Endogenous type II system includes Cas9 protein and two non-coding crRNAs: trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA) arrays, which contain nuclease guide sequences (also referred to as "spacers") spaced by identical direct repeats (DRs). TracrRNA is very important for processing pre-crRNA and forming Cas9 complexes. First, tracrRNA hybridizes with the repeat region of pre-crRNA. Secondly, endogenous RNaseIII cuts the hybridized crRNA-tracrRNA, and the second event removes the 5' end of each spacer, producing a mature crRNA still associated with both tracrRNA and Cas9. Again, each mature complex locates the target double-stranded DNA (dsDNA) sequence, and the nuclease activity of Cas9 is used to cut two chains.

[0177] CRISPR / Cas gene editing system has been developed, which can target and modify specific genes of interest in eukaryotic cells. CRISPR / Cas gene editing system is generally based on Cas9 nuclease guided by RNA from type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system. The engineered CRISPR / Cas system for eukaryotic cells generally involves the reconstruction of crRNA-tracrRNA-Cas9 complex. For example, in human cells, Cas9 amino acid sequence can be codon optimized and modified to include appropriate nuclear localization signals, and crRNA and tracrRNA sequences can be expressed alone or as a single chimeric molecule by RNA polymerase II promoter. Typically, crRNA and tracrRNA sequences are expressed as chimeras, and are collectively referred to as "guide RNA" (gRNA) or single guide RNA (sgRNA). Therefore, the terms "guide RNA", "single guide RNA" and "synthetic guide RNA" are used interchangeably herein, and refer to nucleic acid sequences comprising tracrRNA and pre-crRNA arrays containing guide sequences. The terms "guide sequence", "guide" and "spacer" are used interchangeably herein and refer to a sequence of about 20 nucleotides that specifies a target site within a guide RNA. In the CRISPR / Cas9 system, the guide RNA contains a guide sequence of about 20 nucleotides followed by a protospacer adjacent motif (PAM), which guides Cas9 to the target sequence by Watson-Crick base pairing.

[0178] In some embodiments or the present invention, a system or composition for RNA-guided recombineering using tools from a CRISPR gene editing system is provided. The system comprises: a Cas protein, a nucleic acid molecule comprising a guide RNA sequence complementary to a target DNA sequence, and a recombinant protein. In certain embodiments, the recombinant protein comprises a microbial recombinant protein. In certain embodiments, the recombinant protein comprises a viral recombinant protein. In certain embodiments, the recombinant protein comprises a eukaryotic recombinant protein. In certain embodiments, the recombinant protein comprises a mitochondrial recombinant protein.

[0179] The Cas protein family is described in more detail, for example, Haft et al., PLoS Comput. Biol., 1 (6): e60 (2005), which is incorporated herein by reference. The Cas protein can be any Cas endonuclease. In some embodiments, the Cas protein is Cas9 or Cas12a, also known as Cpf1. In one embodiment, the Cas9 protein is a wild-type Cas9 protein. The Cas9 protein can be obtained from any suitable microorganism, and many bacteria express Cas9 protein orthologs or variants. In some embodiments, Cas9 is from Streptococcus pyogenes or Staphylococcus aureus. Cas9 proteins of other species are known in the art (see, for example, U.S. Patent Application Publication 2017 / 0051312, which is incorporated herein by reference) and can be used in conjunction with the present invention. The amino acid sequences of Cas proteins from various species are publicly available through GenBank and UniProt databases.

[0180] In some embodiments, the Cas9 protein is a Cas9 nickase (Cas9n). Wild-type Cas9 has two catalytic nuclease domains that promote double-stranded DNA breaks. The Cas9 nickase protein is usually engineered by causing one of the catalytic nuclease domains to inactivate a point mutation, causing Cas9 to use the remaining active nuclease domain to only produce a nick or enzymatic break in one of the two DNA chains. Cas9 nickases are known in the art (see, e.g., U.S. Patent Application Publication 2017 / 0051312, which is incorporated herein by reference), and include, for example, Streptococcus pyogenes with point mutations at D10 or H840. In a preferred embodiment, the Cas9 nickase is Streptococcus pyogenes Cas9n (D10A).

[0181] In some embodiments, the Cas protein is a catalytically inactive Cas. For example, the catalytically inactive Cas9 is essentially a DNA binding protein, usually because there are two or more mutations in its catalytic nuclease domain, so that the protein has very little or no catalytic nuclease activity. Streptococcus pyogenes Cas9 can be catalytically inactive by mutations in D10 and at least one of E762, H840, N854, N863 or D986 (usually H840 and / or N863) (see, e.g., U.S. Patent Application Publication 2017 / 0051312, which is incorporated herein by reference). Mutations of corresponding orthologs are known, such as N580 in Staphylococcus aureus Cas9. Typically, such mutations result in catalytically inactive Cas proteins having no more than 3% of the activity of normal nucleases.

[0182] In certain embodiments, the system comprises a nucleic acid molecule comprising a guide RNA sequence complementary to a target DNA sequence. As described above, the guide RNA sequence specifies a target site by a guide sequence of about 20 nucleotides, followed by a protospacer adjacent motif (PAM) that guides Cas9 to the target sequence by Watson-Crick base pairing.

[0183] In certain embodiments, the system comprises a nucleic acid molecule comprising an inactivated guide RNA (dgRNA) sequence complementary to a target DNA sequence. The inactivated guide is shortened or modified so that the CRISPR complex comprising the dgRNA binds to the target DNA but does not cut it or cause it to have a gap. Non-limiting examples include guidance, such as described in WO / 2016 / 094872, to allow the formation of a CRISPR complex and successfully bind to the target, while not allowing successful nuclease activity (e.g., no nuclease activity / no insertion and deletion activity) to be modified. In terms of nuclease activity, the guide nucleic acid can be considered to be catalytically inactive or conformationally inactive (conformationally inactive). The dgRNA with a short target recognition sequence can significantly improve the editing specificity of Cas9-mediated by binding to the active Cas9sgRNA complex and shielding its off-target site. (Rose et al., Suppression of unwanted CRISPR-Cas9editing by co-administration of catalytically inactivating truncated guideRNAs. Nature Communications (2020) vol. 11, article 2697). According to the present invention, shortened / modified dgRNA is used to recruit Cas9-SSAP to achieve long sequence-free knock-in.

[0184] The terms "target DNA sequence", "target nucleic acid", "target sequence" and "target site" are used interchangeably herein to refer to polynucleotides (nucleic acids, genes, chromosomes, genomes, etc.), and guide sequences (e.g., guide RNAs) are designed to have complementarity therewith, wherein hybridization between the target sequence and the guide sequence promotes Cas9 / CRISPR complex formation as long as there are sufficient binding conditions. In some embodiments, the target sequence is a genomic DNA sequence. As used herein, the term "genome" refers to a nucleic acid sequence (e.g., a gene or locus) located on a chromosome in a cell. The target sequence and the guide sequence do not need to show complete complementarity, as long as there is sufficient complementarity to cause hybridization and promote CRISPR complex formation. The target sequence may comprise any polynucleotide, such as DNA or RNA. Suitable DNA / RNA binding conditions include physiological conditions that are normally present in cells. Other suitable DNA / RNA binding conditions (e.g., conditions in a cell-free system) are known in the art; see, e.g., Sambrook, which is cited herein and incorporated herein by reference. The target DNA strand that is complementary to and hybridizes with the DNA-targeting RNA is referred to as the "complementary strand," and the target DNA strand that is complementary to the "complementary strand" (and therefore not complementary to the DNA-targeting RNA) is referred to as the "non-complementary strand" or "non-complementary strand."

[0185] The target genomic DNA sequence can encode a gene product. As used herein, the term "gene product" refers to any biochemical product produced by gene expression. The gene product can be RNA or protein. RNA gene products include non-coding RNA, such as tRNA, rRNA, microRNA (miRNA) and small interfering RNA (siRNA), and coding RNA, such as messenger RNA (mRNA). In some embodiments, the target genomic DNA sequence encodes a protein or polypeptide.

[0186] In some embodiments, for example, when the system includes a Cas9 nickase or a catalytically inactive Cas9, two nucleic acid molecules comprising a guide RNA sequence can be utilized. The two nucleic acid molecules can have the same or different guide RNA sequences, thereby being complementary to the same or different target DNA sequences. In some embodiments, the guide RNA sequences of the two nucleic acid molecules are complementary to the target DNA sequence at the opposite ends (e.g., 3' or 5') and / or opposite strands of the insertion position.

[0187] In some embodiments, the system further comprises a recruitment system comprising at least one aptamer sequence and an aptamer binding protein that is functionally linked to the recombinant protein as part of a fusion protein.

[0188] In some embodiments, the aptamer sequence is an RNA aptamer sequence. In some embodiments, the nucleic acid molecule comprising the guide RNA further comprises one or more RNA aptamers, or can recruit and bind to another molecular species, i.e., different RNA secondary structures or sequences of adapter molecules (e.g., nucleic acids or proteins). Various CRISPR systems are compatible with guide RNA insertion and extension, including but not limited to SpCas9, SaCas9, and LbCas12a (also known as Cpf1). RNA aptamers can be naturally occurring or synthetic oligonucleotides that are engineered by repeated multiple rounds of in vitro selection or SELEX (systemic evolution of ligands by exponential enrichment) to bind to specific target molecule species. In some embodiments, the nucleic acid comprises two or more aptamer sequences. The aptamer sequences may be the same or different, and may target the same or different adapter proteins. In a preferred embodiment, the nucleic acid comprises two aptamer sequences.

[0189] Any known RNA aptamer / aptamer binding protein can be selected and used in conjunction with the present invention (see, e.g., Jayasena, SD, Clinical Chemistry, 1999. 45(9): p. 1628-1650; Gelinas, et al., Current Opinion in Structural Biology, 2016. 36: p. 122-132; and Hasegawa, H., Molecules, 2016; 21(4): p. 421, which are incorporated herein by reference).

[0190] There are many RNA aptamer binding proteins or adaptor proteins, including a variety of phage coat proteins. Examples of such coat proteins include, but are not limited to: MS2, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, and PRR1. In some embodiments, the RNA aptamer binds to the MS2 phage coat protein or a functional derivative, fragment, or variant thereof. RNA aptamers that bind to MS2 generally have a simple stem-loop structure, typically defined by a 19-nucleotide RNA molecule with a single protruding adenine on the 5' leg of the stem (Witherall GW, et al., (1991) Prog. Nucleic Acid Res. Mol. Biol., 40, 185-220, which is incorporated herein by reference). However, many very different primary sequences have been found to be able to bind to the MS2 coat protein (Parrott AM, et al., Nucleic Acids Res. 2000; 28 (2): 489-497, Buenrostro JD, et al. Natura Biotechnology 2014; 32, 562-568, and incorporated herein by reference). Any RNA aptamer sequence known to bind to the MS2 bacteriophage coat protein can be used in the present invention to bind to a fusion protein comprising MS2. In preferred embodiments, the MS2 RNA aptamer sequence comprises: AACAUGAGGAUCACCCAUGUCUGCAG (SEQ ID NO: 145), AGCAUGAGGAUCACCCAUGUCUGCAG (SEQ ID NO: 146), or AGCGUGAGGAUCACCCAUGCCUGCAG (SEQ ID NO: 147).

[0191] The N-protein (Nut-utilization site protein) of the bacteriophage contains a conserved RNA recognition motif rich in arginine of 20 amino acids, called the N-peptide. The RNA aptamer can bind to the bacteriophage N-peptide or its functional derivative, fragment or variant. In some embodiments, the bacteriophage N-peptide is a λ or P22 bacteriophage N-peptide or its functional derivative, fragment or variant.

[0192] In a preferred embodiment, the N peptide is a lambda phage N22 peptide or a functional derivative, fragment or variant thereof. In some embodiments, the N22 peptide comprises an amino acid sequence having at least 70% similarity to the amino acid sequence GNARTRRRERRAEKQAQWKAAN (SEQ ID NO: 149). The N22 peptide is a 22 amino acid RNA binding domain of the lambda phage anti-terminator protein N (λN-(1–22) or λN peptide) that is capable of specifically binding to a specific stem-loop structure, including but not limited to the BoxB stem-loop. See, e.g., Cilley and Williamson, RNA 1997; 3(1): 57-67, which is incorporated herein by reference. Many different BoxB stem-loop primary sequences are known to bind to the N22 peptide, and any of these sequences can be used in the present invention. In some embodiments, the N22 peptide RNA aptamer sequence comprises a nucleotide sequence having at least 70% similarity to an RNA sequence selected from the group consisting of GCCCUGAAAAAGGGC (SEQ ID NO: 150), GCCCUGAAGAAGGGC (SEQ ID NO: 151), GCGCUGAAAAAGCGC (SEQ ID NO: 152), GCCCUGACAAAGGGC (SEQ ID NO: 153), and GCGCUGACAAAGCGC (SEQ ID NO: 154). In some embodiments, the N22 peptide RNA aptamer sequence is selected from the group consisting of SEQ ID NOs: 150-154.

[0193] In a preferred embodiment, the N peptide is a P22 phage N peptide or a functional derivative, fragment or variant thereof. Many different BoxB stem-loop primary sequences are known to bind to the P22 phage N peptide and its variants, and any of these sequences can be used in conjunction with the present invention. See, for example, Cocozaki, Ghattas, and Smith, Journal of Bacteriology 2008; 190(23):7699-7708, which is incorporated herein by reference. In some embodiments, the P22 phage N peptide comprises an amino acid sequence having at least 70% similarity to the amino acid sequence GNAKTRRHERRRKLAIERDTI (SEQ ID NO: 155). In some embodiments, the P22 phage N peptide RNA aptamer sequence comprises a sequence having at least 70% similarity to an RNA sequence selected from the group consisting of GCGCUGACAAAGCGC (SEQ ID NO: 156) and CCGCCGACAACGCGG (SEQ ID NO: 157). In some embodiments, the P22 phage N-peptide RNA aptamer sequence is selected from the group consisting of SEQ ID NOs: 156-157, UGCGCUGACAAAGCGCG (SEQ ID NO: 158), or ACCGCCGACAACGCGGU (SEQ ID NO: 159).

[0194] In certain embodiments, different aptamer / aptamer binding protein pairs can be selected to combine recombinant proteins and functions together.

[0195] In some embodiments, the aptamer sequence is a peptide aptamer sequence. The peptide aptamer can be a naturally occurring or synthetic peptide that is specifically recognized by an affinity agent. Such aptamers include, but are not limited to, c-Myc affinity tags, HA affinity tags, His affinity tags, S affinity tags, methionine-His affinity tags, RGD-His affinity tags, 7×His tags, FLAG octapeptides, strep tags or strep tags II, V5 tags or VSV-G epitopes. The corresponding aptamer binding proteins are well known in the art and include, for example, primary antibodies, biotin, affimers, single domain antibodies and antibody mimics.

[0196] Exemplary peptide aptamers include GCN4 peptide (Tanenbaum et al., Cell 2014; 159(3):635-646, which is incorporated herein by reference).Antibodies or GCN4 binding proteins can be used as aptamer binding proteins.

[0197] In some embodiments, the peptide aptamer sequence is conjugated to the Cas protein. The peptide aptamer sequence can be fused to Cas in any direction (e.g., N-terminus to C-terminus, C-terminus to N-terminus, N-terminus to N-terminus). In a preferred embodiment, the peptide aptamer is fused to the C-terminus of the Cas protein.

[0198] In some embodiments, 1 to 24 peptide aptamer sequences can be conjugated to the Cas protein. The aptamer sequences can be the same or different, and can target the same or different aptamer binding proteins. In a preferred embodiment, 1 to 24 tandem repeats of the same peptide aptamer sequence are conjugated to the Cas protein. In a preferred embodiment, 4 to 18 tandem repeats are conjugated to the Cas protein. A single aptamer can be separated by a joint region. Suitable joint regions are known in the art. The joint can be flexible or configured to allow an affinity agent to bind to an adjacent aptamer without steric hindrance or with reduced steric hindrance. The joint sequence can provide an unstructured or linear region of a polypeptide, for example, including one or more glycine and / or serine residues. The length of the joint sequence can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0199] In some embodiments, the fusion protein comprises a recombinant protein functionally linked to an aptamer binding protein. In some embodiments, the recombinant protein comprises a microbial recombinant protein. In some embodiments, the recombinant protein comprises a recombinase. In certain embodiments, the recombinant protein comprises a 5'-3' exonuclease activity. In certain embodiments, the recombinant protein comprises a 3'-5' exonuclease activity. In certain embodiments, the recombinant protein comprises ssDNA binding activity. In certain embodiments, the recombinant protein comprises ssDNA annealing activity.

[0200] The genetic recombination mechanism encoded by bacteriophage λ (called λred system) includes exo and bet genes, assisted by gam genes, collectively referred to as λred genes. Exo is a 5'-3' exonuclease targeting dsDNA, and Bet is a ssDNA binding protein. Bet functions include protecting ssDNA from degradation and promoting the annealing of complementary ssDNA chains. Another phage system found in Escherichia coli is the Rac prophage system, which includes recE and recT genes similar to exo and bet in function. In some embodiments, the microbial recombinant protein can be RecE, RecT, λ exonuclease (Exo), Bet protein (betA, redB), exonuclease gp6, single-stranded DNA binding protein gp2.5, or its derivatives or variants.

[0201] Recombinant proteins and functional fragments thereof that can be used in the present invention include nucleases, ssDNA binding proteins (SSB) and ssDNA annealing proteins (SSAB). Among microbial proteins, these include but are not limited to E. coli proteins, such as ExoI (xonA; sbcB), ExoIII (xthA), ExoIV (orn), ExoVII (xseA, xseB), ExoIX (ygdG), ExoX (exoX), DNApolI 5'Exo (ExoVI) (polA), DNAPol I 3'Exo (ExoII) (polA), DNA Pol II 3'Exo (polB), DNAPol III 3'Exo (dnaQ, mutD), RecBCD (recB, recC, recD) and RecJ (recJ) and functional fragments thereof.

[0202] Although double-stranded DNA contains genetic information, the use of this information involves single-stranded intermediates. Single-stranded intermediates form secondary structures and are sensitive to chemicals and nucleolytic degradation, while cells encode ssDNA binding proteins (SSBs) that can bind and stabilize ssDNA. Useful SSBs include, but are not limited to, SSBs of prokaryotes, bacteriophages, eukaryotes, mammals, mitochondria, and viruses. Although SSBs are found in each organism, the sequence similarity of the protein itself is unexpectedly small, and the subunit composition and oligomeric state may be different. SSB proteins may contain certain structural features. One feature is the use of an oligonucleotide / oligosaccharide binding (OB) domain to bind ssDNA through a combination of electrostatic and base stacking interactions with the phosphodiester backbone and nucleotide bases. Another feature is oligomerization, which brings together the OB folds that bind to DNA. Eukaryotic SSBs are regulated by phosphorylation of serine and threonine residues. Tyrosine phosphorylation of microbial SSBs has been observed in taxonomically distant bacteria, and significantly increases affinity for ssDNA. The human mitochondrial ssDNA binding protein is structurally similar to the SSB of Escherichia coli (EcoSSB), but lacks the C-terminal disordered domain. The eukaryotic replication protein A (RPA) shares functions with bacterial SSB but does not share sequence homology. Herpes simplex virus (HSV-1) SSB (ICP8) is a nuclear protein that, along with other replication proteins, is essential for viral DNA replication.

[0203] Without being bound by theory, it is believed that the exonuclease activity and ssDNA binding activity of the recombinant protein of the present invention expose (uncover) and protect the single-stranded regions of the template and target DNA, thereby promoting recombination. In addition, targeting can be synergistic, involving target-guided CRISPR-mediated chromosomal DNA incisions, coordinated with the recombination guided by the homologous arms designed into the template DNA. In certain embodiments of the present invention, off-target effects are minimized. For example, although targeted recombination involves CRISPR and recombination functions, there is no homology with the HR template DNA at the off-target site, and incision repair may be facilitated.

[0204] Single-stranded DNA annealing protein (SSAP) is also ubiquitous in each organism and has different sequences, and is classified into families and superfamilies by bioinformatics and experimental analysis. In addition, phage-encoded SSAPs are considered to encode their own SSAP recombinases, which replace classical RecA proteins and work together with host proteins to control DNA metabolism. Steczkiewiz classifies SSAP into seven families (RecA, Gp2.5, RecT / Redβ, Erf, Rad52 / 22, Sak3 and Sak4), which are organized into three superfamilies, including prokaryotes, eukaryotes and phages (Steczkiewicze et al., 2021, Front.Microbiol 12: 644622). Non-limiting examples of SSAPs that can be used according to the present invention are provided in Table 7. Any one or more SSAPs can be used in the present invention.

[0205] In certain embodiments, the microbial recombinant protein is RecE or RecT, or a derivative or variant thereof. The derivative or variant of RecE and RecT is a functionally equivalent protein or polypeptide, which has a function substantially similar to wild-type RecE and RecT. RecE and RecT derivatives or variants include biologically active amino acid sequences similar to wild-type sequences but different due to amino acid substitutions, additions, deletions, truncations, post-translational modifications or other modifications. In some embodiments, derivatives can improve translation, purification, biological half-life, activity, or eliminate or mitigate any undesirable side effects or reactions. Derivatives or variants can be naturally occurring polypeptides, synthetic or chemically synthesized polypeptides or genetically engineered peptide polypeptides. RecE and RecT biological activities are known to those of ordinary skill in the art and are easy to measure, and include, for example, exonucleases and single-stranded nucleic acid binding biological activities, respectively.

[0206] RecE or RecT can be derived from a variety of organisms, including Escherichia coli, Pantoea breeneri, F-type symbiont of Plautia stali, Providencia sp. MGF014, Shigella sonnei, Pseudobacteriovoraxantillogorgiicola, etc. Other non-limiting sources include Desulfotalea psychrophila, Lactococcus lactis, Flavobacterium psychrophilum, Mycobacterium smegmatis, Lactobacillus rhamnosus, Psychrobacter arcticus, Psychrobacter cryohalolentis, Psychromonas ingrahamii, Photobacterium profundum, Psychroflexus torquis, and Caulobacter crescentus. In certain embodiments, the RecE and RecT proteins are derived from Escherichia coli.

[0207] In some embodiments, the fusion protein comprises RecE or a derivative or variant thereof. RecE or a derivative or variant thereof may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8. RecE or a derivative or variant thereof may comprise an amino acid sequence having at least 70% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) similarity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8. In a preferred embodiment, RecE or a derivative or variant thereof comprises an amino acid sequence having at least 90% similarity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-8. In an exemplary embodiment, RecE or a derivative or variant thereof comprises an amino acid sequence having at least 90% similarity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3.

[0208] In some embodiments, the fusion protein comprises RecT or a derivative or variant thereof. RecT or a derivative or variant thereof may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 9-14. RecT or a derivative or variant thereof may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 9-14 having at least 70% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) similarity. In a preferred embodiment, RecT or a derivative or variant thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9-14 having at least 90% similarity. In an exemplary embodiment, RecT or a derivative or variant thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9 having at least 90% similarity.

[0209] In certain embodiments, the fusion protein comprises a recombinant protein comprising an amino acid sequence that is at least 75% similar or at least 75% identical to: a recombinant protein of SEQ ID NO:166 to SEQ ID NO:491, a recombinant protein of Table 9, a recombinant protein of SEQ ID NO:179, SEQ ID NO:185, SEQ ID NO:205, SEQ ID NO:321, SEQ ID NO:353, SEQ ID NO:359, SEQ ID NO:366, SEQ ID NO:424, or SEQ ID NO:479, or a recombinant protein of SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:241, SEQ ID NO:253, SEQ ID NO:290, SEQ ID NO:408, SEQ ID NO:411, or SEQ ID NO:442. In certain embodiments, fusion protein comprises recombinant protein, and this recombinant protein comprises and has at least 80%, at least 85%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% similarity or sequence of identity with the above-mentioned recombinant protein.Can be from C-terminal or N-terminal or both brachymemma.For example, as shown in Example 6 below, a series of different brachymemma from any one end or two ends provides functional product. In some embodiments, one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or more) amino acids can be truncated from the C-terminus, N-terminus, compared to the wild-type sequence.

[0210] In some embodiments, the recombinant protein comprises a tyrosine recombinase or a functional fragment thereof. In some embodiments, the recombinant protein comprises a serine recombinase or a functional fragment thereof. In some embodiments, the recombinant protein comprises an integrase, a resolvase or an invertase or a functional fragment thereof. In some embodiments, the recombinase protein comprises a site-specific recombinase protein or a functional fragment thereof. In some embodiments, the recombinant protein comprises an exonuclease or a functional fragment thereof. In some embodiments, the recombinant protein comprises an ssDNA binding protein or a functional fragment thereof. In certain embodiments, the fusion protein includes but is not limited to Hin, Gin, Tn3, β / six, CinH, Min, ParA, γδ, Bxb1, φC31, TP901-1, TGI, Wβ, φ370.1, φK38, φBT1, R4, φRV1, φFC1, MR11, A118, U153, Bxz2, gp29, Cre, Dre, Vika, Flp, Kw, SprA, HK022, P22, L1 or L5, or any homolog of such protein or its functional fragment. Such recombinases can be classified as integrases, resolvases or invertases in the art, can have a common substructure and activity with exonucleases and SSBs, and are used according to the present invention.

[0211] The present invention provides a system comprising a reverse transcriptase, a guide nucleic acid and a recombination protein, and optionally a Cas protein.

[0212] The term "reverse transcriptase" describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require primers to synthesize DNA transcripts from RNA templates. Historically, reverse transcriptases have been primarily used to transcribe mRNA into cDNA, which can then be cloned into a vector for further manipulation. Avian myoblastosis virus (AMV) reverse transcriptase is the first widely used RNA-dependent DNA polymerase (Verma, Biochim. Biophys. Acta 473: 1 (1977)). The enzyme has 5'-3' RNA-guided DNA polymerase activity, 5-3' DNA-guided DNA polymerase activity and RNase H activity. RNase H is a specific progressive 5' and 3' ribonuclease (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)) to the RNA chain of RNA-DNA hybrids. Errors in transcription cannot be corrected by reverse transcriptase because known viral reverse transcriptases lack the 3'-5' exonuclease activity necessary for proofreading (Saunders and Saunders, Microbial Genetics Applied to Biotechnology, London: Croom Helm (1987)). Berger et al, Biochemistry 22: 2365-2372 (1983) provides a detailed study of AMV reverse transcriptase activity and its associated RNase H activity. Another reverse transcriptase widely used in molecular biology is the reverse transcriptase derived from Moloney murine leukemia virus (M-MLV). See, e.g., Gerard, GR, DNA 5: 271-279 (1986) and Kotewicz, ML et al., Gene 35: 249-258 (1985). M-MLV reverse transcriptases that are substantially lacking RNase H activity have also been described. See, e.g., U.S. Patent No. 5,244,797. The present invention contemplates the use of any such reverse transcriptases or variants or mutants thereof (RT).With regard to RT, and methods of linking or functionally connecting components of embodiments of the invention (e.g., RT systems or compositions of the invention) (and with regard to linkers or methods of functionally connecting components of systems or compositions discussed herein that do not involve RT), reference is made to WO2020 / 191241, WO2020 / 191153, WO2020 / 191245, WO2020 / 191243, WO2020 / 191233, WO2020 / 191246, WO2020 / 191249, WO2020 / 191239, WO2020 / 191234, WO2020 / 191242, WO2020 / 191248, WO2020191171 and WO2021 / 226558, which relate to so-called lead editing and double lead editing. Each of WO2020 / 191241, WO2020 / 191153, WO2020 / 191245, WO2020 / 191243, WO2020 / 191233, WO2020 / 191246, WO2020 / 191249, WO2020 / 191239, WO2020 / 191234, WO2020 / 191242, WO2020 / 191248, WO2020191171 and WO2021 / 226558 are hereby incorporated by reference. The RTs of WO2020 / 191241, WO2020 / 191153, WO2020 / 191245, WO2020 / 191243, WO2020 / 191233, WO2020 / 191246, WO2020 / 191249, WO2020 / 191239, WO2020 / 191234, WO2020 / 191242, WO2020 / 191248, WO2020191171 and WO2021 / 226558 can be used in the practice of the present invention. The linkers or functional connection methods of WO2020 / 191241, WO2020 / 191153, WO2020 / 191245, WO2020 / 191243, WO2020 / 191233, WO2020 / 191246, WO2020 / 191249, WO2020 / 191239, WO2020 / 191234, WO2020 / 191242, WO2020 / 191248, WO2020191171 and WO2021 / 226558 can be used in the practice of the present invention. Reference is also made to U.S. patent publications US2014 / 0349400 and US2018 / 0298391, both of which are incorporated herein by reference, which relate to systems containing Cas9, a reverse transcriptase, a guide RNA, and an RNA for reverse transcriptase activity; and, the reverse transcriptase and other aspects of these early systems can be used in the practice of the present invention.

[0213] WO / 2020 / 191153 describes a system comprising a CRISPR protein (e.g., a Cas9 nickase) and a reverse transcriptase for use with a guide RNA that is specific for a target site and provides a template for synthesizing the desired edit (in the form of a replacement DNA strand) by means of an extension (DNA or RNA) engineered onto the guide nucleic acid (e.g., at the 5' or 3' end, or in an internal portion of the guide RNA). The endogenous strand of the target site is replaced by a newly synthesized replacement strand containing the desired edit through DNA repair and / or replication mechanisms. The present invention provides a single-stranded binding protein (e.g., SSAP or SSB) that is used with a reverse transcriptase for editing without CRISPR-mediated nicking or cutting or target DNA.

[0214] Background on RT systems and compositions: Existing genome editing technologies are limited by low precision editing efficiency and accuracy, resulting in the ability to introduce accurate replacements, deletions, or insertions in mammalian cells using existing tools (e.g., CRISPR systems) to be highly unreliable. Conventional processes involve the delivery of gene editing tools (such as CRISPR) and DNA repair templates to introduce desired changes to genomic sequences. However, the DNA delivered to cells can be non-specifically inserted into off-target genomic loci or unintended targets, which is a major challenge in ensuring safe and accurate gene editing for therapeutic purposes.

[0215] Description of RT systems and compositions: Here, the applicant describes an invention that uses RNA as a molecular entity to mediate gene editing. The applicant has designed and validated components of systems and methods to use RNA as a template (donor) to insert, delete, replace or control genomic DNA sequences, which is mediated by the activity of SSAP (single-stranded annealing protein, such as RecT, λRed, T7gp2.5).

[0216] In the first embodiment, the applicant shows here the efficiency of gene editing by delivering three components into the cell: (1) the applicant uses a CRISPR system consisting of a CRISPR enzyme (for cutting / nicking / R-loop formation, corresponding to Cas9 / Cas9n / dCas9 or Cas12a / nCas12a / dCas12a, respectively) and a guide RNA to introduce local DNA cutting, nicking or R-loop formation, wherein the guide RNA contains an aptamer (e.g., MS2, or PP7, or BoxB) to recruit SSAP proteins; (2) an RNA sequence carrying the desired DNA change having one or more homology arm (HA) regions, which is fused / linked to the guide RNA in (1), or fused / linked to a second guide RNA. The HA region is at least 20 bp and provides a homology region next to the editing site for SSAP-mediated editing. If a second guide RNA is used, the second guide RNA will bind to a nearby genomic site that is located 0 bp to 150 bp from the guide RNA in (1). The second guide RNA then forms a complex with a CRISPR enzyme (e.g., Cas9 / nCas9 / dCas9 and Cas12a / nCas12a / dCas12a), is recruited to the target genomic locus, and provides an RNA template / donor for editing. These enzymes are conventional CRISPR enzymes, or Cas proteins, but can also be nicked or inactivated CRISPR enzymes that only bind to the target locus (dCas9, dCas12a, etc.). The guide is a conventional guide RNA or a shorter guide RNA (usually 2 to 6 bp shorter than a conventional guide RNA, i.e., 14 bp to 18 bp) to allow effective binding but not cutting of the target. (3) SSAP protein, which is fused to an RNA aptamer binding protein (RBP) via a linker. RBP is an MS2 coat protein (MCP), a PP7 coat protein (PCP), or a BoxB binding peptide (λN22 peptide) from λ bacteriophage. For this component, the applicant also identified additional factors that enhance this RNA-templated SSAP gene editing: when the applicant fused the reverse transcriptase (RT) to the SSAP protein through a long peptide linker, a third component RBP-SSAP-RT or RBP-RT-SSAP (- represents the linker) was formed, which further improved the editing efficiency.

[0217] In a second embodiment, Cas9 / nCas9 / dCas9 or Cas12a / nCas12a / dCas12a protein is fused to reverse transcriptase (RT) through a joint. The guide RNA in this design also has a primer binding site (PBS) of at least 14-bp or more, which is complementary to the region at the editing site. The PBS helps to trigger RT activity. Alternatively, another design uses the same guide RNA as the first embodiment, and triggers RT activity by providing cells with a short oligoDNA (14bp or longer) complementary to the region at the editing site. This oligoDNA triggers RT activity and allows SSAP-mediated gene editing.

[0218] In a third embodiment, the Cas9 / nCas9 / dCas9 or Cas12a / nCas12a / dCas12a protein is fused to a reverse transcriptase (RT) from a reverse transcription subsystem via a linker. The guide RNA in this design has a msr / msd sequence from a reverse transcriptase, and one or more homology arms (HA) regions that are complementary to the region at the editing site. The msr / msd sequence helps to trigger RT activity. The HA region helps mediate SSAP gene editing.

[0219] Overall, this set of tools and methods provides novel and non-obvious RNA-mediated / RNA-templated gene editing in eukaryotic / mammalian cells. The applicant further demonstrated that by designing cleavable RNA templates using endogenous tRNA, ribozymes, or direct repeat sequences from the Cas12a system, the applicant also achieved multi-target gene editing using RNA as a template.

[0220] Regarding the characteristics of the RT system and composition: The applicant's RNA-templated SSAP gene editing system has five advantages: (1) because RNA has lower immunogenicity than DNA used in existing gene editing processes, and RNA cannot directly integrate into unintended genomic DNA sites or off-target DNA sites, thereby reducing off-target or toxicity; (2) by using a cleavable RNA template in the applicant's method, multiplexing (multiplex) precise gene editing methods can be easily achieved; (3) the simplicity of RNA delivery to cells, easier production, and lower cost for expanding clinical use; (4) RNA has great engineering potential, which can be combined with other regulatory or combinatorial payloads / components through chemical connection or biochemical coupling to achieve more efficient delivery, editing or to make RNA-templated gene editing more synergistic with other types of gene editing or therapeutic methods; and (5) the efficiency of RNA-templated gene editing can be enhanced by RNA and protein factors, and is orthogonal to conventional DNA repair pathways that may be critical to the health of target cells.

[0221] RNA-guided recombinant protein system. In certain embodiments or the present invention, a system or composition for RNA-guided recombinant engineering is provided, which does not rely primarily on CRISPR protein. In such embodiments, the system or composition comprises: a nucleic acid molecule and a recombinant protein comprising a guide RNA sequence complementary to a target DNA sequence. In certain embodiments, the system or composition can promote R-loop formation. In certain embodiments, the system or composition can be recombined. In certain embodiments, the system or composition does not contain CRISPR protein. In certain embodiments, the recombinant protein comprises a microbial recombinant protein. In certain embodiments, the recombinant protein comprises a viral recombinant protein. In certain embodiments, the recombinant protein comprises a eukaryotic recombinant protein. In certain embodiments, the recombinant protein comprises a mitochondrial recombinant protein. In various embodiments, the recombinant protein comprises a single-stranded DNA annealing protein (SSAP), a single-stranded DNA binding protein (SSB), an exonuclease, or a combination of two or more thereof. In certain embodiments, the system or composition does not include Cas9. In certain embodiments, the system or composition does not include Cas12a. In certain embodiments, the system or composition does not include Cas. In certain embodiments, the system or composition does not include CRISPR.

[0222] Without being bound by theory, the system can be considered to comprise a guide nucleic acid that promotes R-loop formation by binding to the target DNA and a recombination protein that promotes recombination between the target nucleic acid and the donor nucleic acid.

[0223] In certain embodiments, the guide RNA and the recombinant protein are operably connected. In some embodiments, the connection is covalent. In some embodiments, the connection is non-covalent. In certain embodiments, the guide nucleic acid comprises an aptamer sequence and the recombinant protein comprises or is connected to an aptamer binding domain. The following table provides non-limiting examples of R-loop guide nucleic acids used in the present invention.

[0224] The R-loop-guide RNA comprises a guide component and a backbone component arranged in various ways, e.g., a guide-backbone and a backbone-guide configuration. In certain embodiments, the R-loop-guide RNA comprises a guide located at the 5' end of the backbone. In certain embodiments, the R-loop-guide RNA comprises a guide located at the 3' end of the backbone.

[0225] The guide sequence is engineered to bind to the target DNA (genomic target). In certain embodiments, the guide is 17 to 160 bases. The backbone comprises one or more aptamer sequences. The aptamers used in the present invention include, but are not limited to, MS2, PP7, BoxB, etc. In certain embodiments, the fusion protein comprises an RNA binding component and a SSAP protein (e.g., but not limited to, RecT, λRed, T7gp2.5, etc.) that binds to an aptamer, such as those described above.

[0226] The donor nucleic acid can be single-stranded or double-stranded DNA and contains (1) homology arms (HA) of various lengths to match the genomic target region, and (2) a transgene, e.g., a knock-in sequence or a replacement sequence, etc. The size of the transgene is not limited. In this paper, 600-bp ( Fig.70 ) and 800-bp( Fig.71 ) as an example.

[0227] In certain embodiments, the R-loop-guide RNA binds to an RNA binding protein or domain fused to a recombinant protein, such as, but not limited to, a SSAP.

[0228] In various embodiments, the invention provides fusion proteins. In the fusion protein, the recombinant protein can be connected to either end of the aptamer binding protein (e.g., N-terminus to C-terminus, C-terminus to N-terminus, N-terminus to N-terminus) in any direction. In a preferred embodiment, the recombinant protein N-terminus is connected to the aptamer binding protein C-terminus. Therefore, the overall fusion protein from the N-terminus to the C-terminus comprises the aptamer binding protein (N-terminus to C-terminus), which is connected to the recombinant protein (N-terminus to C-terminus).

[0229] In some embodiments, the recombinant protein can be functionally connected to a nuclease as a fusion protein or chimera or chimeric molecule. In some embodiments, the recombinant protein can be functionally connected to an endonuclease as a fusion protein or chimera or chimeric molecule. In some embodiments, the recombinant protein can be functionally connected to an exonuclease as a fusion protein or chimera or chimeric molecule. In some embodiments, the recombinant protein can be functionally connected to a nuclease and / or Cas or dCas as a fusion protein or chimera or chimeric molecule. In some embodiments, the recombinant protein can be functionally connected to an endonuclease and / or Cas or dCas as a fusion protein or chimera or chimeric molecule. In some embodiments, the recombinant protein can be functionally connected to an exonuclease and / or Cas or dCas as a fusion protein or chimera or chimeric molecule. In some embodiments, the recombinant protein can be functionally connected to an exonuclease and / or Cas or dCas as a fusion protein or chimera or chimeric molecule. In some embodiments, the recombinant protein can be expressed independently of the nuclease, rather than a fusion protein with the nuclease. In some embodiments, the recombinant protein can be expressed independently of the endonuclease, rather than a fusion protein with the endonuclease. In some embodiments, the recombinant protein can be expressed independently of the exonuclease, rather than as a fusion protein with the exonuclease. In some embodiments, the recombinant protein can be expressed independently of the nuclease and / or Cas or dCas, rather than as a fusion protein with the nuclease and / or Cas or dCas. In some embodiments, the recombinant protein can be functionally connected to an aptamer and / or aptamer binding protein as a fusion protein or chimera or chimeric molecule. In some embodiments, the recombinant protein can be expressed independently of the aptamer and / or aptamer binding protein, rather than as a fusion protein with an aptamer and / or aptamer binding protein. In some embodiments, the recombinant protein can be functionally connected to a nuclease and / or Cas or dCas and / or an aptamer and / or an aptamer binding protein as a fusion protein or chimera or chimeric molecule. In some embodiments, the recombinant protein can be expressed independently of the nuclease and / or Cas or dCas and / or an aptamer and / or an aptamer binding protein, rather than as a fusion protein with a nuclease and / or Cas or dCas and / or an aptamer and / or an aptamer binding protein. In some embodiments, the aptamer and / or aptamer binding protein is an MCP protein. In some embodiments, the recombinant protein may be a SSAP.

[0230] As used herein, the term "nuclease" refers to a substance, such as a protein or a small molecule, that can cut the phosphodiester bond connecting nucleotide residues in a nucleic acid molecule. In some embodiments, the nuclease is woven, for example, an enzyme that can bind to a nucleic acid molecule and cut the phosphodiester bond connecting nucleotide residues in a nucleic acid molecule. The nuclease can be an endonuclease that cuts the phosphodiester bond in a polynucleotide chain, or an exonuclease that cuts the phosphodiester bond at the end of a polynucleotide chain. In some embodiments, the nuclease is a site-specific nuclease that binds and / or cuts a specific phosphodiester bond within a specific nucleotide sequence, which is also referred to herein as a "recognition sequence," "nuclease target site," or "target site." In some embodiments, the nuclease is an RNA-guided (e.g., RNA programmable) nuclease that is compounded (e.g., bound) with an RNA having a sequence complementary to the target site, thereby providing sequence specificity of the nuclease. In some embodiments, the nuclease recognizes a single-stranded target site, while in other embodiments, the nuclease recognizes a double-stranded target site, for example, a double-stranded DNA target site. The target sites of many naturally occurring nucleases (e.g., many naturally occurring DNA restriction nucleases) are well known to those skilled in the art. In many cases, DNA nucleases (e.g., EcoRI, HindIII, or BamHI) recognize palindromic double-stranded DNA target sites of 4 to 10 base pairs in length, and cut each of the two DNA chains at a specific position within the target site. Some endonucleases symmetrically cut double-stranded nucleic acid target sites, e.g., cut two chains at the same position so that the ends contain base-paired nucleotides, also referred to herein as flat ends. Other endonucleases asymmetrically cut double-stranded nucleic acid target sites, e.g., cut each chain at different positions so that the ends contain unpaired nucleotides. The unpaired nucleotides at the ends of double-stranded DNA molecules are also referred to as "overhangs," e.g., "5'-overhangs" or "3'-overhangs," depending on whether the unpaired nucleotides form the 5' end or the 3' end of the corresponding DNA chain. The ends of double-stranded DNA molecules terminated with unpaired nucleotides are also referred to as sticky ends, so they can "stick" to the ends of other double-stranded DNA molecules containing complementary unpaired nucleotides. Nuclease proteins typically include a "binding domain" that mediates the interaction of the protein with the nucleic acid substrate (and in some cases also specifically binds to the target site) and a "cleavage domain" that catalyzes the cleavage of the phosphodiester bond within the nucleic acid backbone. In some embodiments, the nuclease protein is able to bind and cleave nucleic acid molecules in a monomeric form, while in other embodiments, the nuclease protein must dimerize or otherwise cleave the target nucleic acid molecule. The binding and cleavage domains of naturally occurring nucleases and the mode binding and cleavage domains that can be fused to produce nucleases are well known to those skilled in the art.For example, zinc fingers or transcription activator-like elements can be used as binding domains to specifically bind to a desired target site and fused or conjugated to a cleavage domain (e.g., the cleavage domain of fok1) to create an engineered nuclease that cleaves the target site.

[0231] Non-limiting examples of exonucleases include exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VII, exonuclease VIII, lambda exonuclease, Xrn1, mung bean nuclease, TREX2, exonuclease T, T7 exonuclease, strandase exonuclease, 3'-5' exophosphodiesterase, and Bal31 nuclease.

[0232] In some embodiments, the fusion protein further comprises a joint between the microbial recombinant protein and the aptamer-binding protein. The joint can comprise any amino acid sequence of any length. The joint can be flexible so that they do not constrain any of the two components connected together by them in any particular direction. The joint can basically serve as a spacer. In a preferred embodiment, the joint connects the C-terminus of the recombinant protein to the N-terminus of the aptamer-binding protein. In a preferred embodiment, the joint comprises the amino acid sequence of the XTEN joint SGSETPGTSESATPES (SEQ ID NO: 15) of 16 residues or the EXTEN joint SASGGSSGGSSGSETPGTSESATPESSGGSSGGSGGS (SEQ ID NO: 148) of 37 residues.

[0233] In some embodiments, the fusion protein further comprises a nuclear localization sequence (NLS). The nuclear localization sequence can be located at any position within the fusion protein (e.g., the C-terminus of the aptamer binding protein, the N-terminus of the aptamer binding protein, the C-terminus of the recombinant protein). In a preferred embodiment, the nuclear localization sequence is connected to the C-terminus of the recombinant protein. Many nuclear localization sequences are known in the art (see, e.g., Lange, A., et al., J Biol Chem. 2007; 282 (8): 5101-5105, which is incorporated herein by reference), and can be used in conjunction with the present invention. The nuclear localization sequence can be SV40 NLS, PKKKRKV (SEQ ID NO: 16); Ty1 NLS, NSKKRSLEDNETEIKVSRDTWNTKNMRSLEPPRSKKRIH (SEQ ID NO: 17); c-Myc NLS, PAAKRVKLD (SEQ ID NO: 18); biSV40 NLS, KRTADGSEFESPKKKRKV (SEQ ID NO: 19); and MutNLS, PEKKRRRPSGSVPVLARPSPPKAGKSSCI (SEQ ID NO: 20). In a preferred embodiment, the nuclear localization sequence is SV40 NLS, PKKKRKV (SEQ ID NO: 16).

[0234] Cas proteins and fusion proteins are ideally included separately in a single composition, combined with each other and / or one or more polynucleotides (e.g., vectors) comprising guide RNA sequences and aptamer sequences. Cas proteins and / or fusion proteins may or may not be physically or chemically bound to polynucleotides. Any suitable method known in the art for protein-protein connection or protein-virus connection can be used to bind Cas proteins and / or recombinant proteins to polynucleotides.

[0235] The present invention also provides a composition and a vector comprising a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a recombinant protein functionally linked to an RNA aptamer binding protein.

[0236] The composition or vector may further include at least one or both of a polynucleotide comprising a nucleic acid sequence encoding a Cas protein and a nucleic acid molecule comprising a guide RNA sequence complementary to a target DNA sequence. In some embodiments, the nucleic acid molecule comprising a guide RNA sequence further comprises at least one RNA aptamer sequence. In some embodiments, the polynucleotide comprising a nucleic acid sequence encoding a Cas protein further comprises a sequence encoding at least one peptide aptamer sequence.

[0237] The above descriptions related to the nucleic acid molecules comprising guide RNA sequences, aptamer sequences, Cas proteins, recombinant proteins and aptamer binding proteins related to the system of the present invention also apply to the polynucleotides of the described compositions and vectors.

[0238] The nucleic acid sequence encoding the Cas protein and / or the nucleic acid sequence encoding the fusion protein comprising the recombinant protein functionally connected to the aptamer binding protein can be provided to the cell on the same vector as the nucleic acid molecule comprising the guide RNA sequence and / or the RNA aptamer sequence (e.g., in a cis manner). In such embodiments, a unidirectional promoter can be used to control the expression of each nucleic acid sequence. In another embodiment, a combination of bidirectional and unidirectional promoters can be used to control the expression of multiple nucleic acid sequences.

[0239] In other embodiments, the nucleic acid sequence encoding the Cas protein, the nucleic acid sequence encoding the fusion protein comprising the recombinant protein functionally connected to the aptamer binding protein, and the nucleic acid molecule sequence comprising the guide RNA sequence and / or the RNA aptamer can be provided to the cell on a separate vector (e.g., in a trans manner). Each nucleic acid sequence in each separate vector can contain the same or different expression control sequences. The separate vectors can be provided to the cell simultaneously or sequentially.

[0240] One or more vectors comprising a nucleic acid sequence encoding a Cas protein and a nucleic acid sequence encoding a fusion protein comprising a recombinant protein functionally connected to an aptamer binding protein can be introduced into a host cell capable of expressing the polypeptide encoded thereby, including any suitable prokaryotic or eukaryotic cell. Therefore, the present invention provides a cell comprising a separation of a vector or nucleic acid sequence disclosed herein. Preferred host cells are those that can grow easily and reliably, have a relatively fast growth rate, have a well-characterized expression system, and can be easily and effectively transformed or transfected. Examples of suitable prokaryotic cells include, but are not limited to, cells from the genus Bacillus (Bacillus) (e.g., Bacillus subtilis and Bacillus brevis), Escherichia (Escherichia) (e.g., Escherichia coli), Pseudomonas, Streptomyces, Salmonella, and Envinia. Suitable eukaryotic cells are known in the art, and include, for example, yeast cells, insect cells, and mammalian cells. Examples of suitable yeast cells include yeast cells from Kluyveromyces, Pichia, Rhino-sporidium, Saccharomyces, and Schizosaccharomyces. Exemplary insect cells include Sf-9 and HIS (Invitrogen, Carlsbad, CA), and are described in, for example, Kitts et al., Biotechniques, 14: 810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4: 564-572 (1993); and Lucklow et al., J. Virol., 67: 4566-4579 (1993), which are incorporated herein by reference. Ideally, the host cell is a mammalian cell, and in some embodiments, the host cell is a human cell. Many suitable mammalian and human host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, Va.).Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR-cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573) and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), and CV-1 cell line (ATCC No. CCL70). Other exemplary mammalian host cells include primate, rodent and human cell lines, including transformed cell lines. Normal diploid cells, cell lines derived from primary tissue culture in vitro, and primary explants are also suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, HEK, A549, HepG2, mouse L-929 cells, and BHK or HaK hamster cell lines. Methods for selecting suitable mammalian host cells and methods for cell transformation, culture, expansion, screening, and purification are known in the art.

[0241] Methods for changing target DNA. The present invention also provides methods for changing target DNA. In some embodiments, the method changes the genomic DNA sequence in the cell, although any desired nucleic acid can be modified. When applied to DNA contained in a cell, the method includes introducing the system, composition or vector described herein into a cell containing a target genomic DNA sequence. The description of nucleic acid molecules, Cas proteins, recombinant proteins, recruitment systems and polynucleotides encoding them, cells, target genomic DNA sequences and components thereof related to the above-mentioned systems of the present invention are also applicable to methods for changing target genomic DNA sequences in cells. These systems, compositions or vectors can be introduced in any manner known in the art, including but not limited to chemical transfection, electroporation, microinjection, bioballistic delivery by gene guns or magnetic-assisted transfection, depending on the cell type.

[0242] In certain embodiments, the delivery of the editing system or component includes the delivery of a ribonucleoprotein (RNP) complex. According to the present invention, a targeting nucleic acid (including but not limited to gRNA, dgRNA) can be provided in a complex, such as but not limited to a complex comprising Cas9 or dCas9. In certain embodiments, the RNP complex includes a guide nucleic acid and a Cas9 fusion protein, such as but not limited to a complex comprising dCas9-SSAP. In certain embodiments, the RNP complex includes a guide nucleic acid and a recombinant protein, such as SSAP or SSB, which can be transformed or modified to be combined with a guide nucleic acid. In certain embodiments, the guide nucleic acid and the recombinant protein or the Cas9 fusion protein include a binding element that promotes the formation of the complex. In a non-limiting example, the recombinant protein includes an MCP domain, and the guide RNA includes an MS2 aptamer, whereby the combination of the MS2 aptamer and the MCP domain produces RNP.

[0243] In some embodiments, the guide RNA and Cas and / or recombinant protein polypeptide are incubated together to form a ribonucleoprotein (RNP) complex, and then introduced into the cell, for example, mixed together in a container (vessel) to form an RNP complex, and then the RNP complex is introduced into the cell. In other embodiments, the Cas polypeptide described herein can be an mRNA encoding the Cas polypeptide, and the Cas mRNA is introduced into primary cells as a "full RNA" CRISPR system together with the modified sgRNA.

[0244] In some embodiments, RNP complex and donor nucleic acid or carrier are introduced into cell simultaneously.In other embodiments, RNP complex and donor nucleic acid or carrier are introduced into primary cell sequentially.In some cases, RNP complex is introduced into primary cell before donor.In other cases, donor is introduced into primary cell before RNP complex.For example, RNP complex can be introduced into cell about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes or 240 minutes or longer time before donor nucleic acid or carrier, and vice versa. U.S. Patent No. 11,193,141 describes the introduction of RNP complexes and homologous donor adeno-associated virus (AAV) vectors into cells to mediate targeted integration. The method can be used with the present invention. U.S. Patent Publication No. 2019 / 0093128 describes the introduction of ribonucleoproteins (RNPs) into zygotes, the RNPs comprising Class 2 CRISPR / Cas endonucleases, which are complexed with corresponding CRISPR / Cas guide RNAs that hybridize to target sequences within the genomic DNA of the zygote.

[0245] Non-limiting examples include the use of (1) purified Cas9 or dCas9 protein; (2) synthetic guide RNA with MS2 aptamer; (3) purified MCP-SSAP fusion protein; (4) donor DNA (double-stranded, single-stranded DNA donors for HEK293 and K562; and AAV donors for HSCs) delivered to HEK293, K562, and primary hematopoietic stem cells (mouse and human) for knock-in editing.

[0246] The following table provides exemplary sequences for generating knock-ins including ALB and AAVS1. These sequences can be used in RNPs, nucleic acids, vectors, for expression, etc.

[0247] When the system described herein is introduced into a cell comprising a target genomic DNA sequence, the guide RNA sequence binds to the target genomic DNA sequence in the cell genome, the Cas protein associates with the guide RNA and can induce a double-strand break or a single-strand nick in the target genomic DNA sequence, and the aptamer recruits the recombinant protein to the target genomic DNA sequence through the aptamer binding protein of the fusion protein, thereby changing the target genomic DNA sequence in the cell. When the composition or vector described herein is introduced into a cell, a nucleic acid molecule comprising a guide RNA sequence, a Cas9 protein, and a fusion protein is first expressed in the cell.

[0248] In some embodiments, the cell is in an organism or host, so that introducing the disclosed system, composition, or vector into the cell comprises administering to a subject. The method may include providing or administering to a subject in vivo or by transplanting cells, systems, compositions, or vectors of the system treated ex vivo.

[0249] "Subjects" can be human or non-human, and can include, for example, animal strains or species used as "model systems" for research purposes, such as the mouse model described herein. Similarly, subjects can include adults or adolescents (e.g., children). In addition, subjects can mean any living organism that can benefit from the administration of the composition contemplated herein, preferably mammals (e.g., humans or non-humans). Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates (e.g., chimpanzees), and other apes and monkeys; farm animals, such as cattle, horses, sheep, goats, pigs; livestock, such as rabbits, dogs and cats; experimental animals, including rodents, such as rats, mice and guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc. In one embodiment of the methods and compositions provided herein, the mammal is a human. Plants include, but are not limited to, sugarcane, corn, wheat, rice, oil palm fruit, potatoes, soybeans, vegetables, cassava, beets, tomatoes, barley, bananas, watermelons, onions, sweet potatoes, cucumbers, apples, cotton seeds, oranges, etc.

[0250] As used herein, the terms "providing", "administering", and "introducing" are used interchangeably herein and refer to placing the system of the present invention into a subject by a method or route that results in at least partial localization of the system to a desired site. The system can be administered by any appropriate route that results in delivery to a desired location in a subject.

[0251] As used herein, the phrase "altering a DNA sequence" refers to modifying at least one physical characteristic of a DNA sequence of interest. DNA alterations include, for example, single-strand or double-strand DNA breaks, deletions or insertions of one or more nucleotides, and other modifications that affect the structural integrity of the DNA sequence or the nucleotide sequence. Modification of a target sequence in genomic DNA can result in, for example, gene correction, gene replacement, gene tagging, transgenic insertion, nucleotide deletion, gene disruption, gene mutation, gene knockdown, and the like.

[0252] In some embodiments, the systems and methods described herein can be used to correct one or more defects or mutations in a gene (referred to as "gene correction"). In this case, the target genomic DNA sequence encodes a defective version of the gene, and the system further comprises a donor nucleic acid molecule encoding a wild-type or corrected version of the gene. Therefore, in other words, the target genomic DNA sequence is a "disease-associated" gene. The term "disease-associated gene" refers to any gene or polynucleotide whose gene product is expressed at an abnormal level or in an abnormal form in a cell obtained from an individual affected by the disease, compared to a tissue or cell obtained from an individual not affected by the disease. Disease-associated genes can be expressed at abnormally high levels or abnormally low levels, wherein the altered expression is associated with the occurrence and / or progression of the disease. Disease-associated genes also refer to genes whose mutations or genetic variations directly lead to the cause of the disease or are in linkage disequilibrium with one or more genes that lead to the cause of the disease. Examples of genes that cause such "single gene" or "monogenic" diseases include, but are not limited to, adenosine deaminase, alpha-1 antitrypsin, cystic fibrosis transmembrane conductance regulator (CFTR), beta-hemoglobin (HBB), oculocutaneous albinism II (OCA2), huntingtin (HTT), dystonia protein kinase (DMPK), low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), neurofibromin 1 (NF1), polycystic kidney disease 1 (PKD1), polycystic kidney disease 2 (PKD2), coagulation factor VIII (F8), dystrophin (DMD), phospho-regulatory endopeptidase homolog, X-linked (PHEX), methyl CpG binding protein 2 (MECP2), and ubiquitin-specific peptidase 9Y, Y-linked (USP9Y). Other single genes or monogenic diseases are known in the art and are described in, for example, Chial, H. Rare Genetic Disorders: Learning About Genetic Disease Through Gene Mapping, SNPs, and Microarray Data, Nature Education 1(1):192 (2008), which is incorporated herein by reference; Online Mendelian Inheritance in Man (OMIM); and Human Gene Mutation Database (HGMD).

[0253] The present invention provides knocking in large transgenes at treatment-related loci in the human genome. In certain embodiments, the locus provides cell or tissue-specific expression. In certain embodiments, the present invention includes inserting nucleic acids into the albumin (ALB) locus. The ALB locus provides liver targeting in human hepatocytes, is highly expressed and expressed in a liver-specific manner. In certain embodiments, the present invention includes inserting nucleic acids into the AAVS1 locus. The AAVS1 locus is a safe harbor locus for gene therapy, which is well expressed in certain tissue types and can be used for a variety of treatments, with low expression in the liver. U.S. Patent Publication 2018 / 0214490A1 describes gene therapy for lysosomal storage diseases, including targeting transgenic "safe harbor" loci, such as AAVS1, HPRT and CCR5 genes in human cells, and Rosa26 in mouse cells. U.S. Patent No. 9267154 describes the integration of exogenous nucleic acid sequences into the PPP1R12C locus, which is widely expressed in most tissues. Described is achieving cell-specific expression by targeting a transgene (e.g., encoding a chimeric antigen receptor (CAR)) to the T cell receptor alpha constant (TRAC) locus. For loci that can be targeted according to the present invention, these are exemplary and non-limiting loci.

[0254] In another embodiment, the target genomic DNA sequence can include a gene whose mutations in combination with mutations in other genes lead to a specific disease. Diseases caused by multiple gene causes that lack a simple (e.g., Mendelian) inheritance pattern are referred to in the art as "multifactorial" or "polygenic" diseases. Examples of multifactorial or polygenic diseases include, but are not limited to, asthma, diabetes, epilepsy, hypertension, bipolar disorder, and schizophrenia. Certain developmental abnormalities can also be inherited in a multifactorial or polygenic pattern, including, for example, cleft lip / palate, congenital heart defects, and neural tube defects.

[0255] In another embodiment, the method of altering a target genomic DNA sequence can be used to delete nucleic acids from a target sequence in a cell by cleaving the target sequence and allowing the cell to repair the cleaved sequence in the absence of an exogenously provided donor nucleic acid molecule. Deleting nucleic acid sequences in this manner can be used for a variety of applications, for example, to remove disease-causing trinucleotide repeats in neurons, to generate gene knockouts or knockdowns, and to generate mutations for disease models in research.

[0256] The term "donor nucleic acid molecule" refers to a nucleotide sequence that is inserted into a target DNA (e.g., genomic DNA). As described above, the donor DNA can include, for example, a gene or a portion of a gene, a sequence encoding a tag or a positioning sequence, or a regulatory element. The donor nucleic acid molecule can have any length. In some embodiments, the length of the donor nucleic acid molecule is between 10 and 10,000 nucleotides. For example, the length is between about 100 and 5,000 nucleotides, the length is between about 200 and 2,000 nucleotides, the length is between about 500 and 1,000 nucleotides, the length is between about 500 and 5,000 nucleotides, the length is between about 1,000 and 5,000 nucleotides, or the length is between about 1,000 and 10,000 nucleotides,

[0257] The disclosed systems and methods overcome the challenges encountered in conventional gene editing processes, including low efficiency and off-target events, particularly for nucleic acids on a kilobase scale. In some embodiments, the disclosed systems and methods improve the efficiency of gene editing. For example, as shown in Examples 2, 3, and 5, the disclosed systems and methods are 2 to 10 times more efficient than conventional CRISPR-Cas9 systems and methods. In some embodiments, the increase in efficiency is accompanied by a reduction in off-target events. Compared with conventional CRISPR-Cas9 systems and methods, off-target events can be reduced by more than 50%, for example, Example 3 shows that off-target events are reduced by about 90%. Another aspect of improving the overall accuracy of the gene editing system is to reduce on-target insertion-deletion (indel), which is a byproduct of HDR editing. In some embodiments, compared with conventional CRISPR-Cas9 systems and methods, the disclosed systems and methods reduce on-target insertion-deletion by more than 90%, as shown in Example 3.

[0258] The present invention further provides a kit comprising one or more reagents or other components that are useful, necessary or sufficient for implementing any of the methods described herein. For example, the kit may include CRISPR reagents (Cas proteins, guide RNAs, vectors, compositions, etc.), recombinant engineering reagents (recombinant protein-aptamer binding protein fusion proteins, aptamer sequences, vectors, compositions, etc.), transfection or administration reagents, negative and positive control samples (e.g., cells, template DNA), cells, containers for holding one or more components (e.g., microcentrifuge tubes, boxes), detectable labels, detection and analysis instruments, software, instructions, etc.

[0259] RNA can be delivered using adeno-associated virus (AAV), slow virus, adenovirus or other viral vector types or their combination. RNA can be packaged into one or more viral vectors. In some embodiments, viral vectors are delivered to tissues of interest by, for example, intramuscular injection, and other times viral delivery is by intravenous, percutaneous, intranasal, oral, mucosal or other delivery methods. This delivery can be carried out by single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be delivered herein can vary greatly according to various factors (for example, the general condition of the selected carrier, target cell, organism or tissue, subject to be treated, the degree of conversion / modification sought, route of administration, mode of administration, the type of conversion / modification sought, etc.).

[0260] Such a dose may further contain, for example, a carrier (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, a pharmaceutically acceptable carrier (e.g., phosphate buffered saline), a pharmaceutically acceptable excipient, and / or other compounds known in the art. Those skilled in the art can easily determine such dosage formulations. The dose may further contain one or more pharmaceutically acceptable salts, for example, inorganic acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and salts of organic acids, such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances may also be present, such as wetting agents or emulsifiers, pH buffer substances, gels or gelling materials, flavoring agents, colorants, microspheres, polymers, suspending agents, etc. In addition, one or more other conventional pharmaceutical ingredients may also be present, such as preservatives, wetting agents, suspending agents, surfactants, antioxidants, anti-caking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc., particularly if the dosage form is a resolvable form. Suitable exemplary ingredients include microcrystalline cellulose, sodium carboxymethyl cellulose, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, parachlorophenol, gelatin, albumin and combinations thereof. A comprehensive discussion of pharmaceutically acceptable excipients is found in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991), which is incorporated herein by reference.

[0261] In embodiments herein, delivery is via an adenovirus, which may contain at least 1×10 5 A single booster dose of at least about 1×10 adenoviral vector particles (also referred to as particle units, pu). In the embodiments herein, the dose is preferably at least about 1×10 6 particles (e.g., about 1×10 6 -1×10 12particles), more preferably at least about 1×10 10 particles, more preferably at least about 1×10 8 particles (e.g., about 1×10 8 -1×10 11 particles or about 1×10 8 -1×10 12 particles), and most preferably at least about 1×10 10 particles (e.g., about 1×10 9 -1×10 10 particles or about 1×10 9 -1×10 12 particles), or even at least about 1×10 10 particles (e.g., about 1×10 10 -1×10 12 Optionally, the dose contains no more than about 1×10 14 particles, preferably no more than about 1×10 13 particles, even more preferably no more than about 1×10 12 particles, even more preferably no more than about 1×10 11 particles, and most preferably no more than about 1×10 10 particles (e.g., no more than about 1×10 9 Thus, a dose may contain a single dose of adenoviral vector, for example, about 1×10 6 Particle unit (pu), about 2×10 6 pu, about 4×10 6 pu, about 1×10 7 pu, about 2×10 7 pu, about 4×10 7 pu, about 1×10 8 pu, about 2×10 8 pu, about 4×10 8 pu, about 1×10 9 pu, about 2×10 9 pu, about 4×10 9 pu, about 1×10 10 pu, about 2×10 10 pu, about 4×10 10 pu, about 1×10 11 pu, about 2×10 11 pu, about 4×10 11 pu, about 1×10 12 pu, about 2×10 12 pu or about 4×10 12See, e.g., the adenoviral vectors in U.S. Pat. No. 8,454,972 B2, issued Jun. 4, 2013 to Nabel et al., incorporated herein by reference, and the dosages at col. 29, lines 36-58 thereof. In embodiments herein, the adenovirus is delivered in multiple doses.

[0262] In embodiments herein, delivery is via AAV. A therapeutically effective dose for in vivo delivery of AAV to humans is believed to be in the range of about 20 ml to about 50 ml of saline solution containing about 1×10 10 About 1×10 10 Functional AAV / ml solution. The dosage can be adjusted to balance the therapeutic benefit with any side effects. In the embodiments herein, the AAV dosage is generally about 1×10 5 to 1×10 50 AAV genome, about 1×10 8 to 1×10 20 AAV genome, about 1×10 10 to about 1×10 16 genomes, or about 1×10 11 to about 1×10 16 The human dose can be about 1×10 13 The invention relates to a method for preparing an AAV vector comprising: administering a single-genome AAV. Such concentrations may be delivered in a carrier solution of about 0.001 ml to about 100 ml, about 0.05 ml to about 50 ml, or about 10 ml to about 25 ml. Other effective doses may be readily determined by one of ordinary skill in the art by routine experiments to establish dose-effect curves. See, e.g., U.S. Pat. No. 8,404,658 B2, issued to Hajjar et al. on March 26, 2013, col. 27, lines 45-60.

[0263] In embodiments herein, delivery is by plasmid. In such plasmid compositions, the dosage should be sufficient to elicit a plasmid amount of response. For example, a suitable amount of plasmid DNA in a plasmid composition can be about 0.1 mg to about 2 mg, or about 1 μg to about 10 μg.

[0264] The dosage herein is based on an average 70 kg individual. The frequency of administration is within the scope of a medical or veterinary practitioner (e.g., physician, veterinarian) or a person skilled in the art. The mice used in the experiment were approximately 20 g. From the dosage administered to the 20 g mice, the dosage for a 70 kg individual can be inferred.

[0265] Lentiviruses are complex retroviruses that are able to infect and express their genes in both mitotic and post-mitotic cells. The most common lentivirus is the human immunodeficiency virus (HIV), which utilizes the envelope glycoproteins of other viruses to target a variety of cell types.

[0266] Lentivirus can be prepared as follows. After cloning pCasES10 (which contains the lentiviral transfer plasmid backbone), low-passage (p=5) HEK293FT was inoculated into a T-75 flask one day before transfection to reach 50% confluence in DMEM containing 10% fetal bovine serum and without antibiotics. After 20 hours, the culture medium was replaced with OptiMEM (serum-free) medium, and transfection was completed after 4 hours. Cells were transfected with 10 μg of lentiviral transfer plasmid (pCasES10) and the following packaging plasmids: 5 μg of pMD2.G (VSV-g pseudotype) and 7.5ug of psPAX2 (gag / pol / rev / tat). Transfection was completed in 4 mL of OptiMEM using a cationic lipid delivery agent (50uL Lipofectamine2000 and 100ul Plus reagent). After 6 hours, the culture medium was replaced with DMEM without antibiotics containing 10% fetal bovine serum.

[0267] Lentivirus can be purified as follows. Harvest the viral supernatant after 48 hours. First, clear the supernatant of debris and filter through a 0.45um low protein binding (PVDF) filter. Then, spin them in an ultracentrifuge at 24000rpm for 2 hours. Resuspend the viral pellet in 50ul DMEM at 4°C overnight. Then, aliquot them and freeze immediately at -80°C.

[0268] In another embodiment, minimal non-primate lentiviral vectors based on equine infectious anemia virus (EIAV) are also contemplated, particularly for ocular gene therapy (see, e.g., Balagaan, J Gene Med 2006; 8:275-285, published online at Wiley InterScience on November 21, 2005; available at: interscience.wiley.com. DOI: 10.1002 / jgm.845). In another embodiment, lentiviral gene therapy vectors based on equine infectious anemia virus (EIAV) are also contemplated. It expresses the angiostatin proteins endostatin and angiostatin, is delivered by subretinal injection for the treatment of reticular age-related macular degeneration (see, e.g., Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)), and can be modified according to the system of the present invention.

[0269] Lentiviral vectors have been disclosed for use in treating Parkinson's disease, see, e.g., U.S. Patent Publication No. 20120295960 and U.S. Patent Nos. 7,303,910 and 7,351,585. Lentiviral vectors have also been disclosed for use in treating ocular diseases, see, e.g., U.S. Patent Publication Nos. 20060281180, 20090007284, US20110117189; US20090017543; US20070054961, US20100317109. Lentiviral vectors have also been disclosed for delivery to the brain, see, e.g., U.S. Patent Publication Nos. US20110293571; US20110293571, US20040013648, US20070025970, US20090111106, and U.S. Patent No. 7,259,015.

[0270] Several types of particle delivery systems and / or formulations are known for a variety of biomedical applications. In general, a particle is defined as a small object that behaves as a whole unit in terms of transport and properties. Particles are further classified based on diameter, with coarse particles ranging between 2500 nanometers and 10,000 nanometers. Fine particles range in size from 100 nanometers to 2500 nanometers. Ultrafine particles, or nanoparticles, typically range in size from 1 nanometer to 100 nanometers. The basis for the 100-nm limit is that new properties that distinguish particles from larger bulk materials often develop at critical length scales below 100 nm.

[0271] As used herein, particle delivery systems / preparations are defined as any biological delivery systems / preparations comprising particles according to the present invention. Particles according to the present invention are any entities having a maximum dimension (e.g., diameter) less than 100 microns (μm). In some embodiments, the maximum dimension of particles of the present invention is less than 10. In some embodiments, the maximum dimension of particles of the present invention is less than 2000 nanometers (nm). In some embodiments, the maximum dimension of particles of the present invention is less than 1000 nanometers (nm). In some embodiments, the maximum dimension of particles of the present invention is less than 900nm, 800nm, 700nm, 600nm, 500nm, 400nm, 300nm, 200nm or 100nm. Typically, the maximum dimension (e.g., diameter) of particles of the present invention is 500nm or less. In some embodiments, the maximum dimension (e.g., diameter) of particles of the present invention is 250nm or less. In some embodiments, the maximum dimension (e.g., diameter) of particles of the present invention is 200nm or less. In some embodiments, the maximum dimension (e.g., diameter) of particles of the present invention is 150nm or less. In some embodiments, the maximum size (e.g., diameter) of particles of the present invention is 100 nm or less. In some embodiments of the present invention, smaller particles are used, for example, particles with a maximum size of 50 nm or less. In some embodiments, the maximum size of particles of the present invention ranges from 25 nm to 200 nm.

[0272] Use various different techniques to complete particle characterization (including, for example, characterization morphology, size, etc.). Common techniques are electron microscope (TEM, SEM), atomic force microscope (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visible spectroscopy, dual polarization interferometry and nuclear magnetic resonance (NMR). Characterization (size measurement) can be for natural particles (for example, before loading) or after loading carrier (carrier refers to one or more RNA and / or the carrier encoding it herein, and can include other components, carriers and / or excipients), to provide for any external, in vitro and / or in vivo application of the present invention The particle of the best size of delivery. In some preferred embodiments, particle size (for example, diameter) characterization is based on the measurement using dynamic laser scattering (DLS).

[0273] The particle delivery system within the scope of the present invention can be provided in any form, including but not limited to solid, semi-solid, emulsion or colloidal particles. Therefore, any delivery system described herein, including but not limited to, for example, lipid-based systems, liposomes, micelles, microvesicles, exosomes or gene guns, can be provided as a particle delivery system within the scope of the present invention.

[0274] For the present invention, it is preferred to use nanoparticles or lipid envelopes to deliver one or more components of the system. Nanoparticles or lipid envelopes can be used to deliver CRISPR enzyme mRNA and guide RNA simultaneously. Other delivery systems or carriers can be used in combination with the nanoparticle aspects of the present invention.

[0275] Typically, "nanoparticle" refers to any particle with a diameter less than 1000nm. In certain preferred embodiments, the maximum size (e.g., diameter) of the nanoparticles of the present invention is 500nm or less. In other preferred embodiments, the maximum size range of the nanoparticles of the present invention is between 25nm and 200nm. In other preferred embodiments, the maximum size of the nanoparticles of the present invention is 100nm or less. In other preferred embodiments, the maximum size range of the nanoparticles of the present invention is between 35nm and 60nm.

[0276] Nanoparticles encompassed by the present invention can be provided in different forms, for example, as solid nanoparticles (e.g., metals (e.g., silver, gold, iron, titanium), non-metals, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof. Metallic, dielectric, and semiconductor nanoparticles can be prepared, as well as hybrid structures (e.g., core-shell nanoparticles). If the nanoparticles made of semiconductor materials are small enough (typically less than 10 nm) that quantization of the electronic energy levels can occur, they can also be labeled as quantum dots. Such nanoscale particles are used as drug carriers or imaging agents in biomedical applications and can be suitable for similar purposes in the present invention.

[0277] Semisolid and soft nanoparticles have been produced and are within the scope of the present invention. The prototype nanoparticle of semisolid nature is liposome. Various types of liposome nanoparticles are currently used clinically as delivery systems for anticancer drugs and vaccines. Nanoparticles that are half hydrophilic and the other half hydrophobic are called Janus particles and are particularly effective for stabilizing emulsions. They can self-assemble at the water / oil interface and act as solid surfactants.

[0278] For example, Su X, Fricke J, Kavanagh DG, Irvine DJ ("In vitro and in vivo mRNA delivery using lipid-enveloped pH-responsive polymer nanoparticles" Mol Pharm. 2011 Jun. 6; 8 (3): 774-87. doi: 10.1021 / mp100390w. Epub 2011 Apr. 1) describe biodegradable core-shell nanoparticles having a poly (β-amino ester) (PBAE) core encapsulated by a phospholipid bilayer shell. These are developed for in vivo mRNA delivery. The pH-responsive PBAE component is selected to promote endosomal destruction, while the lipid surface layer is selected to minimize the toxicity of the polycationic core. Therefore, these are preferably used to deliver the RNA of the present invention.

[0279] In one embodiment, nanoparticles based on self-assembling bioadhesive polymers are envisioned, which can be applied to oral delivery of peptides, intravenous delivery of peptides, and nasal delivery of peptides, all of which are delivered to the brain. Other embodiments are also envisioned, such as oral absorption and ocular delivery of hydrophobic drugs. Molecular envelope technology involves engineered polymer envelopes that are protected and delivered to the disease site. (See, for example, Mazza, M. et al. ACS Nano, 2013.7(2):1016-1026; Siew, A., et al. Mol Pharm, 2012.9(1):14-28; Lalatsa, A., et al. J Contr Rel, 2012.161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012.9(6):1665-80; Lalatsa, A., et al. Mol Pharm, 2012.9 (6): 1764-74; Garrett, NL, et al. J Biophotonics, 2012.5 (5-6): 458-68; Garrett, NL, et al. J Raman Spect,2012.43(5):681-688; Ahmad,S.,etal.J Royal Soc Interface 2010.7:S423-33; Uchegbu, IF Expert Opin Drug Deliv, 2006.3(5):629-40; Qu, X., et al. Biomacromolecules, 2006.7(12):3452-9 and Uchegbu, IF, et al. Int J Pharm, 2001.224:185-199). The envisaged dose is about 5 mg / kg, single dose or multiple doses, depending on the target tissue.

[0280] In one embodiment, nanoparticles developed by Dan Anderson's laboratory at MIT that can deliver RNA to cancer cells to prevent tumor growth can be used and / or adapted to the CRISPR Cas system of the present invention. In particular, the Anderson laboratory has developed a fully automated combined system for the synthesis, purification, characterization, and formulation of novel biomaterials and nanoformulations. See, e.g., Alabi et al., Proc Natl Acad Sci USA. 2013 Aug. 6; 110(32): 12881-6; Zhang et al., Adv Mater. 2013 Sep. 6; 25(33): 4641-5; Jiang et al., NanoLett. 2013 Mar. 13; 13(3): 1059-64; Karagiannis et al., ACS Nano. 2012 Oct. 23; 6(10): 8484-7; Whitehead et al., ACS Nano. 2012 Aug. 28; 6(8): 6922-9 and Lee et al., Nat Nanotechnol. 2012 Jun. 3; 7(6): 389-93.

[0281] U.S. Patent Application 20110293703 relates to lipidoid compounds, which are also particularly useful in the administration of polynucleotides and can be used to deliver the CRISPR Cas system of the present invention. In one aspect, amino alcohol lipidoid compounds are combined with reagents to be delivered to cells or subjects to form microparticles, nanoparticles, liposomes or micelles. The reagents delivered by particles, liposomes or micelles can be in the form of gas, liquid or solid, and the reagents can be polynucleotides, proteins, peptides or small molecules. Amino alcohol lipidoid compounds can be combined with other amino alcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form particles. Then, these particles can be optionally combined with pharmaceutical excipients to form pharmaceutical compositions.

[0282] U.S. Patent Publication No. 0110293703 also provides a method for preparing amino alcohol lipidoid compounds. One or more equivalents of amine are reacted with one or more equivalents of epoxide-terminated compounds under suitable conditions to form amino alcohol lipidoid compounds of the present invention. In certain embodiments, all amino groups of amine react completely with epoxide-terminated compounds to form tertiary amines. In other embodiments, all amino groups of amine do not react completely with epoxide-terminated compounds to form tertiary amines, thereby producing primary or secondary amines in amino alcohol lipidoid compounds. These primary or secondary amines remain intact or can react with another electrophilic reagent (e.g., different epoxide-terminated compounds). As will be appreciated by those skilled in the art, reacting amines with less than an excess of epoxide-terminated compounds will produce a variety of different amino alcohol lipidoid compounds with different numbers of tails. Some amines can be fully functionalized with two epoxide-derived compound tails, while other molecules are not fully functionalized with epoxide-derived compound tails. For example, a diamine or polyamine can include one, two, three or four epoxide-derived compound tails branching from various amino moieties of the molecule, producing primary amines, secondary amines and tertiary amines. In certain embodiments, all amino groups are not fully functionalized. In certain embodiments, two epoxide-terminated compounds of the same type are used. In other embodiments, two or more different epoxide-terminated compounds are used. The synthesis of amino alcohol lipidoid compounds is carried out with or without solvent, and the synthesis can be carried out at a higher temperature within the range of 30.-100°C, preferably within the range of about 50.-90°C. The prepared amino alcohol lipidoid compounds can be optionally purified. For example, a mixture of amino alcohol lipidoid compounds can be purified to produce amino alcohol lipidoid compounds with a specific number of epoxide-derived compound tails. Or a mixture can be purified to produce a specific stereoisomer or regioisomer. Amino alcohol lipidoid compounds can also be alkylated using alkyl halides (e.g., methyl iodide) or other alkylating agents, and / or they can be acylated.

[0283] U.S. Patent Publication No. 0110293703 also provides a library of amino alcohol lipid compounds prepared by the method of the present invention. These amino alcohol lipid compounds can be prepared and / or screened using high throughput techniques (involving liquid handlers, robots, microtiter plates, computers, etc.). In certain embodiments, amino alcohol lipid compounds are screened for their ability to transfect polynucleotides or other agents (e.g., proteins, peptides, small molecules) into cells.

[0284] U.S. Patent Publication No. 20130302401 relates to a class of poly (β-amino alcohols) (PBAA) prepared using combinatorial polymerization. The PBAAs of the present invention can be used as coatings (e.g., coatings for films or multilayer films for medical devices or implants), additives, materials, excipients, non-biofouling agents, micropatterning agents, and cell encapsulation agents in biotechnology and biomedical applications. When used as surface coatings, these PBAAs cause varying degrees of inflammation both in vitro and in vivo, depending on their chemical structure. The huge chemical diversity of such materials enables us to identify polymer coatings that inhibit macrophage activation in vitro. In addition, after subcutaneous implantation of carboxylated polystyrene microparticles, these coatings reduce the recruitment of inflammatory cells and reduce fibrosis. These polymers can be used to form polyelectrolyte composite capsules for cell encapsulation. The invention can also have many other biological applications, such as antimicrobial coatings, DNA or siRNA delivery, and stem cell tissue engineering. The teachings of U.S. Patent Publication No. 20130302401 can be applied to the system of the present invention.

[0285] In another embodiment, lipid nanoparticles (LNP) are envisioned. In particular, anti-transthyretin small interfering RNA (see, e.g., Coelho et al., N Engl J Med 2013; 369: 819-29) encapsulated in lipid nanoparticles can be applied to the system of the present invention. It is envisioned that the dosage for intravenous administration is about 0.01 mg / kg body weight to about 1 mg / kg body weight. It is envisioned that drugs are used to reduce the risk of infusion-related reactions, such as dexamethasone, acetaminophen, diphenhydramine or cetirizine, and ranitidine are envisioned. It is also envisioned that multiple doses of about 0.3 mg / kg every 4 weeks, a total of five doses. Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200, and colipids distearoylphosphatidylcholine, cholesterol, and PEG-DMG, and RNA can be formulated using a spontaneous vesicle formation procedure instead of siRNA (see, e.g., Novobrantseva, Molecular Therapy—Nucleic Acids (2012) 1, e4; doi: 10.1038 / mtna.2011.3). The component molar ratio can be about 50 / 10 / 38.5 / 1.5 (DLin-KC2-DMA or C12-200 / distearoylphosphatidylcholine / cholesterol / PEG-DMG). In the case of DLin-KC2-DMA and C12-200 lipid nanoparticles (LNPs), the final lipid: siRNA weight ratio can be ˜12: 1 and 9: 1, respectively. The formulation can have an average particle size of ˜80 nm and an encapsulation efficiency of >90%. A dose of 3 mg / kg is contemplated.

[0286] LNP has been shown to be very effective in delivering siRNA to the liver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol. 3, No. 4, pages 363-470), therefore, it is envisaged to use LNP to deliver CRISPR Cas to the liver. It is envisaged that a dose of about four doses of 6 mg / kg of LNP (or RNA of CRISPR-Cas) every two weeks. Tabernero et al. demonstrated that tumor regression was observed after a 0.7 mg / kg dose of LNP in the first 2 cycles, and by the end of 6 cycles, the patient had achieved a partial response, with complete regression of lymph node metastases and substantial reduction of liver tumors. A complete response was obtained after the patient received 40 doses, and after receiving the dose for more than 26 months, the patient remained in a state of response and completed treatment. Two patients with RCC and disease at extrahepatic sites (including kidney, lung, and lymph nodes) who had progressed after prior treatment with a VEGF pathway inhibitor had stable disease at all sites for approximately 8 to 12 months, and a patient with PNET and liver metastases continued treatment in an 18-month (36 doses) extension study with stable disease.

[0287] However, the charge of LNP must be considered. Because cationic lipids are combined with negatively charged lipids to induce non-double-layer structure, thus promoting intracellular delivery. Because charged LNP is rapidly removed from circulation after intravenous injection, ionizable cationic lipids (see, e.g., Rosin et al, Molecular Therapy, vol.19, no.12, pages 1286-2200, December 2011) with a pKa value lower than 7 have been developed. Negatively charged polymers (e.g., siRNA oligonucleotides) can be loaded into LNP under low pH values ​​(e.g., pH 4), wherein ionizable lipids show positive charge. However, under physiological pH values, LNP shows low surface charge, which is suitable for longer circulation time. Four ionizable cationic lipids have received attention, namely 1,2-dilinoleoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleoyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleoyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA) and 1,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). It has been shown that LNP siRNA systems containing these lipids exhibit significantly different gene silencing properties in hepatocytes in vivo, using a factor VII gene silencing model, with efficacy varying according to the series of DLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011). Dosing at the 1 μg / ml level is envisioned, particularly for formulations containing DLinKC2-DMA. Preparation of LNPs and CRISPR Cas encapsulation may be used and / or adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011.The cationic lipids 1,2-dilinoleoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleoyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleoyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-o-[2″-(methoxypolyethylene glycol 2000)succinyl]-1,2-dimyristoyl-sn-ethylene glycol (PEG-S-DMG), and R-3-[(w-methoxy-poly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-C-DOMG) were prepared by Tekmira. Pharmaceuticals (Vancouver, Canada) or synthesized. Cholesterol can be purchased from Sigma (St Louis, Mo.). Specific CRISPR Cas RNA can be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid: DSPC: CHOL: PEGS-DMG or PEG-C-DOMG in a 40:10:40:10 molar ratio). If necessary, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) was used to assess cellular uptake, intracellular delivery, and biodistribution. Encapsulation can be performed by dissolving a lipid mixture consisting of a cationic lipid: DSPC: cholesterol: PEG-c-DOMG (40:10:40:10 molar ratio) in ethanol to a final lipid concentration of 10 mMol / 1. The ethanol solution of the lipid can be added dropwise to 50 mMol / 1 citrate (pH 4.0) to form multilamellar vesicles, resulting in a final concentration of 30% ethanol vol / vol. Large unilamellar vesicles can be formed after extruding the multilamellar vesicles through two stacked 80 nm Nuclepore polycarbonate filters using an extruder (Northern Lipids, Vancouver, Canada). Encapsulation can be achieved by dissolving 50 mMol / 1 citrate (pH 4.0) containing 30% ethanol vol / vol at 2 mg / ml. RNA in 4.0) was added dropwise to the extruded preformed large unilamellar vesicles and incubated at 31°C for 30 minutes with constant mixing to a final RNA / lipid weight ratio of 0.06 / 1 wt / wt. The ethanol was removed and the formulation buffer was neutralized by dialyzing against phosphate buffered saline (PBS) (pH 7.4) for 16 hours using a Spectra / Por 2 regenerated cellulose dialysis membrane.Nanoparticle size distribution can be determined by dynamic light scattering using NICOMP 370 particle size analyzer, vesicle / intensity mode and Gaussian fitting (Nicomp Particle Sizing, Santa Barbara, Calif.). The particle size of all three LNP systems can be diameter ~ 70nm. The siRNA encapsulation efficiency can be determined by removing free siRNA from samples collected before and after dialysis using VivaPureD MiniH columns (Sartorius Stedim Biotech). Encapsulated RNA can be extracted from the eluted nanoparticles and quantified at 260nm. The ratio of siRNA to lipid is determined by measuring the cholesterol content in the vesicle using the cholesterol E enzyme assay from Wako Chemicals USA (Richmond, Va.). Pegylated liposomes (or LNPs) can also be used for delivery.

[0288] The preparation of large LNPs can be used and / or adapted from Rosin et al, Molecular Therapy, vol.19, no.12, pages 1286-2200, December 2011. A lipid premix solution (20.4 mg / ml total lipid concentration) can be prepared in ethanol containing DLinKC2-DMA, DSPC and cholesterol in a molar ratio of 50:10:38.5. Sodium acetate can be added to the lipid premix at a molar ratio of 0.75:1 (sodium acetate: DLinKC2-DMA). Subsequently, the lipids are hydrated by mixing the mixture with 1.85 volumes of citrate buffer (10 mmol / 1, pH 3.0) and stirring vigorously, resulting in spontaneous formation of liposomes in an aqueous buffer containing 35% ethanol. The liposome solution can be incubated at 37°C to allow a time-dependent increase in particle size. Aliquots can be taken out at different times during the incubation process to study the change of liposome size by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). Once the desired particle size is reached, a PEG lipid aqueous solution (stock solution = 10mg / ml PEG-DMG in 35% (vol / vol) ethanol) can be added to the liposome mixture to produce a total lipid of a final PEG molar concentration of 3.5%. After adding PEG-lipid, the liposome should adjust its size, thereby effectively inhibiting further growth. Then, RNA can be added to empty liposomes with a siRNA and total lipid ratio of about 1: 10 (wt: wt), followed by incubation at 37°C for 30 minutes to form the LNP of the load. Subsequently, the mixture can be dialyzed overnight in PBS and filtered with a 0.45 μm syringe filter.

[0289] Spherical nucleic acids (SNA) are also contemplated. TM ) constructs and other nanoparticles (particularly gold nanoparticles) as a means of delivering CRISPR / Cas systems to the intended target. A large amount of data shows that AuraSense Therapeutics' spherical nucleic acids (SNAs) TM ) constructs are based on nucleic acid-functionalized gold nanoparticles and outperform alternative platforms in terms of several key success factors, such as:

[0290] High in vivo stability. Due to its dense loading, most of the cargo (DNA or siRNA) remains bound to the construct inside the cell, conferring nucleic acid stability and resistance to enzymatic degradation.

[0291] Deliverability. The construct demonstrated 99% transfection efficiency for all cell types studied (eg, neurons, tumor cell lines, etc.) without the need for a carrier or transfection agent.

[0292] Therapeutic targeting. The unique target binding affinity and specificity of the construct allows for precise specificity of the matched target sequence (e.g., limited off-target effects).

[0293] Superior efficacy. This construct significantly outperformed the leading conventional transfection reagents (Lipofectamine 2000 and Cytofectin).

[0294] Low toxicity. The construct can enter a variety of cultured cells, primary cells and tissues without significant toxicity.

[0295] No significant immune response was observed. The construct caused minimal changes in global gene expression as measured by genome-wide microarray studies and cytokine-specific protein assays.

[0296] Chemical Customizability. The surface of the construct can be customized using any number of single or combined agents (eg, proteins, peptides, small molecules).

[0297] This nucleic acid-based therapeutic platform may be applicable to a variety of disease states, including inflammatory and infectious diseases, cancer, dermatological diseases, and cardiovascular diseases.

[0298] Citable references include: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao et al., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970, Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., Nano Lett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012 109:11975-80, Mirkin, Nanomedicine 2012 7:635-638, Zhang et al., J. Am. Chem. Soc. 2012 134:16488-1691,Weintraub,Nature2013 495:S14-S16,Choi et al.,Proc.Natl.Acad.Sci.USA.2013 110(19):7625-7630,Jensen et al. al., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small, doi.org / 10.1002 / smll.201302143.

[0299] Self-assembled nanoparticles with siRNA can be constructed with polyethyleneimine (PEI), which is PEGylated and has an Arg-Gly-Asp (RGD) peptide ligand attached to the distal end of polyethylene glycol (PEG), for example, as a means of targeting tumor neovasculature expressing integrins, and for delivering siRNA that inhibits vascular endothelial growth factor receptor-2 (VEGF R2) expression and thereby inhibits tumor angiogenesis (see, for example, Schiffelers et al., Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanocomplexes can be prepared by mixing equal volumes of aqueous solutions of cationic polymers and nucleic acids to obtain a net molar excess of ionizable nitrogen (polymer) relative to phosphate (nucleic acid) in the range of 2 to 6. The electrostatic interaction between the cationic polymer and the nucleic acid results in the formation of a polymer complex with an average particle size distribution of about 100 nm, so it is referred to as a nanocomplex herein. It is expected that a CRISPR Cas dose of about 100 mg to 200 mg can be delivered in the self-assembled nanoparticles of Schiffelers et al.

[0300] The nanocomplexes of Bartlett et al. (PNAS, Sep. 25, 2007, vol. 104, no. 39) can also be applied to the present invention. The nanocomplexes of Bartlett et al. are prepared by mixing equal volumes of aqueous solutions of cationic polymers and nucleic acids so that the net molar excess of ionizable nitrogen (polymer) relative to phosphate (nucleic acid) is in the range of 2 to 6. The electrostatic interaction between the cationic polymer and the nucleic acid results in the formation of a polymer complex with an average particle size distribution of about 100 nm, and is therefore referred to herein as a nanocomplex. The synthesis of DOTA-siRNA of Bartlett et al. is as follows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester) (DOTA-NHSester) was ordered from Macrocyclos (Dallas, Tex.). The amine-modified RNA sense strand was added to a microcentrifuge tube with a 100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9). The contents were reacted by stirring at room temperature for 4 h. The DOTA-RNA sense strand conjugate was ethanol precipitated, resuspended in water, and annealed with the unmodified antisense strand to produce DOTA-siRNA. All liquids were pretreated with Chelex-100 (Bio-Rad, Hercules, Calif.) to remove trace metal contaminants. Tf-targeted and non-targeted siRNA nanoparticles can be formed using polycations containing cyclodextrin. Typically, nanoparticles are formed in water with a charge ratio of 3 (+ / -) and a siRNA concentration of 0.5 g / liter. One percent of the adamantane-PEG molecules on the surface of the targeted nanoparticles are modified by Tf (adamantane-PEG-Tf). The nanoparticles are suspended in a 5% (wt / vol) glucose carrier solution for injection.

[0301] Davis et al. (Nature, Vol 464, 15 April 2010) conducted a siRNA clinical trial using a targeted nanoparticle delivery system (clinical trial registration number NCT00689065). Patients with solid cancers refractory to standard treatment were administered targeted nanoparticle doses by 30-min intravenous infusion on days 1, 3, 8, and 10 of a 21-day cycle. The nanoparticles consist of a synthetic delivery system containing: (1) a linear cyclodextrin-based polymer (CDP), (2) a human transferrin (TF) targeting ligand displayed on the outside of the nanoparticles for engagement with TF receptors (TFR) on the surface of cancer cells, (3) a hydrophilic polymer (polyethylene glycol (PEG) for promoting nanoparticle stability in biological fluids), and (4) a siRNA designed to reduce RRM2 expression (the sequence used in the clinic was previously denoted as siR2B+5). It has long been known that TFR is upregulated in malignant cells, and RRM2 is an established anticancer target. These nanoparticles (the clinical version is denoted CALAA-01) have been well tolerated in multiple-dose studies in nonhuman primates. Although only one patient with chronic myeloid leukemia has been administered siRNA via liposomal delivery, the clinical trial by Davis et al. is the first human trial to use a targeted delivery system to deliver siRNA systemically and treat solid cancer patients. To determine whether the targeted delivery system can provide effective delivery of functional siRNA to human tumors, Davis et al. studied biopsies from three patients in three different dose groups; Patient A, Patient B, and Patient C all had metastatic melanoma and received CALAA-01 at a dose of 18 mg m -2 , 24mg m -2 and 30 mg m - 2 siRNA. Similar dosages are also envisioned for the CRISPR Cas system of the present invention. Delivery of the present invention can be achieved by nanoparticles containing a linear cyclodextrin-based polymer (CDP), a human transferrin (TF) targeting ligand displayed on the outside of the nanoparticle to engage the TF receptor (TFR) on the surface of the cancer cell, and / or a hydrophilic polymer (e.g., polyethylene glycol (PEG) for promoting nanoparticle stability in biological fluids).

[0302] Delivery or application according to the present invention can be carried out with liposomes. Liposomes are spherical vesicle structures, consisting of a monolayer or multilayer lipid bilayer around an internal aqueous compartment and a relatively impermeable external lipophilic phospholipid bilayer. Liposomes have received considerable attention as drug delivery vehicles because they are biocompatible, nontoxic, can deliver hydrophilic and lipophilic drug molecules, protect their carriers from being degraded by plasma enzymes and transport their loads across biological membranes and blood-brain barrier (BBB) ​​(for review see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155 / 2011 / 469679).

[0303] Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Although the formation of liposomes is spontaneous when the lipid film is mixed with an aqueous solution, the formation of liposomes can also be accelerated by applying force in the form of shaking using a homogenizer, an ultrasonic generator, or an extrusion device (for review, see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155 / 2011 / 469679).

[0304] Several other additives can be added to liposomes to change their structure and properties. For example, cholesterol or sphingomyelin can be added to the liposome mixture to help stabilize the liposome structure and prevent leakage of the liposome internal cargo. In addition, liposomes are prepared from hydrogenated egg phosphatidylcholine or egg phosphatidylcholine, cholesterol and dihexadecyl phosphate, and their average vesicle size is adjusted to about 50nm and 100nm. (For a review, see, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155 / 2011 / 469679).

[0305] Conventional liposome preparations are mainly composed of natural phospholipids and lipids (e.g., 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, and monosialoganglioside). Since the preparation is composed only of phospholipids, liposome preparations have encountered many challenges, one of which is instability in plasma. Some attempts have been made to overcome these challenges, particularly in the manipulation of lipid membranes. One of the attempts focuses on the manipulation of cholesterol. Adding cholesterol to conventional preparations can reduce the rapid release of encapsulated bioactive compounds into plasma, or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases stability (for review, see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155 / 2011 / 469679).

[0306] In a particularly advantageous embodiment, Trojan Horse liposomes (also referred to as molecule Trojan Horses) are desirable and protocols can be found at cshprotocols.cshlp.org / content / 2010 / 4 / pdb.prot5407.long. These particles can deliver transgenes to the entire brain after intravascular injection. Without limitation, it is believed that neutral lipid particles with specific antibodies conjugated to the surface allow for crossing the blood-brain barrier by endocytosis. Applicants assume that the use of Trojan Horse liposomes to deliver CRISPR family nucleases to the brain by intravascular injection will produce whole-brain transgenic animals without the need for embryonic manipulation. It is conceivable that about 1-5g of nucleic acid molecules (e.g., DNA, RNA) will be administered in vivo in liposomes.

[0307] In another embodiment, the system can be administered in a liposome, such as a stable nucleic acid-lipid particle (SNALP) (see, e.g., Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August 2005). It is contemplated that about 1 mg / kg / day, 3 mg / kg / day, or 5 mg / kg / day of a targeted specific CRISPR Cas in SNALP is injected intravenously daily. Daily treatment can last for about three days, followed by weekly treatment for about five weeks. In another embodiment, it is also contemplated that a specific CRISPR Cas (encapsulated SNALP) is administered by intravenous injection at a dose of about 1 mg / kg or 2.5 mg / kg (see, e.g., Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006). The SNALP formulation can contain the lipids 3-N-[(w-methoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA), 1,2-dilinoleyl-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol in a molar percentage ratio of 2:40:10:48 (see, e.g., Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006).

[0308] In another embodiment, it has been shown that stable nucleic acid-lipid particles (SNALP) are effective delivery molecules for highly vascularized HepG2-derived liver tumors, but are not particularly effective for HCT-116-derived liver tumors with a lower degree of vascularization (see, e.g., Li, Gene Therapy (2012) 19, 775-780). SNALP liposomes can be prepared by using 25:1 lipid / siRNA ratios and 48 / 40 / 10 / 2 molar ratios of cholesterol / D-Lin-DMA / DSPC / PEG-C-DMA, preparing D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine (DSPC), cholesterol and siRNA. The resulting SNALP liposome size is about 80-100nm.

[0309] In yet another embodiment, the SNALP may comprise synthetic cholesterol (Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxypropylamine and cationic 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (see, e.g., Geisbert et al., Lancet 2010; 375: 1896-905). A dose of about 2 mg / kg total CRISPR Cas per dose administered, e.g., as a bolus intravenous infusion, may be envisioned.

[0310] In yet another embodiment, SNALP can include synthetic cholesterol (Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG-cDMA and 1,2-dilinoleoyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, e.g., Judge, J. Clin. Invest. 119:661-673 (2009)). The formulation for in vivo studies can include a final lipid / RNA mass ratio of about 9:1.

[0311] Other cationic lipids (e.g., amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA)) can be used to encapsulate CRISPR Cas similar to siRNA (see, e.g., Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533). Preformed vesicles with the following lipid composition can be envisioned: amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and (R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethylene glycol) 2000)propylcarbamate (PEG-lipid), in molar ratios of 40 / 10 / 40 / 10, respectively, and a FVIIsiRNA / total lipid ratio of about 0.05 (w / w). To ensure a narrow particle size distribution in the range of 70-90 nm and a low polydispersity index of 0.11_0.04 (n=56), the particles can be extruded through an 80 nm membrane up to 3 times before adding CRISPR Cas RNA. Particles containing the highly efficient amino lipid 16 can be used, where the molar ratio of the four lipid components 16, DSPC, cholesterol and PEG-lipid (50 / 10 / 38.5 / 1.5) can be further optimized to enhance in vivo activity.

[0312] Any elements of any suitable CRISPR / Cas gene editing system known in the art can be used in the systems and methods described herein as appropriate. CRISPR / Cas gene editing technology is described in detail, for example, in U.S. Patent Application Publication No. 2014 / 0068797; U.S. Patent Nos. 8,697,359; 8,771,945; and 8,945,839; US2010 / 0076057; US2011 / 0189776; US2011 / 0223638; US2013 / 0130248; WO / 2008 / 108989; WO / 2010 / 054108; WO / 2012 / 164565; WO / 2013 / 098244 ; WO / 2013 / 176772; US20150050699; US20150045546; US20150031134; US20150024500; US20140377868; US20140357530; US20140349400; US20140335620; US20140335063; US20140315985; US20140310830; US20140310828; US20140309487; US 20140304853; US20140298547; US20140295556; US20140294773; US20140287938; US20140273234; US20140273232; US20 140273231; US20140273230; US20140271987; US20140256046; US20140248702; US20140242702; US20140242700; US2014 0242699; US20140242664; US20140234972; US20140227787; US20140212869; US20140201857; US20140199767; US20140189896; US20140186958; US20140186919; US20140186843; US20140179770; US20140179006; and US20140170753, which are incorporated herein by reference.

[0313] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

[0314] The present invention will be further described in the following examples, which are given for illustrative purposes only and are not intended to limit the present invention in any way. Example Example 1 - Materials and Methods

[0315] RecE / T homology screening RefSeq non-redundant protein database was downloaded from NCBI on October 29, 2019. The query was performed with E. coli Rac prophage RecT (NP_415865.1) and RecE (NP_415866.1) using position-specific iterative (PSI)-BLAST. 1 Databases were searched to retrieve protein homologs. Hits were clustered using CD-HIT2 and representative sequences were selected from each cluster using MUSCLE 3 Multiple alignments were performed. Then, maximum likelihood tree reconstruction was performed using FastTree4 with default parameters. A diverse set of RecET homologues was selected, synthesized by GenScript, and cloned into the pMPH_MCP vector for testing.

[0316] Plasmid construction pX330, pMPH and pU6-(BbsI)_CBh-Cas9-T2A-BFP plasmids were obtained from Addgene. The effector DNA fragments tested were ordered from IDT, Genewiz and GenScript. These fragments were Gibson assembled into the main chain using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs). All sgRNAs (Table 3) were inserted into the main chain using Golden Gate cloning. All constructs were sequence verified by Sanger sequencing of pre-prepared plasmids.

[0317] Cell culture Human embryonic kidney (HEK) 293T, HeLa, and HepG2 were maintained at 37°C and 5% CO 2 The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) containing 10% fetal bovine serum (FBS, HyClone), 100 U / mL penicillin, and 100 μg / mL streptomycin (Life Technologies).

[0318] hES-H9 cells were maintained at 37°C and 5% CO 2, in mTeSR1 medium (StemCell Technologies). Plates were pre-coated with Matrigel (Corning) 12 hours before use, and cells were supplemented with 10 μM Y27632 (Sigma) for the first 24 hours after passage. The medium was changed every 24 hours.

[0319] Transfection 12-24 hours before transfection, HEK293T cells were seeded into 96-well plates (Corning) at a density of 30,000 cells / well, and 250 ng of total DNA was transfected per well. One day before transfection, HeLa and HepG2 cells were seeded into 48-well plates (Corning) at a density of 50,000 and 30,000 cells / well, respectively, and 400 ng of total DNA was transfected per well. Transfection was performed using Lipofectamine 3000 (Life Technologies) according to the manufacturer's instructions.

[0320] Electroporation For hES-H9 related transfection experiments, P3 primary cells 4D-Nucleofector™ X Kit S (Lonza) was used according to the manufacturer's protocol. For each reaction, 300,000 cells were nucleofected with 4 μg of total DNA using the DC100 Nucleofector program.

[0321] Fluorescence activated cell sorting (FACS) was used to analyze mKate knock-in efficiency on a CytoFLEX flow cytometer (Beckman Coulter; Stanford Stem Cell FACS Core). 72 hours after transfection, cells were washed once with PBS and dissociated with TrypLE Express enzyme (Thermo Fisher Scientific). The cell suspension was then transferred to a 96-well U-bottom plate (Thermo Fisher Scientific) and centrifuged at 300xG for 5 minutes. After removing the supernatant, the pelleted cells were resuspended with 50 μl of PBS containing 4% FBS and sorted within 30 minutes of preparation.

[0322] RFLP plasmid DNA and PCR template were used to transfect HEK293T cells, and genomic DNA was harvested 72 hours later using QuickExtractDNA extraction solution (Biosearch Technologies) according to the manufacturer's protocol. Specific primers outside the homology arms of the PCR template were used to amplify the target genomic region. PCR products were purified using Monarch PCR & DNA Cleanup Kit (New England BioLabs). 300 ng of the purified product was digested with BsrGI (EMX1, New England BioLabs) or XbaI (VEGFA, NEB), and the digestion products were analyzed on a 5% Mini-PROTEAN TBE gel (Bio-Rad).

[0323] Preparation of second-generation sequencing libraries 72 hours after transfection, genomic DNA was extracted using QuickExtract DNA extraction solution (Biosearch Technologies). 200ng of total DNA was used for NGS library preparation. In the first round of PCR reactions, specific primers (Table 4) were used to amplify the gene of interest. Using the primers listed in Table 4, Illumina adapters and index barcodes were added to the fragments by a second round of PCR. The second round of PCR products were purified by gel electrophoresis on a 2% agarose gel using the Monarch DNA gel extraction kit (NEB). The purified products were quantified using the Qubit dsDNA HS assay kit (Thermo Fisher) and sequenced on the Illumina MiSeq according to the manufacturer's instructions.

[0324] High-throughput sequencing data analysis using CRISPPResso2 5 , processed (demultiplexed, trimmed, and merged) sequencing reads were analyzed to determine editing outcomes by aligning the sequencing amplicons to the reference and expected HDR amplicons. The quantification window was increased to 10 bp around the expected cut site to better capture different editing outcomes, but substitutions were ignored to avoid including sequencing errors. Only reads that did not contain mismatches to the expected amplicon were considered for HDR quantification; reads that contained indels that partially matched the expected amplicon were included in the overall reported indel frequency.

[0325] Statistical analysis Unless otherwise stated, all statistical analyses and comparisons were performed using t-tests using the two-stage step-up method of Benjamini, Krieger and Yekutieli with a false discovery rate (FDR) of 1% (Benjamini, Y., et al, Biometrika 93, 491-507 (2006), which is incorporated herein by reference). Unless otherwise stated, all experiments were repeated three times to ensure adequate statistical power in the analysis.

[0326] Determination of editing at predicted Cas9 off-target sites To evaluate RecT / RecE off-target editing activity at known Cas9 off-target sites, the same genomic DNA extracts used for knock-in analysis were used as templates to perform PCR amplification of the most likely predicted off-target sites directed by EMX1 and VEGFA (high scores predicted according to CRISPOR (a web-based analysis tool)), and the primer sequences are listed in Table 4.

[0327] iGUIDE off-target analysis According to the iGUIDE process (Nobles, CL, et al. Genome Biol 20, 14 (2019), which is incorporated herein by reference), a genome-wide unbiased off-target analysis was performed based on the previously invented Guide-seq (Tsai, S., et al. Nat Biotechnol 33, 187–197 (2015), which is incorporated herein by reference). According to the manufacturer's instructions, HEK293T cells were transfected with program DS-150 on a Lonza Nucleofector 4-D in 20uL Lonza SF cell line nuclear transfection agent solution. Transfect 300ng of gRNA-Cas9 plasmid (or 150ng of each gRNACas9n plasmid for double nickase), 150ng of effector plasmid and 5pmol of double-stranded oligonucleotide (dsODN). After 72 hours, cell genomic DNA was harvested using the Agencourt DNAdvance reaction kit. 400 ng of purified gDNA was fragmented to an average of 500 bp and ligated to adapters using the NEBNext Ultra II FSDNA Library Preparation Kit according to the manufacturer's instructions. Two rounds of nested anchor PCR from the oligonucleotide tags to the ligated adapter sequences were performed to amplify the targeted DNA, and the amplified libraries were purified, size-selected, and sequenced using an Illumina Miseq V2 PE300. Sequencing data were analyzed using the published iGUIDE pipeline, with an additional downstream sampling step to ensure unbiased comparison between samples. Example 2

[0328] Compared to mammals, bacteria have convenient recombineering editing tools available, such as bacteriophage λRed and RecE / T. Microbial recombineering has two main steps: the template DNA is chewed back by exonucleases (Exo), and then single-stranded annealing proteins (SSAPs) support homology-directed repair through the template, optionally facilitated by nuclease inhibitors. A system for RNA-guided targeted RecE / T recombineering activity was developed, and kilobase (kb) human gene editing was achieved without cutting DNA.

[0329] Candidate microbial systems with recombineering activity were investigated. Two lines of reasoning guided the search: 1) orthogonality: proteins with minimal similarity to mammalian repair enzymes were prioritized; 2) parsimony: systems with the fewest interdependent components were focused on. Three protein families were identified: λRed, RecE / T, and the bacteriophage T7 gp6 (Exo) and gp2.5 (SSAP) recombination machinery. Based on phylogenetic reconstruction, RecE / T proteins were identified as the most distant from eukaryotic recombination proteins and as one of the most compact (Figure 1). Therefore, the RecE / T system was used for downstream analysis.

[0330] The NCBI protein database was systematically searched for RecE / T homologs. To develop a portable tool, the evolutionary relationships and length ( Figure 2A ). Co-occurrence analysis showed that most RecE / T systems have only one of these two proteins ( Figure 2B ). Because prophage integration can be imprecise, the 11% of species with two homologues were prioritized as evidence of full functionality.

[0331] The top 12 candidates were codon optimized and constructed as MS2 coat protein (MCP) fusions to recruit these RecE / T homologs (hereinafter referred to as "recombinants") to wild-type S. pyogenes Cas9 (wtCas9) via MS2 RNA aptamers. To understand their respective molecular effects as Exo and SSAP, each was tested independently ( Figure 2C Preliminary results showed that the Escherichia coli RecE / T proteins (abbreviated as RecE and RecT) were promising candidates as determined by genomic knock-in assays ( Figure 2D Although RecT is only 269 amino acids (AA) in length, RecE was truncated from AA587 (RecE_587) and the carboxyl terminal domain (RecE_CTD) based on functional studies (Muyrers, JP, Genes Dev. (2000); 14, 1971-1982, which is incorporated herein by reference).

[0332] To validate RecE / T recombineering in human cells, homology-directed repair (HDR) was measured with two templates at five genomic loci. Although RecE variants (RecE_587, RecE_CTD) showed varying degrees of increase in knock-in efficiency, in all cases RecT significantly enhanced HDR, replacing ~16 bp sequences at EMX1 and VEGFA, and knocking in ~1 kb cassettes at HSP90AA1, DYNLT1, AAVS1 ( Figure 3A -E, Figure 4). These results were confirmed by imaging ( Figure 3F ), and the junction sites were sequenced using Sanger sequencing to confirm accurate insertion ( Figure 3G , Figure 4G). To test whether these activities are truly sequence-specific, a no-recruitment control of the PP7 coat protein (PCP) that recognizes the PP7 aptamer but not the MS2 aptamer was used. RecE is active in the absence of recruitment, while RecT shows increased efficiency in a recruitment-dependent manner ( Figure 3H Without being bound by theory, this could be explained by the RecE exonuclease activity acting indiscriminately ( Figure 2C ). The RecE / T Recombineering Editing (REDIT) tool was called REDITv1, with REDITv1_RecT as the preferred variant. Example 3

[0333] REDITv1 was tested in three ways to explore: 1) activity across cell types, 2) optimized design of HDR templates, and 3) specificity. REDITv1 activity was robust across multiple genomic loci in HEK, A549, HepG2, and HeLa cells ( Figure 5A -C. Fig. 6A Notably, in human embryonic stem cells (hESCs), REDITv1 showed consistent increases in kilobase-per-unit knock-in efficiency at HSP90AA1 and OCT4, up to 3.5-fold relative to Cas9-HDR ( Figure 5D -E, Fig.6D-E). Different template designs were also tested. REDITv1 performed efficient kilobase editing using a total HA length as short as 200bp, with longer HAs supporting higher efficiencies. For kb-scale knock-ins, it achieved efficiencies up to 10% (without selection), a 5-fold increase over Cas9-HDR and significantly higher than the typical efficiency of 1-2% (Figure 7). Finally, the accuracy of REDITv1 was determined using deep sequencing and GUIDE-seq of predicted off-target sites (OTS). Although REDITv1 did not increase off-target effects, there were still detectable OTS at the previously reported EMX1 and VEGFA sites ( Fig. 5F -G, Figure 8). In short, REDITv1 demonstrated kilobase-scale genome recombineering but retained the off-target problem, with REDITv1_RecT having the highest efficiency. Example 4

[0334] In order to reduce unwanted editing, a version of REDIT using a non-cutting Cas9 nickase (Cas9n) was evaluated. A similar strategy was previously used (Ran, FA, et al., Cell (2013), 154: 1380-1389, which is incorporated herein by reference) to solve the off-target problem, but the HDR efficiency was low. REDIT was tested to determine whether the system could overcome the limitations of endogenous repair and promote nick-mediated recombination. In fact, the nickase version showed higher efficiency, with single-nick and double-nick Cas9n (D10A) having the best results. This Cas9n (D10A) variant was named REDITv2N ( Fig. 9A ). Using REDITv2N double incisions, 5% to 10% unselected knock-in was observed, which is comparable to REDITv1 using wtCas9 ( Fig. 9A , Fig. 10A ). Ligation sequencing confirmed the precise knock-in of all targets (Figure 11). This result is 6- to 10-fold higher than Cas9n-HDR. Even with the single-nick REDITv2N, an efficiency of 1 kb knock-in was observed to be ~2%, which is significantly higher than the 0.46% HDR efficiency ( ) using conventional single-nick Cas9n and a less challenging 12-bp knock-in template reported previously (Cong, L. et al., Science. 339, 819-823, which is incorporated herein by reference) Fig. 9A ).

[0335] The off-target activity of REDITv2N was investigated using GUIDE-seq. The results showed minimal off-target cleavage and a ~90% reduction in OTS compared to REDITv1 ( Fig. 9B). Specifically, for DYNLT1 targeting guidance, the most abundant KIF6 OTS was significantly enriched in the REDITv1 group but disappeared when using REDITv2N ( Fig. 9C REDITv2N has a high degree of accuracy ( Fig. 9B -C, Figure 12).

[0336] Another byproduct of HDR editing is on-target insertion-deletions (indels). They can significantly reduce the yield of gene editing, especially for long sequences. Indel formation was measured in EMX1 knock-in experiments using deep sequencing. REDITv2N increased HDR to the same efficiency as its counterpart using wtCas9 ( Fig. 12C , top), and undesired on-target indels were reduced by 92% ( Fig. 12C , figure below).

[0337] Concepts from GUIDE-seq, LAM-PCR, and TLA were used to develop NGS-based assays to identify genome-wide insertion sites (GIS) or GIS-seq ( Fig. 30A ). Using GIS-seq, obtain NGS read clusters / peaks representing knock-in insertion sites ( Fig. 30B ), showing representative reads from on-target sites. GIS-seq was applied to the DYNLT1 and ACTB loci to measure knock-in accuracy. Sequencing results showed that REDIT identified fewer off-target insertion sites compared to Cas9 when considering high-confidence sites based on maximum likelihood estimation ( Fig. 30C ). Sanger sequencing of knock-in ligated clones ( Fig. 9C and Figure 12), GUIDE-seq analysis ( Fig. 9B ) and GIS seq results ( Figure 30A-Figure 30C ) collectively suggest that REDIT can be an efficient method capable of inserting kilobase-length sequences with few undesired editing events. Example 5

[0338] The long sequence editing capability of REDIT was examined in the absence of any nicking / cleavage of the target DNA. Notably, precise kilobase cassette genomic knock-in was observed in human cells when REDITv2D was constructed using catalytically inactive Cas9 (dCas9). Fig.9D , above, Figure 13). Although REDITv2D is less efficient than REDITv2N, it achieves programmable DNA damage-free editing at the kilobase scale with an efficiency of 1-2% and without selection ( Fig.9D , Fig. 10B). We speculate that two processes may contribute to REDITv2D recombineering. One possibility is through dCas9 unwinding. If dCas9 is able to unwind DNA while inducing sequence-specific loop formation, dual binding of two dCas9s would be expected to promote RecE / T accessibility to the genome. However, no significant increase was observed after delivery of two guide RNAs ( Fig.9D , lower figure). Another possibility is that DNA unwinding during the cell cycle allows RecE / T to access the target region through dCas9 binding. 1kb knock-in was performed using different REDIT tools under different serum levels (10% normal, 2% reduced serum, and no serum). Since serum starvation prevents cell proliferation, the results suggest that the cell cycle is positively correlated with REDITv2D recombineering ( Fig.9E ). In the absence of serum, only the HDR efficiency of the REDITv2D (dCas9) group decreased, while REDITv1 (wtCas9) and REDITv2N (D10A) were not affected ( Fig.9E , Figure 14 ), supporting DNA unwinding to allow RecE / T to access the target region. Example 6

[0339] Microscopic analysis showed that nuclear targeting of REDITv1 was incomplete, especially for REDITv1_RecT ( Fig.15 Therefore, different designs of protein linkers and nuclear localization signals (NLS) were tested ( Fig.15 A). The extended XTEN-linker with C-terminal SV40-NLS was identified as the preferred configuration, called REDITv3 (Figure 16). REDITv3 further achieved HDR efficiency 2- to 3-fold higher than REDITv2 in the case of various genomic targets and Cas9 variants (wtCas9, Cas9n, dCas9) (Figure 17).

[0340] Finally, REDITv3 was used in hESCs to engineer kilobase knock-in alleles in human stem cells. The REDITv3N single- and double-nicking designs increased HDR efficiency by 5- and 20-fold, respectively, compared to the no-recombinant control ( Fig.9F Efficacy and fidelity were confirmed by a combination of assays described in a previous REDIT version ( Fig.9F -G, Fig.18 ). In addition, REDITv3 cooperates effectively with Staphylococcus aureus Cas9 (SaCas9), which is a compact CRISPR system suitable for in vivo delivery (Figure 19). Example 7

[0341] To further investigate the RecT and RecE_587 variants, respectively, Fig. 20A and Fig.21A As shown, both RecT and RecE_587 were truncated at different lengths. The resulting efficiency was measured using an mKate knock-in assay, where wild-type SpCas9 and Cas9n(D10A) had single and double nicks at the DYNLT1 locus (respectively Fig. 20B -C and Fig. 21B -C). The efficiency of the group without recombination is shown as a control.

[0342] When used with different Cas9, the truncated versions of both RecT and RecE_587 retain significant recombination activity. Specifically, compared with full-length RecT (1-269aa), new truncated versions (such as RecT (93-264aa)) have shrunk by more than 30%, but they have substantially retained the full activity of RecT stimulating recombination in eukaryotic cells. Similarly, compared with full-length RecE (1-280aa), truncated versions (such as RecE_587 (120-221aa) and RecE_587 (120-209aa) have shrunk by more than 60%, but still maintain high recombination activity in human cells. These truncated versions demonstrate the potential for further designing minimal functionalized recombinases using RecE and RecT protein variants, and also provide valuable compact recombineering tools for human genome editing, which are very suitable for in vitro, ex vivo and in vivo delivery due to their small size.

[0343] Overall, REDIT exploits the specificity of CRISPR genome targeting and the efficiency of RecE / RecT recombineering. The disclosed efficient, low-error system is a powerful complement to the existing CRISPR toolkit. The balanced efficiency and accuracy of REDITv3N make it an attractive therapeutic option for large cassette knock-ins in immunity and stem cells. Example 8

[0344] Phylogenetic tree of RecE and RecT reconstructed using eukaryotic recombinases from yeast and human ( Figure 1A and Figure 1B ) shows the evolutionary distance of proteins based on sequence homology. The dotted boxes represent full-length E. coli RecB and E. coli RecE proteins. The catalytic core domains (solid boxes) of E. coli RecB and E. coli RecE proteins were used for comparison. The gene editing activity of these recombinant protein families was measured using the MS2-MCP recruitment system, in which sgRNAs carrying MS2 stem loops were used together with recombinant proteins fused to MCP proteins via peptide linkers and nuclear localization signals.

[0345] Three exonuclease proteins were used: an exonuclease from bacteriophage λ, the RecE587 core domain of the E. coli RecE protein, and an exonuclease from bacteriophage T7 (gene name gp6) ( Fig.22A ). Gene editing activity was measured using the mKate knock-in assay at genomic loci (DYNLT1 and HSP90AA1).

[0346] Similar measurements were performed to test the genome editing efficiency of three single-stranded DNA annealing proteins (SSAPs) from the same three microbial species as the exonucleases, namely the Bet protein from bacteriophage λ, the RecT protein from Escherichia coli, and the SSAP from bacteriophage T7 (gene name gp2.5) ( Fig. 22B ).

[0347] According to these results, the genome recombineering activities of all three major phage / microbial recombination system families (λ phage exonucleases and β proteins; Escherichia coli prophase RecE and RecT proteins, T7 phage exonucleases gp6 and single-stranded binding gp2.5 proteins) were systematically measured and verified in eukaryotic cells. All six proteins from the three systems achieved efficient gene editing, knocking kilobase-long sequences into the mammalian genome at two genomic loci. Overall, the exonucleases showed ~3 times higher recombination efficiency (up to 4% of mKate genome knock-in) compared to the no-recombinant control. The single-stranded annealing protein (SSAP) showed higher activity, with gene editing activity 4 to 8 times higher than that of the control group. This demonstrates the universal applicability and effectiveness of microbial recombination proteins in the exonuclease and SSAP families that can be engineered through a Cas9-based fusion protein system to achieve efficient genome recombination in mammalian cells. Example 9

[0348] To demonstrate the generalizability of the REDIT protein design, alternative recruitment systems were developed and tested. For the more compact REDIT system, the REDIT recombinant protein was fused to the N22 peptide, and the sgRNA included a short known sequence of the N22 peptide, boxB, replacing the MCP ( Fig.23A ). The boxB-N22 system showed comparable editing efficiency at both genomic sites tested in a side-by-side comparison with the MS2-MCP recruitment system. Figure 23B-Figure 23E shown.

[0349] Developed the REDIT system using SunTag recruitment (protein-based recruitment system) Fig.24A and Fig.27A). Since SunTag is a fusion protein-based design, the sgRNA or guide RNA is the same as the wild-type CRISPR system. Specifically, the REDIT recombinant protein is fused to the scFV antibody peptide (replacing the MCP), and the GCN4 peptide is fused to the Cas9 protein in a tandem manner (10 copies of the GCN4 peptide separated by a linker). Therefore, the scFV-REDIT can be recruited to the Cas9 complex by the affinity of GCN4 for scFV.

[0350] mKate knock-in experiment ( Fig. 24B and Fig.27B ) were used to measure the editing efficiency at the DYNLT1 locus and the HSP90AA1 locus, respectively. This SunTag-based REDIT system demonstrated a significant improvement in gene editing knock-in efficiency at the tested DYNLT1 genomic locus. In addition, the SunTag design significantly improved HRD efficiency, which was ~2 times higher than Cas9, but not as high as the MS2 aptamer. Example 10

[0351] To demonstrate the universality of REDIT protein design and develop a versatile REDIT system applicable to a range of CRISPR enzymes, a Cpf1 / Cas12a-based REDIT system was developed using SunTag recruitment design ( Fig.25A As previously shown, two different Cpf1 / Cas12a proteins (Lachnospiraceae bacterium ND2006, LbCpf1, and Acidaminococcus sp. BV3L6) were tested using the mKate knock-in assay ( Fig.25B ).

[0352] These results suggest that recombinase proteins (exonucleases and single-stranded annealing proteins) can be engineered using alternative designs, such as the SunTag recruitment system, for genome editing in eukaryotic cells. These protein-based recruitment systems do not require the use of RNA aptamers or RNA-binding proteins, but instead utilize fusion protein domains directly linked to the CRISPR enzyme to recruit the REDIT protein.

[0353] In addition to the flexibility of the recruitment system design, these results using Cpf1 / Cas12a-type CRISPR enzymes also demonstrate the universal applicability of REDIT proteins to various CRISPR systems (in genome recombination). The Cpf1 / Cas12a enzyme has catalytic residues and DNA recognition mechanisms that are different from those of the Cas9 enzyme. Therefore, REDIT recombination proteins (exonucleases and single-stranded annealing proteins) can function independently of the specific selection of CRISPR enzyme components (Cas9, Cpf1 / Cas12a, etc.). This demonstrates the universality of the REDIT system and provides the possibility of using additional (known and unknown) CRISPR enzymes as components of the REDIT system to achieve precise genome editing in eukaryotic cells. Embodiment 11

[0354] Fifteen different microorganisms with RecE / RecT proteins were selected for screening various RecE and RecT proteins in the microbial community (Table 5). Each protein was codon optimized and synthesized. As previously described for the E. coli RecE / RecT-based REDIT system, each protein was fused to the MCP protein via an E-XTEN linker and had an additional nuclear localization signal. The mKate knock-in gene editing assay was used to measure the DYNLT1 locus ( Fig.26A , Table 6) and HSP90AA1 locus ( Fig.26B , Table 6). These homologs demonstrate the ability to enable and enhance precise gene editing. Example 12

[0355] Next, to benchmark the RecT-based REDIT design, it is compared with three categories of existing HDR enhancement tools ( Fig.28A and Fig.28B ): DNA repair enzyme CtIP fused to Cas9 (Cas9-HE), human synaptonemal functional domain (amino acids 1 to 110) fused to Cas9 (Cas9-Gem), and small molecule HDR enhancer nocodazole that works through cell cycle control. Among the endogenous targets tested, the RecT-based REDIT design has good performance compared to three alternative strategies ( Figure 3C). In addition, the RecT-based REDIT design, which is presumed to work through activity independent of other approaches, may synergize with existing approaches. To test this hypothesis, the RecT-based REDIT design was combined with three different approaches (conveniently via MS2-aptamers) ( Fig.28A , right). The RecT-based REDIT design can indeed further improve the HDR-promoting activity of the tools tested ( Fig.28C ). Embodiment 13

[0356] The effect of template HA length on REDIT editing efficiency was quantified using a canonical HDR donor with at least 100 bp of HA on each side ( Fig.29A , left). As HA length increased, increased HDR rates were observed for both the Cas9 and RecT groups, and REDIT stimulated HDR more efficiently relative to Cas9 when HA lengths as short as ~100bp on each side were used. When provided with longer templates with 600-800bp total HA, RecT achieved over 10% HDR efficiency for kb-scale knock-ins without selection, significantly higher than the 2-3% efficiency when Cas9 alone was used. Recent reports have confirmed that the use of donor DNA with shorter HA (typically between 10bp and 50bp) can significantly stimulate knock-in efficiency, thanks to the high repair activity of the microhomology-mediated end joining (MMEJ) pathway. The knock-in efficiency of the REDIT-based approach was compared with that of Cas9 using donor DNA with 0bp (NHEJ-based), 10bp, or 50bp (MMEJ-based) HA. The results indicate that short HA donors utilizing the MMEJ mechanism produced higher editing efficiencies compared to HDR donors ( Fig.29A , right). At the same time, REDIT was able to improve knock-in efficiency as long as HA was present (no effect on 0bp NHEJ donors). This effect was particularly pronounced in the 10bp donor, which had a significant effect and was selected for further characterization and comparison with the HDR donor.

[0357] Knock-in cells are clonally isolated and the target genomic region is amplified using primers that bind exclusively to the donor DNA for colony Sanger sequencing ( Fig.29B Junction sequencing analysis (~48 clones per gene per condition) revealed varying degrees of indels at 5'- and 3'-knock-in junctions (including single or double junctions). Fig.29C ). Overall, HDR donors had better precision than MMEJ donors, and REDIT improved the knock-in yield to some extent compared to Cas9, although junctional indels were still observed.

[0358] In addition, the efficiency of REDIT and Cas9 was compared when making edits of different lengths. For longer edits, a 2-kb knock-in cassette ( Fig.29D ), for shorter edits, a single-stranded oligonucleotide donor (ssODN) was used. When the knock-in sequence length was increased to ∼2-kb using a dual mKate / GFP template, REDIT maintained its HDR-promoting activity compared to Cas9 across all tested endogenous targets ( Fig.29D ). For ssODN testing, REDIT and Cas9 were used to introduce 12-16 bp of exogenous sequence at two established loci, EMX1 and VEGFA. Since the ssODN templates were short (<100 bp HA on each side), next-generation sequencing (NGS) was used to quantify editing events. Comparable levels of indels were observed between Cas9 and REDIT, and HDR efficiency was improved using REDIT. Embodiment 14

[0359] The sensitivity of REDIT's ability to promote HDR was enhanced in the presence or absence of two different pharmacological inhibitors of RAD51, B02 and RI-1. Fig.31A As expected, inhibition of RAD51 significantly reduced HDR efficiency for Cas9-based editing ( Fig.31B , Fig.31C and Fig.32A Interestingly, inhibition of RAD51 only moderately reduced REDIT and REDITdn efficiency, as both REDIT / REDITdn approaches maintained significantly higher knock-in efficiencies compared to Cas9 / Cas9dn under RAD51 inhibition.

[0360] Mirin, a potent chemical inhibitor of DSB repair, was also used and has been shown to prevent MRN complex formation, MRN-dependent ATM activation, and inhibit Mre11 exonuclease activity. When cells were treated with Mirin, only the editing efficiency of the Cas9 reference experiment was affected by Mirin treatment, while the REDIT version was essentially the same as the vehicle-treated group in all genomic targets ( Fig.32A ).

[0361] To test whether cell cycle inhibition affects recombination, cells were chemically synchronized at the G1 / S boundary using double thymidine block (DTB). The REDIT version showed reduced editing efficiency under DTB treatment, although when Miring RI-1 or B02 was combined with DTB treatment, it maintained higher editing efficiency under DNA repair pathway inhibition compared to the Cas9 reference experiment ( Fig.32B ).

[0362] To validate REDIT in different contexts, REDIT was applied to human embryonic stem cells (hESCs) to test its ability to engineer long sequences in non-transformed human cells. Robust stimulation of HDR was observed at all three genomic loci (HSP90AA1, ACTB, OCT4 / POU5F1) using REDIT and REDITdn. Fig.31D and Fig.31E ). Notably, REDIT and REDITdn editing using donor DNA with 200-bp HA on each side achieved kb-scale gene editing efficiencies of over 5% without selection, compared to ~1% using the non-REDIT approach. In addition, REDIT improved knock-in efficiencies in A549 (lung-derived), HepG2 (liver-derived), and HeLa (cervical-derived) cells, demonstrating kb-scale genome knock-in efficiencies of up to ~15% without selection. This improvement is up to 4-fold higher than that of the Cas9 group, providing support for the potential use of the REDIT approach in different cell types. Embodiment 15

[0363] The in vivo use of dCas9-EcRecT (SAFE-dCas9) was tested using the cut-free dCas9 editor by hydrodynamic tail vein injection. The gene editing vectors and template DNA used were as follows Fig.33A As shown. Gene editing vector (60 μg) and template DNA (60 μg) were injected via hydrodynamic tail vein injection to deliver the components to mice. Successful gene editing of hepatocytes was monitored by expression of transgenic encoded protein at the albumin locus. A schematic diagram of the experimental procedure is shown in Fig.33B shown.

[0364] Approximately seven days after injection, perfused mouse livers were dissected. Liver lobes were homogenized and processed to extract liver genomic DNA from primary hepatocytes. The extracted genomic DNA was used for three different downstream analyses: 1) PCR using knock-in specific primers and agarose gel electrophoresis ( Fig.34A ); 2) Sanger sequencing of knock-in PCR products ( Fig.34B ); 3) high-throughput deep sequencing of knock-in junctions to confirm and quantify the accuracy of gene editing using SAFE-dCas9 in vivo ( Fig.34C ). Each downstream analysis confirmed successful knock-in.

[0365] In addition, adeno-associated virus (AAV) delivery into the lungs of LTC mice was used to test in vivo use. LTC mice include three genomic alleles: 1) Lkb1 (flox / flox) allele allows Lkb1-KO when Cre is expressed; 2) R26 (LSL-TdTom) allele allows detection of AAV-transduced cells by TdTom red fluorescent protein; 3) H11 (LSL-Cas9) allele allows expression of Cas9 in AAV-transduced cells. Schematic diagram of REDI gene editing vector and Cas9 control vector is shown in Fig.35A As shown. Fig.35B As shown in , successful gene editing using a gene editing vector resulted in a Kras allele that drove tumor growth in the lungs of treated mice.

[0366] Approximately fourteen weeks after AAV injection, perfused mouse lungs were dissected. Fixed lung tissue was used for imaging analysis to identify tumor formation resulting from successful gene editing ( Fig.35C Quantification of surface tumor numbers by imaging analysis showed that gene editing efficiency and total tumor numbers were increased in REDIT-treated mice ( Fig.35C ). Escherichia coli RecE amino acid sequence (SEQ ID NO: 1): MSTKPLFLLRKAKKSSGEPDVVLWASNDFESTCATLDYLIVKSGKKLSSYFKAVATNFPVVNDLPAEGEIDFTWSERYQLSKDSMTWELKPGAAPDNAHYQGNTNVNGEDMTEIEENMLLPISGQELPIRWLAQHGSEKPVTHVSRDGLQALHIARAEELPAVTALAVSHKTSLLDPLEIRELHKLVRDTDKVFPNPGNSNLGLITAFFEAYLNADYTDRGLLTKEWMKGNRVSHITRTASGANAGGGNLTDRGEGFVHDLTSLARDVATGVLARSMDLDIYNLHPAHAKRIEEIIAENKPPFSVFRDKFITMPGGLDYSRAIVVASVKEAPIGIEVIPAHVTEYLNKVLTETDHANPDPEIVDIACGRSSAPMPQRVTEEGKQDDEEKPQPSGTTAVEQGEAETMEPDATEHHQDTQPLDAQSQVNSVDAKYQELRAELHEARKNIPSKNPVDDDKLLAASRGEFVDGISDPNDPKWVKGIQTRDCVYQNQPETEKTSPDMNQPEPVVQQEPEIACNACGQTGGDNCPDCGAVMGDATYQETFDEESQVEAKENDPEEMEGAEHPHNENAGSDPHRDCSDETGEVADPVIVEDIEPGIYYGISNENYHAGPGISKSQLDDIADTPALYLWRKNAPVDTTKTKTLDLGTAFHCRVLEPEEFSNRFIVAPEFNRRTNAGKEEEKAFLMECASTGKTVITAEEGRKIELMYQSVMALPLGQWLVESAGHAESSIYWEDPETGILCRCRPDKIIPEFHWIMDVKTTADIQRFKTAYYDYRYHVQDAFYSDGYEAQFGVQPTFVFLVASTTIECGRYPVEIFMMGEEAKLAGQQEYHRNLRTLADCLNTDEWPAIKTLSLPRWAKEYAND Amino acid sequence of Escherichia coli RecE_587 (SEQ ID NO:2): ADPVIVEDIEPGIYYGISNENYHAGPGVSKSQLDDIADTPALYLWRKNAPVDTTKTKTLDLGTAFHCRVLEPEEFSNRFIVAPEFNRRTNSGKEEEKAFLRECASTGKTVITAEEGRKIELMYQSVMALPLGQWLVESAGHAESSIYWEDPETAILCRCRPDKIIPEFHWIMDVKTTADIQRFKTAYYDYRYHVQDAFYSDGYEAQFGVQPTFVFLVASTTIECGRYPVEIFMMGEEAKLAGQLEYHRNLRTLADCLNTDEWPAIKTLSLPRWAKEYAND* Amino acid sequence of CTD_RecE from Escherichia coli (SEQ ID NO:3): GISNENYHAGPGVSKSQLDDIADTPALYLWRKNAPVDTTKTKTLDLGTAFHCRVLEPEEFSNRFIVAPEFNRRTNSGKEEEKAFLRECASTGKTVITAEEGRKIELMYQSVMALPLGQWLVESAGHAESSIYWEDPETAILCRCRPDKIIPEFHWIMDVKTTADIQRFKTAYYDYRYHVQDAFYSDGYEAQFGVQPTFVFLVASTTIECGRYPVEIFMMGEEAKLAGQLEYHRNLRTLADCLNTDEWPAIKTLSLPRWAKEYAND* Amino acid sequence of RecE from Pantoea brenneri (SEQ ID NO:4): MQPGIYYDISNEDYHRGAGISKSQLDDIAISPAIYQWRKHAPVDEEKTAALDLGTALHCLLLEPDEFSKRFQIGPEVNRRTTAGKEKEKEFIERCEAEGITPITHDDNRKLKLMRDSALAHPIARWMLEAQGNAEASIYWNDRDAGVLSRCRPDKIITEFNWCVDVKSTADIMKFQKDFYSYRYHVQDAFYSDGYESHFHETPTFAFLAVSTSIDCGRYPVQVFIMDQQAKDAGRAEYKRNIHTFAECLSRNEWPGIATLSLPFWAKELRNE Amino acid sequence of RecE from the F-type symbiont of Plautia stali (SEQ ID NO:5): MQPGIYYDISNEDYHGGPGISKSQLDDIAISPAIYQWRKHAPVDEEKTAALDLGTALHCLLLEPDEFSKRFEIGPEVNRRTTAGKEKEKEFMERCEAEGVTPITHDDNRKLRLMRDSAMAHPIARWMLEAQGNAEASIYWNDRDTGVLSRCRPDKIITDFNWCVDVKSTADIIKFQKDFYSYRYHVQDAFYSDGYESHFDETPTFAFLAVSTSIDCGRYPVQVFIMDQQAKDAGRAEYKRNIHTFAECLSRNEWPGIATLSLPYWAKELRNE Amino acid sequence of Providencia sp. MGF014 RecE (SEQ ID NO:6): MKEGIYYNISNEDYHNGLGISKSQLDLINEMPAEYIWSKEAPVDEEKIKPLEIGTALHCLLLEPDEYHKRYKIGPDVNRRTNVGKEKEKEFFDMCEKEGITPITHDDNRKLMIMRDSALAHPIAKWCLEADGVSESSIYWTDKETDVLCRCRPDRIITAHNYIIDVKSSGDIEKFDYEYYNYRYHVQDAFYSDGYKEVTGITPTFLFLVVSTKIDCGKYPVRTYVMSEEAKSAGRTAYKHNLLTYAECLKTDEWAGIRTLSLPRWAKELRNE Amino acid sequence of Shigella sonnei RecE (SEQ ID NO:7): DRGLLTKEWRKGNRVSRITRTASGANAGGGNLTDRGEGFVHDLTSLARDIATGVLARSMDVDIYNLHPAHAKRIEEIIAENKPPFSVFRDKFITMPGGLDYSRAIVVASVKEAPIGIEVIPAHVTAYLNKVLTETDHANPDPEIVDIACGRSSAPMPQRVTEEGKQDDEEKLQPSGTTADEQGEAETMEPDATKHHQDTQPLDAQSQVNSVDAKYQELRAELHEARKNIPSKNPVDADKLLAASRGEFVDGISDPNDPKWVKGIQTRDSVYQNQPETEKTSPDMKQPEPVVQQEPEIAFNACGQTGGDNCPDCGAVMGDATYQETFDEENQVEAKENDPEEMEGAEHPHNENAGSDPHRDCSDETGEVADPVIVEDIEPGIYYGISNENYHAGPGVSKSQLDDIADTPALYLWRKNAPVDTTKTKTLDLGTAFHCRVLEPEEFSNRFIVAPEFNRRTNAGKEEEKAFLMECASTGKMVITAEEGRKIELMYQSVMALPLGQWLVESAGHAESSIYWEDPETGILCRCRPDKIIPEFHWIMDVKTTADIQRFKTAYYDYRYHVQDAFYSDGYEAQFGVQPTFVFLVASTTIECGRYPVEIFMMGEEAKLAGQLEYHRNLRTLADCLNTDEWPAIKTLSLPRWAKEYAND Amino acid sequence of RecE from Pseudobacteriovorax antilogorgiicola (SEQ ID NO:8): MSKLSNLKVSNSDVDTLSRIRMKEGVYRDLPIESYHQSPGYSKTSLCQIDKAPIYLKTKVPQKSTKSLNIGTAFHEAMEGVFKDKYVVHPDPGVNKTTKSWKDFVKRYPKHMPLKRSEYDQVLAMYDAARSYRPFQKYHLSRGFYESSFYWHDAVTNSLIKCRPDYITPDGMSVIDFKTTVDPSPKGFQYQAYKYHYYVSAALTLEGIEAVTGIRPKEYLFLAVSNSAPYLTALYRASEKEIALGDHFIRRSLLTLKTCLESGKWPGLQEEILELGLPFSGLKELREEQEVEDEFMELVG Amino acid sequence of Escherichia coli RecT (SEQ ID NO:9): MTKQPPIAKADLQKTQGNRAPAAVKNSDVISFINQPSMKEQLAAALPRHMTAERMIRIATTEIRKVPALGNCDTMSFVSAIVQCSQLGLEPGSALGHAYLLPFGNKNEKSGKKNVQLIIGYRGMIDLARRSGQIASLSARVVREGDEFSFEFGLDEKLIHRPGENEDAPVTHVYAVARLKDGGTQFEVMTRKQIELVRSLSKAGNNGPWVTHWEEMAKKTAIRRLFKYLPVSIEIQRAVSMDEKEPLTIDPADSSVLTGEYSVIDNSEE* Amino acid sequence of Pantoea brenneri RecT (SEQ ID NO:10): MSNQPPIASADLQKTQQSKQVANKTPEQTLVGFMNQPAMKSQLAAALPRHMTADRMIRIVTTEIRKTPQLAQCDQSSFIGAVVQCSQLGLEPGSALGHAYLLPFGNGRSKSGQSNVQLIIGYRGMIDLARRSGQIVSLSARVVRADDEFSFEYGLDENLVHRPGENEDAPITHVYAVARLKDGGTQFEVMTVKQVEKVKAQSKASSNGPWVTHWEEMAKKTVIRRLFKYLPVSIEMQKAVVLDEKAESDVDQDNASVLSAEYSVLESGDEATN F-type symbiont RecT amino acid sequence of Plautia stali (SEQ ID NO:11): MSNQPPIASADLQKTQQSKQVANKTPEQTLVGFMNQPAMKSQLAAALPRHMTADRMIRIVTTEIRKTPALATCDQSSFIGAVVQCSQLGLEPGSALGHAYLLPFGNGRSKSGQSNVQLIIGYRGMIDLARRSGQIVSLSARVVRADDEFSFEYGLDENLIHRPGDNEDAPITHVYAVARLKDGGTQFEVMTAKQVEKVKAQSKASSNGPWVTHWEEMAKKTVIRRLFKYLPVSIEMQKAVVLDEKAESDVDQDNASVLSAEYSVLEGDGGE RecT amino acid sequence of Providencia sp. MGF014 (SEQ ID NO:12): MSNPPLAQSDLQKTQGTEVKVKTKDQQLIQFINQPSMKAQLAAALPRHMTPDRMIRIVTTEIRKTPALATCDMQSFVGAVVQCSQLGLEPGNALGHAYLLPFGNGKAKSGQSNVQLIIGYRGMIDLARRSNQIISISARTVRQGDNFHFEYGLNEDLTHTPSENEDSPITHVYAVARLKDGGVQFEVMTYNQVEKVRASSKAGQNGPWVSHWEEMAKKTVIRRLFKYLPVSIEMQKAVVLDEKAEANVDQENATIFEGEYEEVGTDGN RecT amino acid sequence of Shigella sonnei (SEQ ID NO:13): MTKQPPIAKADLQKTQENRAPAAIKNNDVISFINQPSMKEQLAAALPRHMTAERMIRIATTEIRKVPALGNCDTMSFVSAIVQCSQLGLEPGSALGHAYLLPFGNKNEKSGKKNVQLIIGYRGMIDLARRSGQIASLSARVVREGDEFNFEFGLDEKLIHRPGENEDAPVTHVYAVARLKDGGTQFEVMTRRQIELVRSQSKAGNNGPWVTHWEEMAKKTAIRRLFKYLPVSIEIQRAVSMDEKEPLTIDPADSSVLTGEYSVIDNSEE Pseudobacteriovorax antilogorgiicola RecT amino acid sequence (SEQ ID NO: 14): MGHLVSKTEQDYIKQHYAKGATDQEFEHFIGVCRARGLNPAANQIYFVKYRSKDGPAKPAFILSIDSLRLIAHRTGDYAGCSEPIFTDGGKACTVTVRRNLKSGETGNFSGMAFYDEQVQQ KNGRPTSFWQSKPRTMLEKCAEAKALRKAFPQDLGQFYIREEMPPQYDEPIQVHKPKALEEPRFSKSDLSRRKGLNRKLSALGVDPSRFDEVATFLDGTPDRELGQKLKLWLKEAGYGVNQ SV40 NLS amino acid sequence (SEQ ID NO: 16): PKKKRKV Ty1 NLS amino acid sequence (SEQ ID NO:17): NSKKRSLEDNETEIKVSRDTWNTKNMRSLEPPRSKKRIH c-Myc NLS amino acid sequence (SEQ ID NO: 18): PAAKRVKLD biSV40 NLS amino acid sequence (SEQ ID NO: 19): KRTADGSEFESPKKKRKV Mut NLS amino acid sequence (SEQ ID NO:20): PEKKRRRPSGSVPVLARPSPPKAGKSSCI Template DNA sequence (underline marks the replaced or inserted editing sequence) EMX1 HDR template sequence (SEQ ID NO:79): CATTCTGCCTCTCTGTATGGAAAAGAGCATGGGGCTGGCCCGTGGGGTGGTGTCCACTTTAGGCCCTGTGGGAGATCATGGGAACCCACGCAGTGGGTcataggctctctcatttactactcacatccactctgtgaagaagcgattatgatctctcctctagaaaCTCGTAGAGTCCCATGTCTGCCGGCTTCCAGAGCCTGCACTCCTCCACCTTGGCTTGGCTTTGCTGGGGCTAGAGGAGCTAGGATGCACAGCAGCTCTGTGACCCTTTGTTTGAGAGGAACAGGAAAACCACCCTTCTCTCTGGCCCACTGTGTCCTCTTCCTGCCCTGCCATCCCCTTCTGTGAATGTTAGACCCATGGGAGCAGCTGGTCAGAGGGGACCCCGGCCTGGGGCCCCTAACCCTATGTAGCCTCAGTCTTCCCATCAGGCTCTCAGCTCAGCCTGAGTGTTGAGGCCCCAGTGGCTGCTCTGGGGGCCTCCTGAGTTTCTCATCTGTGCCCCTCCCTCCCTGGCCCAGGTGAAGGTGTGGTTCCAGAACCGGAGGACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTGGCGCATTGCCACGAAGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAATGACT CGGATGTACACGGTCTGCAACCACAAACCCACGAGGGCAGAGTGCTGCTTGCTGCTGGCCAGGCCCCTGCGTGGGCCCAAGCTGGACTCTGGCCACTCCCTGGCCAGGCTTTGGGGAGGCCTGGAGTCATGGCCCCACAGGGCTTGAAGCCCGGGGCCGCCATTGACAGAGGGACAAGCAATGGGCTGGCTGAGGCCTGGGACCACTTGGCCTTCTCCTCGGAGAGCCTGCCTGCCTGGGCGGGCCCGCCCGCCACCGCAGCCTCCCAGCTGCTCTCCGTGTCTCCAATCTCCCTTTTGTTTTGATGCATTTCTGTTTTAATTTATTTTCCAGGCACCACTGTAGTTTAGTGATCCCCAGTGTCCCCCTTCCCTATGGGAATAATAAAAGTCTCTCTCTTAATGACACGGGCATCCAGCTCCAGCCCCAGAGCCTGGGGTGGTAGATTCCGGCTCTGAGGGCCAGTGGGGGCTGGTAGAGCAAACGCGTTCAGGGCCTGGGAGCCTGGGGTGGGGTACTGGTGGAGGGGGTCAAGGGTAATTCATTAACTCCTCTCTTTTGTTGGGGGACCCTGGTCTCTACCTCCAGCTCCACAGCAGGAGAAACAGGCTAGACATAGGGAAGGGCCATCCTGTATCTTGAGGGAGGACAGGCCCAGGTCTTTCTTAACGTATTGAGAGGTGGGAATCAGGCCCAGGTAGTTCAATGGG VEGFA HDR template sequence (SEQ ID NO:80): AGGTTTGAATCATCACGCAGGCCCTGGCCTCCACCCGCCCCCACCAGCCCCCTGGCCTCAGTTCCCTGGCAACATCTGGGGTTGGGGGGGCAGCAGGAACAAGGGCCTCTGTCTGCCCAGCTGCCTCCCCCTTTGGGTTTTGCCAGACTCCACAGTGCATACGTGGGCTCCAACAGGTCCTCTTCCCTCCCAGTCACTGACTAACCCCGGAACCACACAGCTTCCCGTTctcagctccacaaacttggtgccaaattcttctcccctgggaagcatccctggacacttcccaaaggaccccagtcactccagcctgttggctgccgctcactttgatgtctgcaggccagatgagggctccagatggcacattgtcagagggacacactgtggcccctgtgcccagccctgggctctctgtacatgaagcaactccagtcccaaatatgtagctgtttgggaggtcagaaatagggggtccaggagcaaactccccccaccccctttccaaagcccattccctctttagccagagccggggtgtgcagacggcagtcactagggggcgctcggccaccacagggaagctgggtgaatggagcgagcagcgtcttcgagagtgaggacgtgtgtgtctgtgtgggtgagtgagtgtg CgcACTCTAGAGgtgtCgTgttgagggcgttggagcggggagaaggccaggggtcactccaggattccaatagatctgtgtgtccctctccccacccgtccctgtccggctctccgccttcccctgcccccttcaatattcctagcaaagagggaacggctctcaggccctgtccgcacgtaacctcactttcctgctccctcctcgccaatgccccgcgggcgcgtgtctctggacagagtttccgggggcggatgggtaattttcaggctgtgaaccttggtgggggtcgagcttccccttcattgcggcgggctGCGGGCCAGGCTTCACTGAGCGTCCGCAGAGCCCGGGCCCGAGCCGCGTGTGGAAGGGCTGAGGCTCGCCTGTccccgccccccggggcgggccgggggcggggtcccggcggggcggAGCCATGCGCCCCCCCCttttttttttAAAAGTCGGCTGGTAGCGGGGAGGATCGCGGAGGCTTGGGGCAGCCGGGTAGCTCGGAGGTCGTGGCGCTGGGGGCTAGCACCAGCGCTCTGTCGGGAGGCGCAGCGGTTAGGTGGACCGGTCAGCGGACTCACCGGCCAGGGCGCTCGGTGCTGGAATTTGATATTCATTGATCCGGGttttatccctcttcttttttcttaaacatttttttttAAAACTGTATTGTTTCTCGTTTTAATTTATTTTTGCTTGCCATTCCCCACTTGAAT DYNLT1 HDR template sequence (SEQ ID NO:81): AGTGACCTGTGTAATTATGCAGAAGAATGGAGCTGGATTACACACAGCAAGTTCCTGCTTCTGGGACAGCTCTACTGACGGTATGATTTTCATTCATGTTTGTGAAGTTTTGTTGTGTGAAATATATGACTGGAAGTTTCCTATCTTTGAATGCAATGCATGTTTATCACCTTTTAAAACATTTAATAATAGACTTGCCAAGGTTCTTTGTGTAGCATAGAGATGGGTACTTGAATGTTGGCCTTATTGTGAGTAAAACGTCGTCCCCCAGCTTTCCCTGCCGTAAATGCTGCTCTCTTCCCTCCCGCAGGGAGCTGCACTGTGCGATGGGAGAATAAGACCATGTACTGCATCGTCAGTGCCTTCGGACTGTCTATT GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTgcca ccatggtgagcgagctgattaaggagaacatgcacatgaagctgtacatggagggcaccgtgaacaaccaccactt caagtgcacatccgagggcgaaggcaagccctacgagggcacccagaccatgagaatcaaggcggtcgagggcggc cctctccccttcgccttcgacatcctggctaccagcttcatgtacggcagcaaaaccttcatcaaccacacccagg gcatccccgacttctttaagcagtccttccccgagggcttcacatggggagagagtcaccacatacgaagatggggg cgtgctgaccgctacccaggacaccagcctccaggacggctgcctcatctacaacgtcaagatcagaggggtgaac ttcccatccaacggccctgtgatgcagaagaaaacactcggctgggaggcctccaccgagacactgtaccccgctg acggcggcctggaaggcagagccgacatggccctgaagctcgtgggcgggggccacctgatctgcaaccttaagac cacatacagatccaagaaacccgctaagaacctcaagatgcccggcgtctactatgtggacaggagactggaaaga atcaaggaggccgacaaagagacatacgtcgagcagcacgaggtggctgtggccagatactgcgacctccctagca aactggggcacaaacttaattcc TAACCaGCtGTCCtGCCTATGGCCTTTCTCCTTTTGTCTCTAGTTCATCCTCTAACCACCAGCCATGAATTCAGTGAACTCTTTTCTCATTCTCTTTGTTTTGTGGCACTTTCACAATGTAGAGGAAAAAACCAAATGACCGCACTGTGATGTGAATGGCACCGAAGTCAGATGAGTATCCCTGTAGGTCACCTGCAGCCTGCGTTGCCACTTGTCTTAACTCTGAATATTTCATTTCAAAGGTGCTAAAATCTGAAATCTGCTAGTGTGAAACTTGCTCTACTCTCTGAAATGATTCAAATACACTAATTTTCCATACTTTATACTTTTGTTAGAATAAATTATTCAAATCTAAAGTCTGTTGTGTTCTTCATAGTCTGCATAGTATCATAAACG HSP90AA1 HDR template sequence (SEQ ID NO:82): GCAGCAAAGAAACACCTGGAGATAAACCCTGACCATTCCATTATTGAGACCTTAAGGCAAAAGGCAGAGGCTGATAAGAACGACAAGTCTGTGAAGGATCTGGTCATCTTGCTTTATGAAACTGCGCTCCTGTCTTCTGGCTTCAGTCTG GAAGATCCCCAGACACATGCTAACAGGATCTACAGGATGATCAAACTTGGTCTGGGTAAGCCTTATACTATGTAATGTTAAAAGAAAATAAACACACGTGACATTGAAGAAAATGGTGAACTTTCAGTTATCCAAACTTGGAGCACCTT GTCCTGCTTGCTGCTTGGAGGTATTAAAGTATGttttttttAGGGATAAGTAAGGTCTTACAAGAGCAAAGAAATGAAATTGAGACTCATATGTCCTGTAATACTGTCTTGAAAGCAGATAGAAACCAAGAGTATTACCCTAATAGCTGGCTTTAAGAAATCTTTGTAATATGAGGATTTTATTTTGGAAACAGGTATTGATGAAGATGACCCTACTGCTGATGATACCAGTGCTGCTGTAACTGAAGAAATGCCACCCCTTGAAGGAGATGACGACACATCACGCATGGAAGAAGTAGAC GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTgtgagcgag ctgattaaggagaacatgcacatgaagctgtacatggagggcaccgtgaacaaccaccacttcaagtgcacatccg agggcgaaggcaagccctacgagggcacccagaccatgagaatcaaggcggtcgagggcggccctctccccttcgc cttcgacatcctggctaccagcttcatgtacggcagcaaaaccttcatcaaccacacccagggcatccccgacttc tttaagcagtccttccccgagggcttcacatgggagagagtcaccacatacgaagatgggggcgtgctgaccgcta cccaggacaccagcctccaggacggctgcctcatctacaacgtcaagatcagaggggtgaacttcccatccaacgg ccctgtgatgcagaagaaaacactcggctgggaggcctccaccgagacactgtaccccgctgacggcggcctggaa ggcagagccgacatggccctgaagctcgtgggcgggggccacctgatctgcaaccttaagaccacatacagatcca agaaacccgctaagaacctcaagatgcccggcgtctactatgtggacaggagactggaaagaatcaaggaggccga caaagagacatacgtcgagcagcacgaggtggctgtggccagatactgcgacctccctagcaaactggggcacaaa cttaattccTAa ATCTgTGGCTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATATTTTCAAGGATGTTTTTCTTTATTTTTGTTAATATTAAAAAGTCTGTATGGCATGACAACTACTTTAAGGGGAAGATAAGATTTCTGTCTACTAAGTGATGCTGTGATACCTTAGGCACTAAAGCAGAGCTAGTAATGCTTTTTGAGTTTCATGTTGGTTTATTTTCACAGATTGGGGTAACGTGCACTGTAAGACGTATGTAACATGATGTTAACTTTGTGGTCTAAAGTGTTTAGCTGTCAAGCCGGATGCCTAAGTAGACCAAATCTTGTTATTGAAGTGTTCTGAGCTGTATCTTGATGTTTAGAAAAGTATTCGTTACATCTTGTAGGATCTACTTTTTGAACTTTTCATTCCCTGTAGTTGACAATTCTGCATGTACTAGTCCTCTAGAAATAGGTTAAACTGAAGCAACTTGATGGAAGGATCTCTCCACAGGGCTTGTTTTCCAAAGAAAAGTATTGTTTGGAGGAGCAAAGTTAAAAGCCTACCTAAGCATATCGTAAAGCTGTTCAAAAATAACTCAGACCCAGTCTTGTGGA AAVS1 HDR template sequence (SEQ ID NO:83): gatgctctttccggagcacttccttctcggcgctgcaccacgtgatgtcctctgagcggatcctccccgtgtctgggtcctctccgggcatctctcctccctcacccaaccccatgccgtcttcactcgctgggttcccttttccttctccttctggggcctgtgccatctctcgtttcttaggatggccttctccgacggatgtctcccttgcgtcccgcctccccttcttgtaggcctgcatcatcaccgtttttctggacaaccccaaagtaccccgtctccctggctttagccacctctccatcctcttgctttctttgcctggacaccccgttctcctgtggattcgggtcacctctcactcctttcatttgggcagctcccctaccccccttacctctctagtctgtgctagctcttccagccccctgtcatggcatcttccaggggtccgagagctcagctagtcttcttcctccaacccgggcccctatgtccacttcaggacagcatgtttgctgcctccagggatcctgtgtccccgagctgggaccaccttatattcccagggccggttaatgtggctctggttctgggtacttttatctgtcccctccaccccacagtggggcaagctt ctgacctcttctcttcctcccacagggcctcgaga gatctggcagcggaGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGG ACCTgtgagcgagctgattaaggagaacatgcacatgaagctgtacatggagggcaccgtgaacaaccaccacttc aagtgcacatccgagggcgaaggcaagccctacgagggcacccagaccatgagaatcaaggcggtcgagggcggcc ctctccccttcgccttcgacatcctggctaccagcttcatgtacggcagcaaaaccttcatcaaccacacccaggg catccccgacttctttaagcagtccttccccgagggcttcacatgggagagagtcaccacatacgaagatgggggc gtgctgaccgctacccaggacaccagcctccaggacggctgcctcatctacaacgtcaagatcagaggggtgaact tcccatccaacggccctgtgatgcagaagaaaacactcggctgggaggcctccaccgagacactgtaccccgctga cggcggcctggaaggcagagccgacatggccctgaagctcgtgggcgggggccacctgatctgcaaccttaagacc acatacagatccaagaaacccgctaagaacctcaagatgcccggcgtctactatgtggacaggagactggaaagaa tcaaggaggccgacaaagagacatacgtcgagcagcacgaggtggctgtggccagatactgcgacctccctagcaa actggggcacaaacttaattccTA aactagggacaggattggtgacagaaaagccccatccttaggcctcctccttcctagtctcctgatattgggtctaacccccacctcctgttaggcagattccttatctggtgacacacccccatttcctggagccatctctctccttgccagaacctctaaggtttgcttacgatggagccagagaggatcctgggagggagagcttggcagggggtgggagggaagggggggatgcgtgacctgcccggttctcagtggccaccctgcgctaccctctcccagaacctgagctgctctgacgcggctgtctggtgcgtttcactgatcctggtgctgcagcttccttacacttcccaagaggagaagcagtttggaaaaacaaaatcagaataagttggtcctgagttctaactttggctcttcacctttctagtccccaatttatattgttcctccgtgcgtcagttttacctgtgagataaggccagtagccagccccgtcctggcagggctgtggtgaggaggggggtgtccgtgtggaaaactccctttgtgagaatggtgcgtcctaggtgttcaccaggtcgtggccgcctctactccctttctctttctccatccttctttccttaaagagtccccagtgctatctgggacatattcctccgcccagagcagggtcccgcttccctaaggccctgctctgggcttctgggtttgagtccttggc OCT4 HDR template sequence (SEQ ID NO:84): GCGACTATGCACAACGAGAGGATTTTGAGGCTGCTGGGTCTCCTTTCTCAGGGGGACCAGTGTCCTTTCCTCTGGCCCCAGGGCCCCATTTTGGTACCCCAGGCTATGGGAGCCCTCACTTCACTGCACTGTACTCCTCGGTCCCTTTCCCTGAGGGGGAAGCCTTTCCCCCTGTCTCCGTCACCACTCTGGGCTCTCCCATGCATTCAAAt GGAAGCGG AGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTgccaccatggtgagcgag ctgattaaggagaacatgcacatgaagctgtacatggagggcaccgtgaacaaccaccacttcaagtgcacatccg agggcgaaggcaagccctacgagggcacccagaccatgagaatcaaggcggtcgagggcggccctctccccttcgc cttcgacatcctggctaccagcttcatgtacggcagcaaaaccttcatcaaccacacccagggcatccccgacttc tttaagcagtccttccccgagggcttcacatggggagagtcaccacatacgaagatgggggcgtgctgaccgcta cccaggacaccagcctccaggacggctgcctcatctacaacgtcaagatcagaggggtgaacttcccatccaacgg ccctgtgatgcagaagaaaacactcggctgggaggcctccaccgagacgaccacatacagatccaagaaacccgct aagaacctcaagatgcccggcgtctactatgtggacaggagactggaaagaatcaaggaggccgacaaagagacat acgtcgagcagcacgaggtggctgtggccagatactgcgacctccctagcaaactggggcacaaacttaattccTA a TGACTAGGAATGGGGGACAGGGGGAGGGGAGGAGCTAGGGAAAGAAAACCTGGAGTTTGTGCCAGGGTTTTTGGGATTAAGTTCTTCATTCACTAAGGAAGGAATTGGGAACACAAAGGGTGGGGGCAGGGGAGTTTGGGGCAACTGGTTGGAGGGAAGGTGAAGTTCAATGATGCTCTTGATTTTAATCCCACATCATGTATCACTTTTTTCTTAAATAAAGAAGCCTGGGACACAGTAGATAGACACACTT Pantoea stewartii RecT DNA (SEQ ID NO:85): AGCAACCAGCCCCCTATCGCCTCCGCCGATCTGCAGAAGGCCAACACCGGCAAGCAGGTGGCCAATAAGACCCCTGAGCAGACACTGGTGGGCTTCATGAATCAGCCAGCAATGAAGAGCCAGCTGGCCGCCGCCCTGCCAAGGCACATGACAGCCGATCGGATGATCAGAATCGTGACCACAGAGATCCGCAAGACCCCCGCCCTGGCCACATGCGACCAGAGCTCCTTCATCGGCGCCGTGGTGCAGTGTTCTCAGCTGGGCCTGGAGCCTGGCAGCGCCCTGGGCCACGCCTACCTGCTGCCATTTGGCAACGGCCGGAGCAAGTCCGGACAGTCCAATGTGCAGCTGATCATCGGCTATAGAGGCATGATCGATCTGGCCCGGAGATCTGGCCAGATCGTGTCTCTGAGCGCCAGGGTGGTGCGCGCAGACGATGAGTTCTCCTTTGAGTACGGCCTGGATGAGAACCTGATCCACCGGCCAGGCGAGAATGAGGACGCACCCATCACCCACGTGTATGCAGTGGCAAGACTGAAGGACGGAGGCACCCAGTTCGAAGTGATGACAGTGAAGCAGATCGAGAAGGTGAAGGCCCAGTCCAAGGCCTCTAGCAACGGACCCTGGGTGACCCACTGGGAGGAGATGGCCAAGAAAACCGTGATCAGGCGCCTGTTTAAGTACCTGCCCGTGAGCATCGAGATGCAGAAGGCCGTGATCCTGGATGAGAAGGCCGAGTCTGACGTGGATCAGGACAATGCCTCCGTGCTGTCTGCCGAGTATAGCGTGCTGGACGGCTCCTCTGAGGAG Pantoea stewartii RecE DNA (SEQ ID NO:86): CAGCCCGGCGTGTACTATGACATCTCCAACGAGGAGTATCACGCCGGCCCTGGCATCAGCAAGTCCCAGCTGGACGACATCGCCGTGTCCCCAGCCATCTTCCAGTGGAGAAAGTCTGCCCCCGTGGACGATGAGAAAACCGCCGCCCTGGACCTGGGCACAGCCCTGCACTGCCTGCTGCTGGAGCCTGATGAGTTCTCCAAGAGGTTTATGATCGGCCCAGAGGTGAACCGGAGAACCAATGCCGGCAAGCAGAAGGAGCAGGACTTCCTGGATATGTGCGAGCAGCAGGGCATCACCCCTATCACACACGACGATAACCGGAAGCTGAGACTGATGAGGGACTCTGCCTTTGCCCACCCAGTGGCCAGATGGATGCTGGAGACAGAGGGCAAGGCCGAGGCCTCTATCTACTGGAATGACAGGGATACACAGATCCTGAGCAGGTGCCGCCCCGACAAGCTGATCACCGAGTTCTCTTGGTGCGTGGACGTGAAGAGCACAGCCGACATCGGCAAGTTCCAGAAGGACTTCTACAGCTATCGCTACCACGTGCAGGACGCCTTCTATTCCGATGGCTACGAGGCCCAGTTTTGCGAGGTGCCAACCTTCGCCTTTCTGGTGGTGAGCTCCTCTATCGATTGTGGCCGGTATCCCGTGCAGGTGTTTATCATGGACCAGCAGGCAAAGGATGCAGGAAGGGCCGAGTATAAGCGGAACCTGACCACATACGCCGAGTGCCAGGCAAGGAATGAGTGGCCTGGCATCGCCACACTGAGCCTGCCTTACTGGGCCAAGGAGATCCGGAATGTG Pantoea brennerii RecT DNA (SEQ ID NO:87): AGCAACCAGCCCCCTATCGCCTCCGCCGATCTGCAGAAAACCCAGCAGTCCAAGCAGGTGGCCAACAAGACCCCTGAGCAGACACTGGTGGGCTTCATGAATCAGCCAGCAATGAAGAGCCAGCTGGCCGCCGCCCTGCCAAGGCACATGACCGCCGATCGGATGATCAGAATCGTGACCACAGAGATCCGCAAGACACCACAGCTGGCCCAGTGCGACCAGAGCTCCTTCATCGGCGCCGTGGTGCAGTGTTCTCAGCTGGGCCTGGAGCCTGGCAGCGCCCTGGGCCACGCCTACCTGCTGCCATTTGGCAACGGCCGGTCCAAGTCTGGCCAGAGCAATGTGCAGCTGATCATCGGCTATAGAGGCATGATCGATCTGGCCCGGAGATCCGGACAGATCGTGAGCCTGTCCGCCAGGGTGGTGCGCGCAGACGATGAGTTCTCTTTTGAGTACGGCCTGGATGAGAACCTGGTGCACCGGCCAGGCGAGAATGAGGACGCACCCATCACCCACGTGTATGCAGTGGCAAGACTGAAGGACGGAGGCACCCAGTTCGAAGTGATGACAGTGAAGCAGGTGGAGAAGGTGAAGGCCCAGTCCAAGGCCTCTAGCAATGGCCCCTGGGTGACCCACTGGGAGGAGATGGCCAAGAAAACCGTGATCAGGCGCCTGTTTAAGTACCTGCCCGTGAGCATCGAGATGCAGAAGGCCGTGGTGCTGGATGAGAAGGCCGAGTCTGACGTGGATCAGGACAACGCCTCTGTGCTGAGCGCCGAGTATTCCGTGCTGGAGTCTGGCGACGAGGCCACAAAT Pantoea brennerii RecE DNA (SEQ ID NO:88): CAGCCTGGCATCTACTATGACATCAGCAACGAGGATTATCACAGGGGAGCAGGCATCAGCAAGTCCCAGCTGGACGACATCGCCATCTCCCCAGCCATCTACCAGTGGAGAAAGCACGCCCCCGTGGACGAGGAGAAAACCGCCGCCCTGGATCTGGGCACAGCCCTGCACTGCCTGCTGCTGGAGCCTGACGAGTTCTCTAAGAGGTTTCAGATCGGCCCAGAGGTGAACCGGAGAACCACAGCCGGCAAGGAGAAGGAGAAGGAGTTCATCGAGCGGTGCGAGGCAGAGGGAATCACCCCAATCACACACGACGATAATAGGAAGCTGAAGCTGATGAGGGATTCCGCCCTGGCCCACCCAATCGCAAGGTGGATGCTGGAGGCACAGGGAAACGCAGAGGCCTCTATCTATTGGAATGACAGAGATGCCGGCGTGCTGAGCAGGTGCCGCCCCGACAAGATCATCACCGAGTTCAACTGGTGCGTGGACGTGAAGTCCACAGCCGACATCATGAAGTTCCAGAAGGACTTCTACTCTTACAGATACCACGTGCAGGACGCCTTCTATTCCGATGGCTACGAGTCTCACTTTCACGAGACACCCACATTCGCCTTTCTGGCCGTGTCTACCAGCATCGACTGCGGCAGGTATCCTGTGCAGGTGTTTATCATGGACCAGCAGGCAAAGGATGCAGGAAGGGCCGAGTACAAGAGAAACATCCACACCTTCGCCGAGTGTCTGAGCAGGAATGAGTGGCCTGGCATCGCCACACTGTCCCTGCCTTTTTGGGCCAAGGAGCTGCGCAATGAG Pantoea dispersa RecT DNA (SEQ ID NO:89): TCCAACCAGCCACCTCTGGCCACCGCAGATCTGCAGAAAACCCAGCAGTCTAACCAGGTGGCCAAGACCCCTGAGCAGACACTGGTGGGCTTCATGAATCAGCCAGCAATGAAGAGCCAGCTGGCCGCCGCCCTGCCAAGGCACATGACCGCCGATCGGATGATCAGAATCGTGACCACAGAGATCCGCAAGACACCCGCCCTGGCCCAGTGCGACCAGAGCTCCTTCATCGGAGCAGTGGTGCAGTGTAGCCAGCTGGGCCTGGAGCCTGGCTCCGCCCTGGGCCACGCCTACCTGCTGCCATTTGGCAACGGCCGGTCCAAGTCTGGCCAGAGCAATGTGCAGCTGATCATCGGCTATAGAGGCATGATCGATCTGGCCCGGAGATCCGGACAGATCGTGAGCCTGTCCGCCAGGGTGGTGCGCGCAGACGATGAGTTCTCTTTTGAGTACGGCCTGGATGAGAACCTGATCCACCGGCCAGGCGACAATGAGTCCGCCCCCATCACCCACGTGTATGCAGTGGCAAGACTGAAGGACGGAGGCACCCAGTTCGAAGTGATGACAGCCAAGCAGGTGGAGAAGGTGAAGGCCCAGTCCAAGGCCTCTAGCAACGGACCCTGGGTGACCCACTGGGAGGAGATGGCCAAGAAAACCGTGATCAGGCGCCTGTTTAAGTACCTGCCCGTGAGCATCGAGATGCAGAAGGCCGTGGTGCTGGACGAGAAGGCCGAGAGCGACGTGGATCAGGACAATGCCTCTGTGCTGAGCGCCGAGTATTCCGTGCTGGAGTCTGGCACAGGCGAG Pantoea dispersa RecE DNA (SEQ ID NO:90): GAGCCAGGCATCTACTATGACATCAGCAACGAGGCCTACCACTCCGGCCCCGGCATCAGCAAGTCCCAGCTGGACGACATCGCCAGGAGCCCTGCCATCTTCCAGTGGCGCAAGGACGCCCCAGTGGATACCGAGAAAACCAAGGCCCTGGACCTGGGCACCGATTTCCACTGCGCCGTGCTGGAGCCAGAGAGGTTTGCAGACATGTATCGCGTGGGCCCTGAAGTGAATCGGAGAACCACAGCCGGCAAGGCCGAGGAGAAGGAGTTCTTTGAGAAGTGTGAGAAGGATGGAGCCGTGCCCATCACCCACGACGATGCACGGAAGGTGGAGCTGATGAGAGGCTCCGTGATGGCCCACCCTATCGCCAAGCAGATGATCGCAGCACAGGGACACGCAGAGGCCTCTATCTACTGGCACGACGAGAGCACAGGCAACCTGTGCCGGTGTAGACCCGACAAGTTTATCCCTGATTGGAATTGGATCGTGGACGTGAAAACCACAGCCGATATGAAGAAGTTCAGGCGCGAGTTTTACGATCTGCGGTATCACGTGCAGGACGCCTTCTACACCGATGGCTATGCCGCCCAGTTTGGCGAGCGGCCTACCTTCGTGTTTGTGGTGACATCCACCACAATCGACTGCGGCAGATACCCCACCGAGGTGTTCTTTCTGGATGAGGAGACAAAGGCCGCCGGCAGGTCTGAGTACCAGAGCAACCTGGTGACCTATTCCGAGTGTCTGTCTCGCAATGAGTGGCCAGGCATCGCCACACTGTCTCTGCCCCACTGGGCCAAGGAGCTGAGGAACGTG F-type symbiont RecT DNA of Plautia stali (SEQ ID NO:91): TCCAACCAGCCCCCTATCGCCTCTGCCGATCTGCAGAAAACCCAGCAGTCTAAGCAGGTGGCCAACAAGACCCCTGAGCAGACACTGGTGGGCTTCATGAATCAGCCAGCAATGAAGTCCCAGCTGGCCGCCGCCCTGCCAAGGCACATGACAGCCGATCGGATGATCAGAATCGTGACCACAGAGATCCGCAAGACCCCCGCCCTGGCCACATGCGACCAGAGCTCCTTCATCGGAGCAGTGGTGCAGTGTAGCCAGCTGGGCCTGGAGCCTGGCTCCGCCCTGGGCCACGCCTACCTGCTGCCATTTGGCAACGGCCGGTCCAAGTCTGGCCAGTCTAATGTGCAGCTGATCATCGGCTATAGAGGCATGATCGACCTGGCCCGGAGAAGCGGACAGATCGTGAGCCTGTCCGCCAGGGTGGTGCGCGCAGACGATGAGTTCTCCTTTGAGTACGGCCTGGATGAGAACCTGATCCACCGGCCAGGCGATAATGAGGACGCCCCCATCACCCACGTGTATGCAGTGGCAAGACTGAAGGACGGAGGCACCCAGTTCGAAGTGATGACAGCCAAGCAGGTGGAGAAGGTGAAGGCCCAGAGCAAGGCCTCTAGCAACGGACCCTGGGTGACCCACTGGGAGGAGATGGCCAAGAAAACCGTGATCAGGCGCCTGTTTAAGTACCTGCCCGTGAGCATCGAGATGCAGAAGGCCGTGGTGCTGGATGAGAAGGCCGAGAGCGACGTGGATCAGGACAATGCCTCTGTGCTGAGCGCCGAGTATTCCGTGCTGGAGGGCGACGGCGGCGAG F-type symbiont RecE DNA of Plautia stali (SEQ ID NO:92): CAGCCTGGCATCTACTATGACATCAGCAACGAGGATTATCACGGCGGCCCTGGCATCAGCAAGTCCCAGCTGGACGACATCGCCATCTCCCCAGCCATCTACCAGTGGAGGAAGCACGCCCCCGTGGACGAGGAGAAAACCGCCGCCCTGGATCTGGGCACAGCCCTGCACTGCCTGCTGCTGGAGCCTGACGAGTTCTCTAAGAGATTTGAGATCGGCCCAGAGGTGAACCGGAGAACCACAGCCGGCAAGGAGAAGGAGAAGGAGTTCATGGAGAGGTGTGAGGCAGAGGGAGTGACCCCTATCACACACGACGATAATCGGAAGCTGAGACTGATGAGGGATAGCGCAATGGCCCACCCAATCGCCAGATGGATGCTGGAGGCACAGGGAAACGCAGAGGCCTCTATCTATTGGAATGACAGGGATACCGGCGTGCTGAGCAGGTGCCGCCCCGACAAGATCATCACCGACTTCAACTGGTGCGTGGACGTGAAGTCCACAGCCGACATCATCAAGTTCCAGAAGGACTTTTACTCTTATCGCTACCACGTGCAGGACGCCTTCTATTCCGATGGCTACGAGTCTCACTTTGACGAGACACCAACATTCGCCTTTCTGGCCGTGTCTACAAGCATCGATTGCGGCCGGTATCCCGTGCAGGTGTTCATCATGGACCAGCAGGCAAAGGATGCAGGAAGGGCCGAGTACAAGCGGAACATCCACACCTTTGCCGAGTGTCTGAGCCGCAATGAGTGGCCTGGCATCGCCACACTGTCCCTGCCTTACTGGGCCAAGGAGCTGCGGAATGAG Providencia stuartii RecT DNA (SEQ ID NO:93): AGCAACCCACCTCTGGCCCAGGCAGACCTGCAGAAAACCCAGGGCACAGAGGTGAAGGAGAAAACCAAGGATCAGATGCTGGTGGAGCTGATCAATAAGCCTTCCATGAAGGCACAGCTGGCCGCCGCCCTGCCAAGGCACATGACACCCGACCGGATGATCAGAATCGTGACCACAGAGATCAGAAAGACCCCCGCCCTGGCCACATGCGATATGCAGAGCTTCGTGGGAGCAGTGGTGCAGTGTTCCCAGCTGGGCCTGGAGCCTGGCAACGCCCTGGGACACGCCTACCTGCTGCCTTTTGGCAACGGCAAGTCTAAGAGCGGCCAGTCTAATGTGCAGCTGATCATCGGCTATCGGGGCATGATCGACCTGGCCCGGAGAAGCGGCCAGATCGTGTCCATCTCTGCCAGGACCGTGCGCCAGGGCGATAACTTCCACTTTGAGTACGGCCTGAACGAGAATCTGACCCACGTGCCTGGCGAGAATGAGGACTCTCCAATCACACACGTGTACGCAGTGGCAAGGCTGAAGGATGGAGGCGTGCAGTTCGAAGTGATGACCTATAACCAGATCGAGAAGGTGCGCGCCAGCTCCAAGGCAGGACAGAATGGACCCTGGGTGAGCCACTGGGAGGAGATGGCCAAGAAAACCGTGATCAGGCGCCTGTTCAAGTACCTGCCCGTGTCTATCGAGATGCAGAAGGCCGTGATCCTGGACGAGAAGGCCGAGGCCAACATCGATCAGGAGAATGCCACCATCTTTGAGGGCGAGTATGAGGAAGTGGGCACAGACGGCAAG Providencia stuartii RecE DNA (SEQ ID NO:94): GAGGGCATCTACTATAACATCAGCAATGAGGACTACCACAACGGCCTGGGCATCTCCAAGTCTCAGCTGGATCTGATCAATGAGATGCCTGCCGAGTATATCTGGTCCAAGGAGGCCCCCGTGGACGAGGAGAAGATCAAGCCTCTGGAGATCGGCACCGCCCTGCACTGCCTGCTGCTGGAGCCAGACGAGTACCACAAGAGATATAAGATCGGCCCCGATGTGAACCGGAGAACAAATGCCGGCAAGGAGAAGGAGAAGGAGTTCTTTGATATGTGCGAGAAGGAGGGCATCACCCCCATCACACACGACGATAACCGGAAGCTGATGATCATGAGAGACTCTGCCCTGGCCCACCCTATCGCCAAGTGGTGTCTGGAGGCCGATGGCGTGAGCGAGAGCTCCATCTACTGGACCGACAAGGAGACAGATGTGCTGTGCAGGTGTCGCCCAGACCGCATCATCACCGCCCACAACTACATCGTGGATGTGAAGTCTAGCGGCGACATCGAGAAGTTCGATTACGAGTACTACAACTACAGATACCACGTGCAGGACGCCTTTTACTCCGATGGCTATAAGGAGGTGACCGGCATCACCCCTACATTCCTGTTTCTGGTGGTGTCTACCAAGATCGACTGCGGCAAGTACCCCGTGCGGACCTACGTGATGAGCGAGGAGGCAAAGTCCGCCGGAAGGACCGCCTACAAGCACAACCTGCTGACCTATGCCGAGTGTCTGAAAACCGATGAGTGGGCCGGCATCAGGACACTGTCTCTGCCCAGATGGGCAAAGGAGCTGCGGAATGAG Providencia sp. MGF014 RecT DNA (SEQ ID NO:95): TCTAACCCCCCTCTGGCCCAGAGCGACCTGCAGAAAACCCAGGGCACAGAGGTGAAGGTGAAAACCAAGGATCAGCAGCTGATCCAGTTCATCAATCAGCCTTCTATGAAGGCACAGCTGGCCGCCGCCCTGCCAAGGCACATGACACCCGACCGGATGATCAGAATCGTGACCACAGAGATCAGAAAGACCCCCGCCCTGGCCACATGCGATATGCAGTCCTTCGTGGGCGCCGTGGTGCAGTGTTCTCAGCTGGGCCTGGAGCCTGGCAACGCCCTGGGACACGCCTACCTGCTGCCTTTTGGCAACGGCAAGGCCAAGTCCGGCCAGTCTAATGTGCAGCTGATCATCGGCTATCGGGGCATGATCGACCTGGCCCGGAGATCCAACCAGATCATCTCTATCAGCGCCAGGACCGTGCGCCAGGGCGATAACTTCCACTTTGAGTACGGCCTGAATGAGGACCTGACCCACACACCTAGCGAGAATGAGGATTCCCCAATCACCCACGTGTACGCAGTGGCAAGGCTGAAGGACGGAGGCGTGCAGTTTGAAGTGATGACATATAACCAGGTGGAGAAGGTGCGCGCCAGCTCCAAGGCAGGACAGAATGGACCCTGGGTGAGCCACTGGGAGGAGATGGCCAAGAAAACCGTGATCAGGCGCCTGTTCAAGTACCTGCCCGTGTCCATCGAGATGCAGAAGGCAGTGGTGCTGGACGAGAAGGCAGAGGCCAACGTGGATCAGGAGAATGCCACCATCTTTGAGGGCGAGTATGAGGAAGTGGGCACAGATGGCAAT Providencia sp. MGF014 RecE DNA (SEQ ID NO:96): AAGGAGGGCATCTACTATAACATCAGCAATGAGGACTACCACAACGGCCTGGGCATCTCCAAGTCTCAGCTGGATCTGATCAATGAGATGCCTGCCGAGTATATCTGGTCCAAGGAGGCCCCCGTGGACGAGGAGAAGATCAAGCCTCTGGAGATCGGCACCGCCCTGCACTGCCTGCTGCTGGAGCCAGACGAGTACCACAAGAGATATAAGATCGGCCCCGATGTGAACCGGAGAACAAATGTGGGCAAGGAGAAGGAGAAGGAGTTCTTTGATATGTGCGAGAAGGAGGGCATCACCCCCATCACACACGACGATAACCGGAAGCTGATGATCATGAGAGACTCTGCCCTGGCCCACCCTATCGCCAAGTGGTGTCTGGAGGCCGATGGCGTGAGCGAGAGCTCCATCTACTGGACCGACAAGGAGACAGATGTGCTGTGCAGGTGTCGCCCAGACCGCATCATCACCGCCCACAACTACATCATCGATGTGAAGTCTAGCGGCGACATCGAGAAGTTCGATTACGAGTACTACAACTACAGATACCACGTGCAGGACGCCTTTTACTCCGATGGCTATAAGGAGGTGACCGGCATCACCCCTACATTCCTGTTTCTGGTGGTGTCTACCAAGATCGACTGCGGCAAGTACCCCGTGCGGACCTACGTGATGAGCGAGGAGGCAAAGTCCGCCGGAAGGACCGCCTACAAGCACAACCTGCTGACCTATGCCGAGTGTCTGAAAACCGATGAGTGGGCCGGCATCAGGACACTGTCTCTGCCCAGATGGGCAAAGGAGCTGCGGAATGAG Shewanella putrefaciens RecT DNA (SEQ ID NO:97): CAGACCGCACAGGTGAAGCTGAGCGTGCCCCACCAGCAGGTGTACCAGGACAACTTCAATTATCTGAGCTCCCAGGTGGTGGGCCACCTGGTGGATCTGAACGAGGAGATCGGCTACCTGAACCAGATCGTGTTTAATTCTCTGAGCACCGCCTCTCCCCTGGACGTGGCAGCACCTTGGAGCGTGTACGGCCTGCTGCTGAACGTGTGCCGGCTGGGCCTGTCCCTGAATCCAGAGAAGAAGCTGGCCTATGTGATGCCCTCCTGGTCTGAGACAGGCGAGATCATCATGAAGCTGTACCCCGGCTATAGGGGCGAGATCGCCATCGCCTCTAACTTCAATGTGATCAAGAACGCCAATGCCGTGCTGGTGTATGAGAACGATCACTTCCGCATCCAGGCAGCAACCGGCGAGATCGAGCACTTTGTGACAAGCCTGTCCATCGACCCTAGGGTGCGCGGAGCATGCAGCGGAGGCTACTGTCGGTCCGTGCTGATGGATAATACAATCCAGATCTCTTATCTGAGCATCGAGGAGATGAACGCCATCGCCCAGAATCAGATCGAGGCCAACATGGGCAATACCCCTTGGAACTCCATCTGGCGGACAGAGATGAATAGAGTGGCCCTGTACCGGAGAGCAGCAAAGGACTGGAGGCAGCTGATCAAGGCCACCCCAGAGATCCAGTCCGCCCTGTCTGATACAGAGTAT Shewanella putrefaciens RecE DNA (SEQ ID NO:98): Bacillus sp. MUM 116 RecT DNA (SEQ ID NO:99): AGCAAGCAGCTGACCACAGTGAATACCCAGGCCGTGGTGGGCACATTCTCCCAGGCCGAGCTGGATACCCTGAAGCAGACAATCGCCAAGGGCACCACAAACGAGCAGTTCGCCCTGTTTGTGCAGACCTGCGCCAACTCTAGGCTGAATCCATTTCTGAACCACATCCACTGTATCGTGTATAACGGCAAGGAGGGCGCCACCATGAGCCTGCAGATCGCAGTGGAGGGCATCCTGTACCTGGCACGCAAGACAGACGGCTATAAGGGCATCGAGTGCCAGCTGATCCACGAGAATGACGAGTTCAAGTTTGATGCCAAGTCCAAGGAGGTGGATCACCAGATCGGATTCCCCAGGGGCAACGTGATCGGAGGATATGCAATCGCAAAGAGGGAGGGCTTTGACGATGTGGTGGTGCTGATGGAGTCTAACGAGGTGGACCACATGCTGAAGGGCCGGAATGGCCACATGTGGAGAGACTGGTTCAACGATATGTTTAAGAAGCACATCATGAAGCGGGCCGCCAAGCTGCAGTACGGCATCGAGATCGCAGAGGACGAGACAGTGAGCAGCGGACCTAGCGTGGATAATATCCCAGAGTATAAGCCACAGCCCCGGAAGGACATCACACCCAACCAGGACGTGATCGATGCCCCCCCTCAGCAGCCTAAGCAGGACGATGAGGCCGCCAAGCTGAAGGCCGCCAGATCTGAGGTGAGCAAGAAGTTCAAGAAGCTGGGCATCGTGAAGGAGGATCAGACCGAGTACGTGGAGAAGCACGTGCCTGGCTTCAAGGGCACACTGTCCGACTTTATCGGCCTGTCTCAGCTGCTGGATCTGAATATCGAGGCCCAGGAGGCCCAGTCCGCCGACGGCGATCTGCTGGAC Bacillus sp. MUM 116 RecE DNA (SEQ ID NO:100): Shigella sonnei RecT DNA (SEQ ID NO:101): ACCAAGCAGCCCCCTATCGCCAAGGCCGACCTGCAGAAAACCCAGGAGAACAGGGCACCAGCAGCCATCAAGAACAATGATGTGATCTCCTTTATCAATCAGCCCTCTATGAAGGAGCAGCTGGCCGCCGCCCTGCCTAGGCACATGACCGCCGAGAGGATGATCCGCATCGCCACCACAGAGATCCGCAAGGTGCCTGCCCTGGGCAACTGCGACACAATGAGCTTCGTGAGCGCCATCGTGCAGTGTAGCCAGCTGGGCCTGGAGCCAGGCTCCGCCCTGGGCCACGCCTACCTGCTGCCCTTCGGCAACAAGAATGAGAAGTCCGGCAAGAAGAATGTGCAGCTGATCATCGGCTATAGGGGCATGATCGATCTGGCCCGGAGATCTGGCCAGATCGCCTCTCTGAGCGCCAGAGTGGTGCGGGAGGGCGACGAGTTCAACTTTGAGTTCGGCCTGGATGAGAAGCTGATCCACCGGCCTGGCGAGAATGAGGACGCCCCAGTGACCCACGTGTACGCAGTGGCCAGACTGAAGGATGGCGGCACCCAGTTTGAAGTGATGACAAGGCGCCAGATCGAGCTGGTGAGGTCCCAGTCTAAGGCCGGCAACAATGGCCCTTGGGTGACCCACTGGGAGGAGATGGCCAAGAAAACCGCCATCCGGAGACTGTTCAAGTACCTGCCAGTGTCTATCGAGATCCAGCGCGCCGTGAGCATGGACGAGAAGGAGCCACTGACCATCGACCCCGCCGATAGCTCCGTGCTGACAGGCGAGTATTCTGTGATCGATAACAGCGAGGAG Shigella sonnei RecE DNA (SEQ ID NO:102): Salmonella enterica RecT DNA (SEQ ID NO:103): ACCAAGCAGCCCCCTATCGCCAAGGCCGACCTGCAGAAAACCCAGGGAAACAGGGCACCTGCAGCAGTGAATGACAAGGATGTGCTGTGCGTGATCAACAGCCCTGCCATGAAGGCACAGCTGGCCGCCGCCCTGCCAAGGCACATGACCGCCGAGAGGATGATCCGCATCGCCACCACAGAGATCAGGAAGGTGCCAGAGCTGCGCAACTGCGACAGCACCAGCTTCATCGGCGCCATCGTGCAGTGTTCTCAGCTGGGCCTGGAGCCCGGCAGCGCCCTGGGCCACGCCTACCTGCTGCCTTTTGGCAATGGCAAGGCCAAGAACGGCAAGAAGAATGTGCAGCTGATCATCGGCTATCGGGGCATGATCGATCTGGCCCGGAGATCTGGCCAGATCATCTCCCTGAGCGCCAGAGTGGTGCGGGAGTGTGACGAGTTCTCCTACGAGCTGGGCCTGGATGAGAAGCTGGTGCACCGGCCAGGCGAGAACGAGGACGCACCCATCACCCACGTGTATGCCGTGGCCAAGCTGAAGGATGGCGGCGTGCAGTTTGAAGTGATGACCAAGAAGCAGGTGGAGAAGGTGAGAGATACACACTCCAAGGCCGCCAAGAATGCCGCCTCTAAGGGCGCCAGCTCCATCTGGGACGAGCACTTCGAGGATATGGCCAAGAAAACCGTGATCCGGAAGCTGTTTAAGTACCTGCCCGTGAGCATCGAGATCCAGAGAGCCGTGAGCATGGACGGCAAGGAGGTGGAGACAATCAACCCAGACGACATCAGCGTGATCGCCGGCGAGTATTCCGTGATCGATAATCCCGAGGAG Salmonella enterica RecE DNA (SEQ ID NO:104): Acetobacter sp. RecT DNA (SEQ ID NO: 105): AACGCCCCCCAGAAGCAGAATACCAGAGCCGCCGTGAAGAAGATCAGCCCTCAGGAGTTCGCCGAGCAGTTTGCCGCCATCATCCCACAGGTGAAGTCCGTGCTGCCCGCCCACGTGACCTTCGAGAAGTTTGAGCGGGTGGTGAGACTGGCCGTGCGGAAGAACCCTGACCTGCTGACATGCTCCCCAGCCTCTCTGTTCATGGCATGTATCCAGGCAGCCTCCGACGGCCTGCTGCCTGATGGAAGGGAGGGAGCAATCGTGAGCCGGTGGAGCTCCAAGAAGAGCTGCAACGAGGCCTCCTGGATGCCAATGGTGGCCGGCCTGATGAAGCTGGCCCGGAACAGCGGCGACATCGCCAGCATCTCTAGCCAGGTGGTGTTCGAGGGCGAGCACTTTAGAGTGGTGCTGGGCGACGAGGAGAGGATCGAGCACGAGCGCGATCTGGGCAAGACCGGCGGCAAGATCGTGGCAGCCTACGCCGTGGCAAGGCTGAAGGACGGCAGCGATCCAATCCGCGAGATCATGTCCTGGGGCCAGATCGAGAAGATCAGAAACACAAATAAGAAGTGGGAGTGGGGACCCTGGAAGGCCTGGGAGGACGAGATGGCCAGAAAGACCGTGATCCGGAGACTGGCCAAGAGACTGCCCATGTCTACAGATAAGGAGGGAGAGAGGCTGCGCAGCGCCATCGAGAGGATCGACTCCCTGGTGGACATCTCTGCCAACGTGGACGCACCTCAGATCGCAGCAGACGATGAGTTTGCCGCCGCCGCCCACGGCGTGGAGCCACAGCAGATCGCAGCACCTGACCTGATCGGCCGCCTGGCCCAGATGCAGTCCCTGGAGCAGGTGCAGGACATCGAGCCCCAGGTGTCTCACGCCATCCAGGAGGCCGACAAGAGGGGCGACAGCGATACAGCCAATGCCCTGGATGCCGCCCTGCAGAGCGCCCTGTCCCGCACCTCTACAGCCAAGGAGGAGGTGCCTGCC Acetobacter sp. RecE DNA (SEQ ID NO:106): GTGATCTCTAAGAGCGGCATCTACGACCTGACCAACGAGCAGTATCACGCCGATCCTTGCCCAGAGATGTCCCTGAGCTCCTCTGGAGCCAGGGACCTGCTGAGCTCCTGTCCTGCCAAGTTCATCGCCGCCAAGCAGCTGCCACAGCAGAATAAGAGGTGCTTTGACATCGGCTCTGCCGGACACCTGATGGTGCTGGAGCCACACCTGTTCGACCAGAAGGTGTGCGAGATCAAGCACCCTGATTGGCGCACAAAGGCAGCAAAGGAGGAGCGGGACGCCGCCTACGCCGAGGGAAGAATCCCCCTGCTGAGCCGCGAGGTGGAGGACATCAGGGCAATGCACTCCGTGGTGTGGAGAGATTCTCTGGGAGCCAGGGCCTTCAGCGGAGGCAAGGCAGAGCAGTCCCTGGTGTGGCGCGACGAGGAGTTTGGCATCTGGTGCCGGCTGCGGCCCGATTACGTGCCTAACAATGCCGTGCGGATCTTCGACTATAAGACCGCCACAAACGGCTCCCCCGATGCCTTTATGAAGGAGATCTACAATCGGGGCTATCACCAGCAGGCCGCCTGGTATCTGGACGGATATGAGGCAGTGACCGGCCACAGGCCACGCGAGTTCTGGTTTGTGGTGCAGGAGAAAACCGCCCCCTTCCTGCTGTCTTTCTTTCAGATGGATGAGATGAGCCTGGAGATCGGCCGGACCCTGAACAGACAGGCCAAGGGCATCTTTGCCTGGTGCCTGCGCAACAATTGTTGGCCAGGCTATCAGCCCGAGGTGGATGGCAAGGTGAGATTCTTTACCACATCTCCCCCTGCCTGGCTGGTGAGGGAGTACGAGTTTAAGAATGAGCACGGCGCCTATGAGCCACCCGAGATCAAGCGGAAGGAGGTGGCC Salmonella enterica subsp. enterica serovar Javiana str. 10721 RecT DNA (SEQ ID NO: 107): CCAAAGCAGCCCCCTATCGCCAAGGCAGACCTGCAGAAAACCCAGGGAGCACGGACCCCAACAGCAGTGAAGAACAATAACGATGTGATCTCCTTTATCAATCAGCCTTCTATGAAGGAGCAGCTGGCCGCCGCCCTGCCAAGGCACATGACCGCCGAGCGGATGATCAGAATCGCCACCACAGAGATCAGGAAGGTGCCCGCCCTGGGCGACTGCGATACAATGTCTTTTGTGAGCGCCATCGTGCAGTGTAGCCAGCTGGGCCTGGAGCCTGGCGGCGCCCTGGGCCACGCCTACCTGCTGCCTTTCGGCAATCGGAACGAGAAGTCCGGCAAGAAGAATGTGCAGCTGATCATCGGCTATAGAGGCATGATCGACCTGGCCCGGAGATCCGGACAGATCGCCAGCCTGTCCGCCAGGGTGGTGCGCGAGGGCGACGATTTCTCTTTTGAGTTCGGCCTGGAGGAGAAGCTGGTGCACAGGCCAGGCGAGAACGAGGACGCCCCCGTGACCCACGTGTACGCAGTGGCACGCCTGAAGGATGGAGGCACCCAGTTTGAAGTGATGACACGGAAGCAGATCGAGCTGGTGAGAGCCCAGTCTAAGGCCGGCAATAACGGCCCTTGGGTGACCCACTGGGAGGAGATGGCCAAGAAAACCGCCATCAGGCGCCTGTTCAAGTACCTGCCCGTGAGCATCGAGATCCAGAGGGCCGTGAGCATGGATGAGAAGGAGACACTGACAATCGACCCAGCCGATGCCAGCGTGATCACCGGCGAGTATTCCGTGGTGGAGAATGCCGGCGTGGAGGAGAACGTGACAGCC Salmonella enterica subsp. enterica serovar Javiana str. 10721 RecE DNA (SEQ ID NO: 108): TACTATGACATCCCAAACGAGGCCTACCACGCAGGCCCCGGCGTGTCTAAGAGCCAGCTGGACGACATCGCCGATACCCCCGCCATCTGTGGGCGGAAGAATGCCCCTGTGGACACCGAGAAAACCAAGTCCCTGGATACCGGCACAGCCTTCCACTGCAGGGTGCTGGAGCCAGGGAGTCAGGTTTCCACT CATCGCCCCCGAGTCCAACCGGAGAACCTCCGCCGGCAAGGAGGAGGAGAAAACCTTCCTGGAGGAGTGTACCCGGACAGGCAGAACCGTGCTGAGCCGAGGGCAGGAAGATCGAGCTGATGTACCA ATTGGGAGGACCCTGAGACAGGCATCCCTGTGCCGGTGTAGACCCGATAAGATCATCCCTGAGTTCCACTGGATCATGGACGTGAAAACCACAGCCGACAGGTTTCGCACCGCCTACTATGACTACAGATACCAGGTGCAGGACGCCTTCTACTGATGGCTATAGAGCCCAGCCGATTGCCTT GTGTTTCTGGTGGCCAGCACCACAGCAGAGTGCGGCAGATACCCCGTGGAGATCTTTATGGGGAGAGGACGCAAAAGCTGGCCGGACAGCGCGAGTATAGGCCAATCTGCAGACCCTGGCCGAGTGTCTGAACAATGATGAGTGGCCTGCCATCAAGACACTGTCTCTGCCAGCCAGGCCGAGGGACC Pseudobacterivorax antillogorgiicola RecT DNA(SEQ ID NO:109): GGCCACCTGGTGAGCAAGACCGAGCAGGATTACATCAAGCAGCACTATGCCAAGGGCGCCACAGACCAGGAGTTCGAGCACTTTATCGGCGTGTGCAGGGCCAGAGGCCTGAACCCAGCCGCCAATCAGATCTACTTCGTGAAGTATCGGTCCAAGGATGGACCAGCAAAGCCAGCCTTTATCCTGTCTATCGACAGCCTGAGGCTGATCGCACACCGCACCGGCGATTACGCAGGATGCTCTGAGCCCATCTTCACAGACGGCGGCAAGGCCTGTACCGTGACAGTGCGGAGAAACCTGAAGAGCGGCGAGACAGGCAATTTCTCCGGCATGGCCTTTTATGACGAGCAGGTGCAGCAGAAGAACGGCCGGCCTACCTCCTTTTGGCAGTCTAAGCCAAGAACAATGCTGGAGAAGTGTGCAGAGGCAAAGGCCCTGAGGAAGGCCTTCCCTCAGGATCTGGGCCAGTTTTACATCAGAGAGGAGATGCCCCCTCAGTATGACGAGCCTATCCAGGTGCACAAGCCAAAGGCCCTGGAGGAGCCCAGGTTCAGCAAGTCCGATCTGTCCAGGCGCAAGGGCCTGAACAGGAAGCTGTCTGCCCTGGGAGTGGACCCCAGCCGCTTCGATGAGGTGGCCACCTTTCTGGACGGCACACCTGATCGCGAGCTGGGCCAGAAGCTGAAGCTGTGGCTGAAGGAGGCCGGCTACGGCGTGAATCAG Pseudobacteriovorax antillogorgiicola RecE DNA(SEQ ID NO:110): AGCAAGCTGTCCAACCTGAAGGTGTCTAATAGCGACGTGGATACACTGAGCCGGATCAGAATGAAGGAGGGCGTGTATCGGGACCTGCCAATCGAGAGCTACCACCAGTCCCCCGGCTATTCTAAGACCAGCCTGTGCCAGATCGATAAGGCCCCTATCTACCTGAAAACCAAGGTGCCACAGAAGTCCACAAAGTCTCTGAACATCGGCACCGCCTTCCACGAGGCTATGGAGGGCGTGTTTAAGGACAAGTATGTGGTGCACCCCGATCCTGGCGTGAATAAGACCACAAAGTCTTGGAAGGACTTCGTGAAGAGGTATCCTAAGCACATGCCACTGAAGCGCAGCGAGTACGACCAGGTGCTGGCCATGTACGATGCCGCCCGGTCTTATAGACCTTTTCAGAAGTACCACCTGAGCCGGGGCTTCTACGAGAGCTCCTTTTATTGGCACGATGCCGTGACAAACAGCCTGATCAAGTGCAGACCCGACTATATCACCCCTGATGGCATGAGCGTGATCGACTTCAAGACCACAGTGGACCCCAGCCCCAAGGGCTTTCAGTACCAGGCCTACAAGTATCACTACTACGTGAGCGCCGCCCTGACCCTGGAGGGAATCGAGGCAGTGACCGGCATCAGGCCAAAGGAGTACCTGTTCCTGGCCGTGTCCAATTCTGCCCCATACCTGACCGCCCTGTATCGCGCCTCTGAGAAGGAGATCGCCCTGGGCGACCACTTTATCCGGCGGAGCCTGCTGACCCTGAAAACCTGTCTGGAGTCTGGCAAGTGGCCCGGCCTGCAGGAGGAGATCCTGGAGCTGGGCCTGCCTTTCTCCGGCCTGAAGGAGCTGAGAGAGGAGCAGGAGGTGGAGGATGAGTTTATGGAGCTGGTGGGC Photobacterium sp. JCM 19050 RecT DNA (SEQ ID NO:111): AACACCGACATGATCGCCATGCCCCCTTCTCCAGCCATCAGCATGCTGGACACAAGCAAGCTGGATGTGATGGTGCGGGCAGCAGAGCTGATGTCCCAGGCCGTGGTCATGGTGCCCGACCACTTCAAGGGCAAGCCAGCCGATTGCCTGGCAGTGGTCATGCAGGCAGACCAGTGGGGCATGAACCCCTTTACCGTGGCCCAGAAAACCCACCTGGTGAGCGGCACCCTGGGATACGAGTCCCAGCTGGTGAATGCCGTGATCAGCTCCTCTAAGGCCATCAAGGGCCGGTTCCACTATGAGTGGTCTGATGGCTGGGAGAGACTGGCCGGCAAGGTGCAGTACGTGAAGGAGTCTCGGCAGAGAAAGGGCCAGCAGGGCAGCTATCAGGTGACCGTGGCCAAGCCAACATGGAAGCCAGAGGACGAGCAGGGCCTGTGGGTGCGGTGTGGAGCCGTGCTGGCCGGAGAGAAGGACATCACATGGGGCCCTAAGCTGTACCTGGCCAGCGTGCTGGTGCGGAACAGCGAGCTGTGGACCACAAAGCCCTACCAGCAGGCCGCCTATACCGCCCTGAAGGATTGGTCCCGCCTGTATACACCTGCCGTGATGCAGGGCTCTATGACCGGCAAGAGCTGGTCCCTGACAGGCAGGCTGATCAGCCCCCGC Photobacterium sp. JCM 19050 RecE DNA (SEQ ID NO:112): GCCGAGCGGGTGAGAACCTATCAGCGGGACGCCGTGTTCGCACACGAGCTGAAGGCCGAGTTTGATGAGGCCGTGGAGAACGGCAAGACCGGCGTGACACTGGAGGACCAGGCCAGGGCCAAGAGGATGGTGCACGAGGCCACCACAAACCCCGCCTCTCGGAATTGGTTCAGATACGACGGAGAGCTGGCCGCATGCGAGAGGAGCTATTTTTGGCGCGATGAGGAGGCAGGCCTGGTGCTGAAGGCCAGGCCTGACAAGGAGATCGGCAACAATCTGATCGATGTGAAGTCCATCGAGGTGCCAACCGACGTGTGCGCCTGTGATCTGAACGCCTATATCAATCGGCAGATCGAGAAGAGAGGCTACCACATCTCCGCCGCCCACTATCTGTCTGGCACAGGCAAGGACCGCTTCTTTTGGATCTTCATCAATAAGGTGAAGGGCTACGAGTGGGTGGCAATCGTGGAGGCCTCTCCCCTGCACATCGAGCTGGGCACCTATGAGGTGCTGGAGGGCCTGCGGAGCATCGCCAGCTCCACAAAGGAGGCAGATTACCCAGCACCTCTGTCCCACCCTGTGAACGAGAGAGGCATCCCACAGCCCCTGATGTCTAATCTGAGCACATACGCCATGAAGAGGCTGGAGCAGTTTCGCGAGCTG Providencia alcalifaciens DSM 30120 RecT DNA (SEQ ID NO:113): AAGGCACAGCTGGCCGCCGCCCTGCCTAAGCACATCACCAGCGACCGGATGATCAGAATCGTGTCCACCGAGATCAGAAAGACCCCATCTCTGGCCAACTGCGACATCCAGAGCTTCATCGGCGCCGTGGTGCAGTGTTCTCAGCTGGGCCTGGAGCCAGGCAACGCCCTGGGACACGCCTACCTGCTGCCCTTTGGCAATGGCAAGTCCGACAACGGCAAGTCTAATGTGCAGCTGATCATCGGCTATCGGGGCATGATCGATCTGGCCCGGAGAAGCGGCCAGATCATCTCTATCAGCGCCAGGACCGTGCGCCAGGGCGACAACTTCCACTTTGAGTACGGCCTGAACGAGAATCTGACCCACATCCCCGAGGGCAATGAGGACTCCCCTATCACACACGTGTACGCAGTGGCACGGCTGAAGGATGAGGGCGTGCAGTTCGAAGTGATGACATATAACCAGATCGAGAAGGTGAGAGATAGCTCCAAGGCCGGCAAGAATGGCCCCTGGGTGACCCACTGGGAGGAGATGGCCAAGAAAACCGTGATCAGGCGCCTGTTTAAGTACCTGCCCGTGAGCATCGAGATGCAGAAGGCCGTGATCCTGGACGAGAAGGCCGAGGCCAATATCGAGCAGGATCACTCCGCCATCTTCGAGGCCGAGTTTGAGGAGGTGGACTCTAACGGCAAT Providencia alcalifaciens DSM 30120 RecE DNA (SEQ ID NO:114): AACGAGGGCATCTACTATGACATCTCTAATGAGGACTATCACCACGGCCTGGGCATCTCTAAGAGCCAGCTGGATCTGATCGACGAGAGCCCCGCCGATTTCATCTGGCACCGGGATGCCCCTGTGGACAACGAGAAAACCAAGGCCCTGGATTTTGGCACAGCCCTGCACTGCCTGCTGCTGGAGCCAGACGAGTTCCAGAAGAGGTTTCGCATCGCCCCCGAGGTGAACCGGAGAACAAATGCCGGCAAGGAGCAGGAGAAGGAGTTCCTGGAGATGTGCGAGAAGGAGAATATCACCCCCATCACAAACGAGGATAATAGGAAGCTGTCTCTGATGAAGGACAGCGCAATGGCCCACCCTATCGCCCGCTGGTGTCTGGAGGCCAAGGGCATCGCCGAGAGCTCCATCTATTGGAAGGACAAGGATACAGACATCCTGTGCCGGTGTAGACCAGACAAGCTGATCGAGGAGCACCACTGGCTGGTGGATGTGAAGTCCACCGCCGACATCCAGAAGTTCGAGCGGTCTATGTACGAGTATAGATACCACGTGCAGGATTCCTTTTATTCTGACGGCTACAAGAGCCTGACAGGCGAGATGCCCGTGTTCGTGTTCCTGGCCGTGTCCACCGTGATCAACTGCGGCAGATACCCCGTGCGGGTGTTCGTGCTGGACGAGCAGGCAAAGTCCGTGGGACGGATCACCTATAAGCAGAATCTGTTTACATACGCCGAGTGTCTGAAAACCGACGAGTGGGCCGGCATCAGAACCCTGAGCCTGCCCTCCTGGGCAAAGGAGCTGAAGCACGAGCACACCACAGCCTCT Pantoea stewartii RecT protein (SEQ ID NO:115): MSNQPPIASADLQKANTGKQVANKTPEQTLVGFMNQPAMKSQLAAALPRHMTADRMIRIVTTEIRKTPALATCDQSSFIGAVVQCSQLGLEPGSALGHAYLLPFGNGRSKSGQSNVQLIIGYRGMIDLARRSGQIVSLSARVVRADDEFSFEYGLDENLIHRPGENEDAPITHVYAVARLKDGGTQFEVMTVKQIEKVKAQSKASSNGPWVTHWEEMAKKTVIRRLFKYLPVSIEMQKAVILDEKAESDVDQDNASVLSAEYSVLDGSSEE Pantoea stewartii RecE protein (SEQ ID NO:116): MQPGVYYDISNEEYHAGPGISKSQLDDIAVSPAIFQWRKSAPVDDEKTAALDLGTALHCLLLEPDEFSKRFMIGPEVNRRTNAGKQKEQDFLDMCEQQGITPITHDDNRKLRLMRDSAFAHPVARWMLETEGKAEASIYWNDRDTQILSRCRPDKLITEFSWCVDVKSTADIGKFQKDFYSYRYHVQDAFYSDGYEAQFCEVPTFAFLVVSSSIDCGRYPVQVFIMDQQAKDAGRAEYKRNLTTYAECQARNEWPGIATLSLPYWAKEIRNV Pantoea brenneri RecT protein (SEQ ID NO:117): MSNQPPIASADLQKTQQSKQVANKTPEQTLVGFMNQPAMKSQLAAALPRHMTADRMIRIVTTEIRKTPQLAQCDQSSFIGAVVQCSQLGLEPGSALGHAYLLPFGNGRSKSGQSNVQLIIGYRGMIDLARRSGQIVSLSARVVRADDEFSFEYGLDENLVHRPGENEDAPITHVYAVARLKDGGTQFEVMTVKQVEKVKAQSKASSNGPWVTHWEEMAKKTVIRRLFKYLPVSIEMQKAVVLDEKAESDVDQDNASVLSAEYSVLESGDEATN Pantoea brenneri RecE protein (SEQ ID NO:118): MQPGIYYDISNEDYHRGAGISKSQLDDIAISPAIYQWRKHAPVDEEKTAALDLGTALHCLLLEPDEFSKRFQIGPEVNRRTTAGKEKEKEFIERCEAEGITPITHDDNRKLKLMRDSALAHPIARWMLEAQGNAEASIYWNDRDAGVLSRCRPDKIITEFNWCVDVKSTADIMKFQKDFYSYRYHVQDAFYSDGYESHFHETPTFAFLAVSTSIDCGRYPVQVFIMDQQAKDAGRAEYKRNIHTFAECLSRNEWPGIATLSLPFWAKELRNE Pantoea dispersa RecT protein (SEQ ID NO:119): MSNQPPLATADLQKTQQSNQVAKTPEQTLVGFMNQPAMKSQLAAALPRHMTADRMIRIVTTEIRKTPALAQCDQSSFIGAVVQCSQLGLEPGSALGHAYLLPFGNGRSKSGQSNVQLIIGYRGMIDLARRSGQIVSLSARVVRADDEFSFEYGLDENLIHRPGDNESAPITHVYAVARLKDGGTQFEVMTAKQVEKVKAQSKASSNGPWVTHWEEMAKKTVIRRLFKYLPVSIEMQKAVVLDEKAESDVDQDNASVLSAEYSVLESGTGE Pantoea dispersa RecE protein (SEQ ID NO:120): MEPGIYYDISNEAYHSGPGISKSQLDDIARSPAIFQWRKDAPVDTEKTKALDLGTDFHCAVLEPERFADMYRVGPEVNRRTTAGKAEEKEFFEKCEKDGAVPITHDDARKVELMRGSVMAHPIAKQMIAAQGHAEASIYWHDESTGNLCRCRPDKFIPDWNWIVDVKTTADMKKFRREFYDLRYHVQDAFYTDGYAAQFGERPTFVFVVTSTTIDCGRYPTEVFFLDEETKAAGRSEYQSNLVTYSECLSRNEWPGIATLSLPHWAKELRNV F-type symbiont RecT protein of Plautia stali (SEQ ID NO:121): MSNQPPIASADLQKTQQSKQVANKTPEQTLVGFMNQPAMKSQLAAALPRHMTADRMIRIVTTEIRKTPALATCDQSSFIGAVQCSQLGLEPGSALGHAYLLPFGNGRSKSGQSNVQLIIGYRGMIDLARRSGQI VSLSARVVRADDEFSFEYGLDENLIHRPGDNEDAPITHVYAVARLKDGGTQFEVMTAKQVEKVKAQSKASSNGPWVTHWEEMAKKTVIRRLFKYLPVSIEMQKAVVLDEKAESDVDQDNASVLSAEYSVLEGDGGE Plautia steel frame RecE (SEQ ID NO:122): MQPGIYYDISNEDYHGGPGISKSQLDDIAISPAIYQWRKHAPVDEEKTAALDLGTALHCLLLEPDEFSKRFEIGPEEVNRRTTAGKEKEKEFMERCEAEGVTPITHDDNRKLRLMRDSAMAHPIARWMLEAQGNAEA SIYWNDRDTGVLSRCRPKIITDFNWCVDVKSTADIIKFQKDFYSYRYHVQDAFYSDGYESHFDETPTFAFLAVSTSIDCGRYPVQVFIMDQQQDAGRAEYKRNIHTFAECLSRNEWPGIATLSLPYWAKELRNE Receipt of RecT (SEQ ID NO:123): MSNPPLAQADLQKTQGTEVKEKTKDQMLVELINKPSMKAQLAAALPRHMTPDRMIRIVTTEIRKTPALATCDMQSFVGAVVQCSQLGLEPGNALGHAYLLPFGNGKSKSGQSNVQLIIGYRGMIDLARRSGQIV SISARTVRQGDNFHFEYGLNENLTHVPGENEDSPITHVYAVARLKDGGVQFEVMTYNQIEKVRASSKAGQNGPWVSHWEEMAKKTVIRRLFKYLPVSIEMQKAVILDEKAEANIDQENATIFEYEEVGTDGK RecE (SEQ ID NO:124): EGIYYNISNEDYHNGLGISKSQLDLINEMPAEYIWSKEAPVDEEKIKPLEIGTALHCLLLEPDEYHKRYKIGPDVNRRTNAGKEKEKEFFDMCEKEGITPITHDDNRKLMIMRDSALAHPIAKWCLEADGVSESSIYWTDKETDVLCRCRPDRIITAHNYIVDVKSSGDIEKFDYEYYNYRYHVQDAFYSDGYKEVTGITPTFLFLVVSTKIDCGKYPVRTYVMSEEAKSAGRTAYKHNLLTYAECLKTDEWAGIRTLSLPRWAKELRNE Providencia sp. MGF014 RecT protein (SEQ ID NO: 125): MSNPPLAQSDLQKTQGTEVKVKTKDQQLIQFINQPSMKAQLAAALPRHMTPDRMIRIVTTEIRKTPALATCDMQSFVGAVVQCSQLGLEPGNALGHAYLLPFGNGKAKSGQSNVQLIIGYRGMIDLARRSNQIISISARTVRQGDNFHFEYGLNEDLTHTPSENEDSPITHVYAVARLKDGGVQFEVMTYNQVEKVRASSKAGQNGPWVSHWEEMAKKTVIRRLFKYLPVSIEMQKAVVLDEKAEANVDQENATIFEGEYEEVGTDGN Providencia sp. MGF014 RecE protein (SEQ ID NO: 126): MKEGIYYNISNEDYHNGLGISKSQLDLINEMPAEYIWSKEAPVDEEKIKPLEIGTALHCLLLEPDEYHKRYKIGPDVNRRTNVGKEKEKEFFDMCEKEGITPITHDDNRKLMIMRDSALAHPIAKWCLEADGVSESSIYWTDKETDVLCRCRPDRIITAHNYIIDVKSSGDIEKFDYEYYNYRYHVQDAFYSDGYKEVTGITPTFLFLVVSTKIDCGKYPVRTYVMSEEAKSAGRTAYKHNLLTYAECLKTDEWAGIRTLSLPRWAKELRNE Shewanella putrefaciens RecT protein (SEQ ID NO: 127): MQTAQVKLSVPHQQVYQDNFNYLSSQVVGHLVDLNEEIGYLNQIVFNSLSTASPLDVAAPWSVYGLLLNVCRLGLSLNPEKKLAYVMPSWSETGEIIMKLYPGYRGEIAIASNFNVIKNANAVLVYENDHFRIQAATGEIEHFVTSLSIDPRVRGACSGGYCRSVLMDNTIQISYLSIEEMNAIAQNQIEANMGNTPWNSIWRTEMNRVALYRRAAKDWRQLIKATPEIQSALSDTEY Shewanella putrefaciens RecE protein (SEQ ID NO:128): MGTALAQTISLDWQDTIQPAYTASGKPNFLNAQGEIVEGIYTDLPNSVYHALDAHSSTGIKTFAKGRHHYFRQYLSDVCRQRTKQQEYTFDAGTYGHMLVLEPENFHGNFMRNPVPDDFPDIELIESIPQLKAALAKSNLPVSGAKAALIERLYAFDPSLPLFEKMREKAITDYLDLRYAKYLRTDVELDEMATFYGIDTSQTREKKIEEILAISPSQPIWEKLISQHVIDHIVWDDAMRVERSTRAHPKADWLISDGYAELTIIARCPTTGLLLKVRFDWLRNDAIGVDFKTTLSTNPTKFGYQIKDLRYDLQQVFYCYVANLAGIPVKHFCFVATEYKDADNCETFELSHKKVIESTEEMFDLLDEFKEALTSGNWYGHDRSRSTWVIEV Bacillus sp. MUM 116 RecT protein (SEQ ID NO:129): MSKQLTTVNTQAVVGTFSQAELDTLKQTIAKGTTNEQFALFVQTCANSRLNPFLNHIHCIVYNGKEGATMSLQIAVEGILYLARKTDGYKGIECQLIHENDEFKFDAKSKEVDHQIGFPRGNVIGGYAIAKREGFDDVVVLMESNEVDHMLKGRNGHMWRDWFNDMFKKHIMKRAAKLQYGIEIAEDETVSSGPSVDNIPEYKPQPRKDITPNQDVIDAPPQQPKQDDEAAKLKAARSEVSKKFKKLGIVKEDQTEYVEKHVPGFKGTLSDFIGLSQLLDLNIEAQEAQSADGDLLD Bacillus sp. MUM 116 RecE protein (SEQ ID NO:130): MTYAADETLVQLLLSVDGKQLLLGRGLKKGKAQYYINEVPSKAKEFEEIRDQLFDKDLFMSLFNPSYFFTLHWEKQRAMMLKYVTAPVSKEVLKNLPEAQSEVLERYLKKHSLVDLEKIHKDNKNKQDKAYISAQSRTNTLKEQLMQLTEEKLDIDSIKAELAHIDMQVIELEKQMDTAFEKNQAFNLQAQIRNLQDKIEMSKERWPSLKNEVIEDTCRTCKRPLDEDSVEAVKADKDNRIAEYKAKHNSLVSQRNELKEQLNTIEYIDVTELREQIKELDESGQPLREQVRIYSQYQNLDTQVKSAEADENGILQDLKASIFILDSIKAFRGKEAEMQAEKVQALFTTLSVRLFKQNKGDGEIKPDFEIEMNDKPYRTLSLSEGIRAGLELRDVLSQQSELVTPTFVDNAESITSFKQPNGQLIISRVVAGQELKIEAVSE Shigella sonnei RecT protein (SEQ ID NO:131): MTKQPPIAKADLQKTQENRAPAAIKNNDVISFINQPSMKEQLAAALPRHMTAERMIRIATTEIRKVPALGNCDTMSFVSAIVQCSQLGLEPGSALGHAYLLPFGNKNEKSGKKNVQLIIGYRGMIDLARRSGQIASLSARVVREGDEFNFEFGLDEKLIHRPGENEDAPVTHVYAVARLKDGGTQFEVMTRRQIELVRSQSKAGNNGPWVTHWEEMAKKTAIRRLFKYLPVSIEIQRAVSMDEKEPLTIDPADSSVLTGEYSVIDNSEE Shigella sonnei RecE protein (SEQ ID NO:132): DRGLLTKEWRKGNRVSRITRTASGANAGGGNLTDRGEGFVHDLTSLARDIATGVLARSMDVDIYNLHPAHAKRIEEIIAENKPPFSVFRDKFITMPGGLDYSRAIVVASVKEAPIGIEVIPAHVTAYLNKVLTETDHANPDPEIVDIACGRSSAPMPQRVTEEGKQDDEEKLQPSGTTADEQGEAETMEPDATKHHQDTQPLDAQSQVNSVDAKYQELRAELHEARKNIPSKNPVDADKLLAASRGEFVDGISDPNDPKWVKGIQTRDSVYQNQPETEKTSPDMKQPEPVVQQEPEIAFNACGQTGGDNCPDCGAVMGDATYQETFDEENQVEAKENDPEEMEGAEHPHNENAGSDPHRDCSDETGEVADPVIVEDIEPGIYYGISNENYHAGPGVSKSQLDDIADTPALYLWRKNAPVDTTKTKTLDLGTAFHCRVLEPEEFSNRFIVAPEFNRRTNAGKEEEKAFLMECASTGKMVITAEEGRKIELMYQSVMALPLGQWLVESAGHAESSIYWEDPETGILCRCRPDKIIPEFHWIMDVKTTADIQRFKTAYYDYRYHVQDAFYSDGYEAQFGVQPTFVFLVASTTIECGRYPVEIFMMGEEAKLAGQLEYHRNLRTLADCLNTDEWPAIKTLSLPRWAKEYAND Salmonella enterica RecT protein (SEQ ID NO: 133): MTKQPPIAKADLQKTQGNRAPAAVNDKDVLCVINSPAMKAQLAAALPRHMTAERMIRIATTEIRKVPELRNCDSTSFIGAIVQCSQLGLEPGSALGHAYLLPFGNGKAKNGKKNVQLIIGYRGMIDLARRSGQIISLS ARVVRECDEFSYELGLDEKLVHRPGENEDAPITHVYAVAKLKDGGVQFEVMTKKQVEKVRDTTHSKAAKNAASKGASSIWDEHFEDMAKKTVIRKLFKYLPVSIEIQRAVSMDGKEVETINPDDISVIAGEYSVIDNPEE Salmonella enterica RecE protein (SEQ ID NO: 134): DRGLLTKEWRKGNRVSRITRTASGANAGGGNLTDRGEGFVHDLTSLARDVATGVLARSMDVDIYNLHPAHAKRVEEIIAENKPPFSVFRDKFITMPGGLDYSRAIVVASVKEAPIGIEVIPAHVTEYLNKVLTETDHANPDPEIVDIACGRSSAPMPQRVTEEGKQDDEEKPQPSGAMADEQATAETVEPNATEHHQNTQPLDAQSQVNSVDAKYQELRAELQEARKNIPSKNPVDADKLLAASRGEFVDGISDPNDPKWVKGIQTRDSVYQNQPETEKISPDAKQPEPVVQQEPETVCNACGQTGGDNCPDCGAVMGDATYQETFGEENQVEAKEKDPEEMEGAEHPHNENAGSDPHRDCSDETGEVADPVIVEDIEPGIYYGISNENYHAGPGVSKSQLDDIADTPALYLWRKNAPVDTTKTKTLDLGTAFHCRVLEPEEFSNRFIVAPEFNRRTNAGKEEEKAFLMECASTGKTVITAEEGRKIELMYQSVMALPLGQWLVESAGHAESSIYWEDPETGILCRCRPDKIIPEFHWIMDVKTTADIQRFKTAYYDYRYHVQDAFYSDGYEAQFGVQPTFVFLVASTTVECGRYPVEIFMMGEEAKLAGQQEYHRNLRTLADCLNTDEWPAIKTLSLPRWAKEYAND Acetobacter RecT protein (SEQ ID NO: 135): MNAPQKQNTRAAVKKISPQEFAEQFAAIIPQVKSVLPAHVTFEKFERVVRLAVRKNPDLLTCSPASLFMACIQAASDGLLPDGREGAIVSRWSSKKSCNEASWMPMVAGLMKLARNSGDIASISSQVVFEGEHFRVVLGDEERIEHERDLGKTGGKIVAAYAVARLKDGSDPIREIMSWGQIEKIRNTNKKWEWGPWKAWEDEMARKTVIRRLAKRLPMSTDKEGERLRSAIERIDSLVDISANVDAPQIAADDEFAAAAHGVEPQQIAAPDLIGRLAQMQSLEQVQDIEPQVSHAIQEADKRGDSDTANALDAALQSALSRTSTAKEEVPA Acetobacter RecE protein (SEQ ID NO:136): MVISKSGIYDLTNEQYHADPCPEMSL...

Claims

1. A system or composition comprising: (i) a nucleic acid molecule comprising a guide RNA sequence complementary to a target DNA sequence; and (ii) recombinant protein, wherein the recombinant protein comprises an exonuclease, a single-stranded DNA annealing protein (SSAP) or a single-stranded DNA binding protein (SSB), or a combination of two or more thereof; or, (iii) encoding or delivering one or more nucleic acid molecules of (i) and / or (ii) for in vivo expression in a cell; or, (iv) one or more vectors containing one or more nucleic acid molecules of (iii) for in vivo expression in cells.

2. The system or composition of claim 1, wherein the system does not comprise a CRISPR protein, or does not comprise a Cas protein, or does not comprise a Cas9 protein, or does not comprise a Cas12a protein.

3. The system or composition according to claim 1 or 2, further comprising a recruitment system, wherein the recruitment system comprises at least one aptamer; and The aptamer binding protein is functionally linked to the recombinant protein as part of a fusion protein.

4. The system or composition of claim 3, wherein the at least one aptamer comprises an RNA aptamer or a peptide aptamer.

5. The system or composition of claim 4, wherein the nucleic acid molecule or the plurality of nucleic acid molecules comprises two RNA aptamer sequences.

6. The system or composition of claim 5, wherein the two RNA aptamer sequences comprise the same sequence.

7. The system or composition of any one of claims 3-6, wherein the aptamer binding protein comprises an MS2 coat protein, or a functional derivative or variant thereof.

8. The system or composition of any one of claims 3-6, wherein the aptamer binding protein comprises a bacteriophage N peptide, or a functional derivative or variant thereof.

9. The system or composition of claim 3 or 4, wherein the at least one aptamer is linked to the guide RNA.

10. The system or composition of claim 9, wherein the guide RNA sequence comprises 1 to 24 aptamer sequences.

11. The system or composition of claim 9 or 10, wherein two or more aptamer sequences comprise the same sequence.

12. The system or composition of any one of claims 3, 4, or 9-11, wherein the aptamer sequence comprises a GCN4 peptide sequence.

13. The system or composition of any one of claims 3-12, wherein the N-terminus of the recombinant protein is linked to the C-terminus of the aptamer binding protein.

14. The system or composition of any one of claims 3-13, further comprising a linker between the recombinant protein and the aptamer binding protein.

15. The system or composition of claim 14, wherein the linker comprises the amino acid sequence of SEQ ID NO:

15.

16. The system or composition of any one of claims 1-15, comprising at least one nuclear localization sequence (NLS), optionally wherein the NLS is linked to the recombinant protein.

17. The system or composition of claim 16, wherein the nuclear localization sequence comprises the amino acid sequence of SEQ ID NO:

16.

18. The system or composition of claim 16 or 17, wherein the nuclear localization sequence is located at the C-terminus or the recombinant N-terminus of the recombinant protein.

19. The system or composition of any one of claims 1-18, wherein the recombinant protein comprises a microbial recombinant protein or an active portion thereof.

20. The system or composition of any one of claims 1-18, wherein the recombinant protein comprises a mitochondrial recombinant protein or an active portion thereof.

21. The system or composition of any one of claims 1-18, wherein the recombinant protein comprises a viral recombinant protein or an active portion thereof.

22. The system or composition of any one of claims 1-18, wherein the recombinant protein comprises a eukaryotic recombinant protein or an active portion thereof.

23. The system or composition of any one of claims 1-18, wherein the recombinant protein comprises a recombinant protein of Table 12 or a derivative or variant or functional portion thereof.

24. The system or composition of claim 23, wherein the recombinant protein, or a derivative or variant thereof, comprises an amino acid sequence having at least 70% similarity or identity to the amino acid sequence of Table 12.

25. The system or composition of claim 19, wherein the fusion protein comprises RecE, RecT, or a derivative or variant thereof.

26. The system or composition of claim 19, wherein the RecE, RecT, or a derivative or variant thereof comprises an amino acid sequence having at least 70% identity or similarity or identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14.

27. The system or composition of any one of claims 1-26, further comprising a donor nucleic acid.

28. The system or composition of any one of claims 1-26, wherein the target DNA sequence is a genomic DNA sequence in a host cell.

29. The system or composition of any one of claims 3-28, wherein the aptamer binding protein and the recombinant protein are functionally linked to each other and constitute a fusion protein.

30. A cell or eukaryotic cell comprising the system or composition of any one of claims 1-29.

31. A method for altering a target genomic DNA sequence in a cell comprising the target genomic DNA sequence, comprising introducing the system or composition of any one of claims 1-29 into the cell.

32. The cell or eukaryotic cell of claim 30 or the method of claim 31 , wherein the cell or eukaryotic cell is a mammalian cell.

33. The cell or eukaryotic cell or method of claim 32, wherein the cell is a human cell.

34. The cell or eukaryotic cell or method according to any one of claims 30-33, wherein the cell or eukaryotic cell or mammalian cell is a stem cell.

35. The method of any one of claims 31-34, wherein the target genomic DNA sequence encodes a gene product.

36. The method of any one of claims 31-34, wherein introducing the cells comprises administering to a subject.

37. The method of claim 36, wherein the subject is a mammalian non-human animal or a human.

38. The method of claim 36 or 37, wherein the administering comprises administering in vivo.

39. The method of any one of claims 31-34, wherein the cell or eukaryotic cell or mammalian cell is an ex vivo or in vitro cell.

40. The method of claim 39, further comprising administering the ex vivo or in vitro cells to a subject after the introducing step.

41. The method of claim 36, wherein the subject is a mammalian non-human animal or a human.

42. Use of the system or composition of any one of claims 1-29 for altering a target DNA sequence in a cell.

43. A system or composition comprising: (i) one or more nucleic acid polymerases; (ii) a nucleic acid molecule comprising a guide RNA sequence complementary to a target DNA sequence and an RNA for reverse transcription, or a plurality of nucleic acid molecules comprising a guide RNA sequence complementary to a target DNA sequence and an RNA for reverse transcription; and (iii) recombinant protein, wherein the recombinant protein comprises an exonuclease, a single-stranded DNA annealing protein (SSAP) or a single-stranded DNA binding protein (SSB), or a combination of two or more thereof; or, (iv) encoding or delivering one or more nucleic acid molecules of (i) and / or (ii) and / or (iii) for in vivo expression in a cell; or, (v) one or more vectors containing one or more nucleic acid molecules of (iv) for in vivo expression in a cell.

44. A system or composition according to claim 43, wherein (i), (ii) and (iii) further comprise a Cas protein; or (iv) comprises one or more nucleic acid molecules encoding or delivering (i) and / or (ii) and / or (iii) and / or Cas proteins for in vivo expression in cells; or (v) one or more vectors additionally contain one or more nucleic acid molecules encoding Cas proteins.

45. The system or composition according to claim 43 or 44, further comprising a recruitment system, the recruitment system comprising at least one aptamer; and The aptamer binding protein is functionally linked to the recombinant protein as part of a fusion protein.

46. ​​The system or composition of claim 45, wherein the at least one aptamer is an RNA aptamer or a peptide aptamer.

47. The system or composition of claim 46, wherein the nucleic acid molecule or the plurality of nucleic acid molecules comprises two RNA aptamers.

48. The system or composition of claim 47, wherein two RNA aptamer sequences comprise the same sequence.

49. The system or composition of any one of claims 45-47, wherein the aptamer binding protein comprises an MS2 coat protein, or a functional derivative or variant thereof.

50. The system or composition of any one of claims 45-47, wherein the aptamer binding protein comprises a bacteriophage N peptide, or a functional derivative or variant thereof.

51. The system or composition of claim 45, wherein the at least one aptamer sequence is linked to the Cas protein.

52. The system or composition of claim 45, wherein the at least one aptamer sequence is linked to the guide RNA.

53. The system or composition of claim 45, wherein the recruitment system comprises 1 to 24 aptamers.

54. The system or composition of any one of claims 51 to 53, wherein two or more aptamers comprise the same sequence.

55. The system or composition of any one of claims 45, 46, or 51-54, wherein the aptamer comprises a GCN4 peptide sequence.

56. The system or composition of any one of claims 45-55, wherein the recombinant protein N-terminus is linked to the aptamer binding protein C-terminus.

57. The system or composition of any one of claims 45-56, further comprising a linker between the recombinant protein and the aptamer binding protein.

58. The system or composition of claim 57, wherein the linker comprises the amino acid sequence of SEQ ID NO:

15.

59. The system or composition of any one of claims 43-58, comprising at least one nuclear localization sequence (NLS), optionally wherein the NLS is linked to the recombinant protein or the Cas protein or the reverse transcriptase, or at least one NLS is on each of at least two or three of the recombinant protein, the reverse transcriptase or the Cas protein.

60. The system or composition of claim 59, wherein the nuclear localization sequence comprises the amino acid sequence of SEQ ID NO:

16.

61. The system or composition of claim 59 or 60, comprising an NLS on one or more of the recombinant protein C-terminus, the recombinant protein N-terminus, or the Cas protein.

62. The system or composition of any one of claims 43-61, wherein the recombinant protein comprises a microbial recombinant protein or an active portion thereof.

63. The system or composition of any one of claims 43-61, wherein the recombinant protein comprises a mitochondrial recombinant protein or an active portion thereof.

64. The system or composition of any one of claims 43-61, wherein the recombinant protein comprises a viral recombinant protein or an active portion thereof.

65. The system or composition of any one of claims 43-61, wherein the recombinant protein comprises a eukaryotic recombinant protein or an active portion thereof.

66. The system or composition of any one of claims 43-61, wherein the recombinant protein comprises a recombinant protein of Table 12 or a derivative or variant or functional portion thereof.

67. The system or composition of claim 66, wherein the recombinant protein, or a derivative or variant thereof, comprises an amino acid sequence having at least 70% similarity or identity to the amino acid sequence of Table 12.

68. The system or composition of claim 62, wherein the fusion protein comprises RecE, RecT, or a derivative or variant thereof.

69. The system or composition of claim 62, wherein the RecE, RecT, or a derivative or variant thereof comprises an amino acid sequence having at least 70% identity or similarity or identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14.

70. The system or composition of any one of claims 44-69, wherein the Cas protein is catalytically inactive (less than 5% nuclease activity compared to a wild-type or non-mutated Cas protein) or catalytically inactive.

71. The system or composition of any one of claims 44-70, wherein the Cas protein comprises Cas9 or Cas12a.

72. The system or composition of any one of claims 70-71, wherein the Cas9 protein comprises wild-type Streptococcus pyogenes Cas9 or wild-type Staphylococcus aureus Cas9.

73. The system or composition of any one of claims 44-69, 71 or 72, wherein the Cas protein comprises a nickase.

74. The system or composition of claim 73, wherein the nickase comprises wild-type Streptococcus pyogenes Cas9 having an amino acid substitution D10A at position 10.

75. The system or composition of any one of claims 43-74, further comprising a donor nucleic acid.

76. The system or composition of any one of claims 43-75, wherein the target DNA sequence is a genomic DNA sequence in a host cell.

77. The system or composition of any one of claims 43-76, wherein the nucleic acid polymerase comprises reverse transcriptase activity.

78. The system or composition of any one of claims 43-76, wherein the nucleic acid polymerase comprises a reverse transcriptase RT.

79. The system or composition of any one of claims 43-78, wherein the nucleic acid polymerase and the recombinant protein are functionally linked to each other and constitute a fusion protein.

80. The system or composition of any one of claims 44-78, wherein the nucleic acid polymerase and the Cas protein are functionally linked to each other and constitute a fusion protein.

81. The system or composition of any one of claims 45-78, wherein the aptamer binding protein and the recombinant protein are functionally linked to each other and constitute a fusion protein.

82. The system or composition of any one of claims 44-78, wherein the recombinant protein and the Cas protein are functionally linked to each other and constitute a fusion protein.

83. The system or composition of any one of claims 44-78, wherein the RT and the Cas protein and the recombinant protein are functionally linked to each other and constitute a fusion protein.

84. A cell or eukaryotic cell comprising the system or composition of any one of claims 43-83.

85. A method for altering a target genomic DNA sequence in a cell comprising the target genomic DNA sequence, comprising introducing the system or composition of any one of claims 43-83 into the cell.

86. The cell or eukaryotic cell of claim 84 or the method of claim 85, wherein the cell or eukaryotic cell is a mammalian cell.

87. The cell or eukaryotic cell or method of claim 86, wherein the cell is a human cell.

88. The cell or eukaryotic cell or method of any one of claims 84-87, wherein the cell or eukaryotic cell or mammalian cell is a stem cell.

89. The method of any one of claims 85-88, wherein the target genomic DNA sequence encodes a gene product.

90. The method of any one of claims 85-88, wherein introducing the cells comprises administering to a subject.

91. The method of claim 90, wherein the subject is a mammalian non-human animal or a human.

92. The method of claim 90 or 91, wherein the administering comprises administering in vivo.

93. The method of any one of claims 85-88, wherein the cell or eukaryotic cell or mammalian cell is an ex vivo or in vitro cell.

94. The method of claim 93, further comprising administering the ex vivo or in vitro cells to a subject after the introducing step.

95. The method of claim 90, wherein the subject is a mammalian non-human animal or a human.

96. Use of the system or composition of any one of claims 43-83 for altering a target DNA sequence in a cell.

97. A recombinant method comprising providing in a cell a system or composition comprising: (i) a nucleic acid molecule comprising a guide RNA sequence that is complementary to a target DNA sequence; wherein the target DNA sequence comprises a genomic DNA sequence in the cell, and (ii) recombinant protein, wherein the recombinant protein comprises an exonuclease, a single-stranded DNA annealing protein (SSAP) or a single-stranded DNA binding protein (SSB), or a combination of two or more thereof; or, (iii) encoding or delivering one or more nucleic acid molecules of (i) and / or (ii) for in vivo expression in a cell; or, (iv) one or more vectors containing one or more nucleic acid molecules of (iii) for in vivo expression in cells.

98. A method according to claim 97, wherein (i) and (ii) further comprise Cas protein or reverse transcriptase (RT) or Cas protein and RT; or (iii) comprises one or more nucleic acid molecules encoding or delivering (i) and / or (ii) and / or Cas protein and / or nucleic acid polymerase for in vivo expression in cells; or (iv) one or more vectors additionally contain one or more nucleic acid molecules encoding Cas protein and / or RT.

99. The method of claim 97 or 98, wherein the target DNA sequence comprises the genomic sequence of albumin (ALB), AAVS1, HSP90AA1, DYNLT1, ACTB, BCAP31, HIST1H2BK, CLTA or RAB11A.

100. The method according to any one of claims 97 to 99, further comprising a recruitment system comprising at least one aptamer; and The aptamer binding protein is functionally linked to the recombinant protein as part of a fusion protein.

101. The method of claim 100, wherein the at least one aptamer is an RNA aptamer or a peptide aptamer.

102. The method of claim 101, wherein the nucleic acid molecule or the plurality of nucleic acid molecules comprises two RNA aptamers.

103. The method of claim 102, wherein the two RNA aptamer sequences comprise the same sequence.

104. The method of any one of claims 100-102, wherein the aptamer binding protein comprises an MS2 coat protein, or a functional derivative or variant thereof.

105. The method of any one of claims 100-102, wherein the aptamer binding protein comprises a bacteriophage N peptide, or a functional derivative or variant thereof.

106. The method of claim 100, wherein the at least one aptamer is linked to the Cas protein.

107. The method of claim 100, wherein the at least one aptamer is linked to the guide RNA.

108. The method of claim 106, wherein the recruitment system comprises 1 to 24 aptamers.

109. The method of claim 106 or 108, wherein two or more aptamers comprise the same sequence.

110. The method of any one of claims 100, 101, or 106-109, wherein the aptamer comprises a GCN4 peptide.

111. The method of any one of claims 100-110, wherein the N-terminus of the recombinant protein is linked to the C-terminus of the aptamer binding protein.

112. The method of any one of claims 100-111, further comprising a linker between the recombinant protein and the aptamer binding protein.

113. The method of claim 112, wherein the linker comprises the amino acid sequence of SEQ ID NO:

15.

114. The method of any one of claims 97-113, comprising at least one nuclear localization sequence (NLS), optionally wherein the NLS is linked to the recombinant protein or the Cas protein or the reverse transcriptase, or at least one NLS is on each of at least two or three of the recombinant protein, the reverse transcriptase or the Cas protein.

115. The method of claim 114, wherein the nuclear localization sequence comprises the amino acid sequence of SEQ ID NO:

16.

116. The method of claim 114 or 115, wherein the system comprises an NLS on one or more of the recombinant protein C-terminus, the recombinant protein N-terminus, or the Cas protein.

117. The method of any one of claims 97-116, wherein the recombinant protein comprises a microbial recombinant protein or an active portion thereof.

118. The method of any one of claims 97-116, wherein the recombinant protein comprises a mitochondrial recombinant protein or an active portion thereof.

119. The method of any one of claims 97-116, wherein the recombinant protein comprises a viral recombinant protein or an active portion thereof.

120. The method of any one of claims 97-116, wherein the recombinant protein comprises a eukaryotic recombinant protein or an active portion thereof.

121. The method of any one of claims 97-116, wherein the recombinant protein comprises a recombinant protein of Table 12 or a derivative or variant or functional portion thereof.

122. The method of claim 121, wherein the recombinant protein, or a derivative or variant thereof, comprises an amino acid sequence having at least 70% similarity or identity to the amino acid sequence of Table 12.

123. The method of claim 117, wherein the fusion protein comprises RecE, RecT, or a derivative or variant thereof.

124. The method of claim 117, wherein the RecE, RecT, or a derivative or variant thereof comprises an amino acid sequence having at least 70% identity or similarity or identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14.

125. The method of any one of claims 98-124, wherein the Cas protein is catalytically inactive (less than 5% nuclease activity compared to a wild-type or non-mutated Cas protein) or catalytically inactive.

126. The method of any one of claims 98-125, wherein the Cas protein comprises Cas9 or Cas12a.

127. The method of any one of claims 98-126, wherein the Cas9 protein comprises wild-type Streptococcus pyogenes Cas9 or wild-type Staphylococcus aureus Cas9.

128. The method of any one of claims 98-124, 126 or 127, wherein the Cas protein comprises a nickase.

129. The method of claim 128, wherein the nickase comprises wild-type Streptococcus pyogenes Cas9 having an amino acid substitution D10A at position 10.

130. The method of any one of claims 97-129, further comprising a donor nucleic acid.

131. The method of any one of claims 97-130, wherein the target DNA sequence is a genomic DNA sequence in a host cell.

132. The method of any one of claims 98-131, wherein the nucleic acid polymerase comprises reverse transcriptase activity.

133. The method of any one of claims 98-131, wherein the nucleic acid polymerase comprises a reverse transcriptase RT.

134. The method according to any one of claims 98-133, wherein the RT and the recombinant protein are functionally linked to each other and constitute a fusion protein.

135. The method according to any one of claims 100-134, wherein the aptamer binding protein and the recombinant protein are functionally linked to each other and constitute a fusion protein.

136. The method of any one of claims 98-135, wherein the RT and the Cas protein are functionally linked to each other and constitute a fusion protein.

137. The method of any one of claims 98-136, wherein the recombinant protein and the Cas protein are functionally linked to each other and constitute a fusion protein.

138. The method of any one of claims 98-137, wherein the RT and the Cas protein and the recombinant protein are functionally linked to each other and constitute a fusion protein.

139. The method of any one of claims 97-138, wherein the target genomic DNA sequence encodes a gene product.

140. The method of any one of claims 97-139, wherein the cell or eukaryotic cell is a mammalian cell.

141. The method of any one of claims 97-140, wherein the cell is a human cell.

142. The method of any one of claims 97-141, wherein the cell or eukaryotic cell or mammalian cell or human cell is a stem cell.

143. A recombinant cell or organism produced by the method of any one of claims 97-142.

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