Systems, methods, and compositions for site-specific genetic engineering using programmable addition via site-specific targeting elements (paste)

Abstract

This disclosure provides systems, methods, and compositions for site-specific genetic engineering using Programmable Addition via Site-Specific Targeting Elements (PASTE). PASTE comprises the addition of an integration site into a target genome followed by the insertion of one or more genes of interest or one or more nucleic acid sequences of interest at the site. PASTE combines gene editing technologies and integrase technologies to achieve unidirectional incorporation of genes in a genome for the treatment of diseases and diagnosis of disease.

1 . A method of site-specific integration of a nucleic acid into a cell genome or target nucleic acid, the method comprising:
 (a) incorporating an integration site at a desired location in the cell genome or target nucleic acid by introducing into a cell:
 i. a DNA binding nuclease domain linked to a reverse transcriptase domain, wherein the DNA binding nuclease domain comprises a nickase activity; and 
 ii. a guide RNA (gRNA) comprising a primer binding targeting sequence linked to a complement of an integration sequence, wherein the gRNA interacts with the DNA binding nuclease domain and targets the desired location in the cell genome genome or target nucleic acid, wherein the DNA binding nuclease domain nicks a strand of the cell genome or target nucleic acid and the reverse transcriptase domain incorporates the integration sequence of the gRNA into the nicked site, thereby providing the integration site at the desired location of the cell genome or target nucleic acid; and 
   (b) integrating the nucleic acid into the cell genome or target nucleic acid by introducing into the cell:
 i. a DNA or RNA strand comprising the nucleic acid linked to a sequence that is complementary or associated to the integration site; and 
 ii. an integration enzyme, wherein the integration enzyme incorporates the nucleic acid into the cell genome or target nucleic acid at the integration site by integration, recombination, or reverse transcription of the sequence that is complementary or associated to the integration site, thereby introducing the nucleic acid into the desired location of the cell genome or target nucleic acid of the cell. 
   
     
     
         2 . The method of  claim 1 , wherein the gRNA hybridizes to a complementary strand of the cell genome to the genomic strand that is nicked by the DNA binding nuclease domain. 
     
     
         3 . The method of  claim 1 , wherein:
 the integration enzyme is introduced as a polypeptide or a nucleic acid encoding the integration enzyme; and/or   the DNA binding nuclease domain is introduced as a polypeptide or a nucleic acid encoding the DNA binding nuclease.   
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the DNA or RNA strand comprising the nucleic acid is introduced into the cell as a minicircle, a plasmid, mRNA or a linear DNA, optionally wherein:
 the DNA or RNA strand comprising the nucleic acid is between 1000 bp and 36,000 bp;   the DNA or RNA strand comprising the nucleic acid is more than 36,000 bp; and/or   the DNA or RNA strand comprising the nucleic acid is less than 1000 bp.   
     
     
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         9 . The method of  claim 1 , wherein the DNA comprising the nucleic acid is introduced into the cell as a minicircle, optionally wherein the minicircle does not comprise a sequence of a bacterial origin. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein the DNA binding nuclease linked to a reverse transcriptase domain and the integration enzyme are linked via a linker, optionally wherein:
 the linker is cleavable;   the linker is non-cleavable; or   the linker can be replaced by two associating binding domains of the DNA binding nuclease linked to a reverse transcriptase.   
     
     
         12 . (canceled) 
     
     
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         15 . The method of  claim 1 , wherein:
 the integration enzyme is selected from the group consisting of Cre, Dre, Vika, Bxb1, φC31, RDF, FLP, φBT1, R1, R2, R3, R4, R5, TP901-1, A118, φFC1, φC1, MR11, TG1, q 370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, retrotransposases encoded by R2, L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1), and Minos, and any mutants thereof;   the integration site is an attB site, an attP site, an attL site, an attR site, a lox71 site a Vox site, or a FRT site;   the DNA binding nuclease comprising a nickase activity is selected from Cas9-D10A, Cas9-H840A, and Cas12a/b nickase; and/or   the reverse transcriptase domain is selected from the group consisting of Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase domain, transcription xenopolymerase (RTX), avian myeloblastosis virus reverse transcriptase (AMV-RT), and Eubacterium rectale maturase RT (MarathonRT), optionally wherein:   the reverse transcriptase domain comprises a mutation relative to the wild-type sequence; and/or   the M-MLV reverse transcriptase domain comprises one or more mutations selected from the group consisting of D200N, T306K, W313F, T330P and L603W.   
     
     
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         22 . The method of  claim 1 , further comprising introducing a nicking guide RNA (ngRNA). 
     
     
         23 . The method of  claim 1 , wherein:
 the gRNA, the nucleic acid encoding the DNA binding nuclease, the reverse transcriptase, the DNA comprising nucleic acid linked to a complementary or associated integration site, the integration enzyme, and optionally the ngRNA, are introduced into a cell in a single reaction; and/or   the gRNA, the nucleic acid encoding the DNA binding nuclease, the reverse transcriptase, the DNA comprising nucleic acid linked to a complementary integration site, the integration enzyme, and optionally the ngRNA, are introduced using a virus, a RNP, an mRNA, a lipid, or a polymeric nanoparticle.   
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 1 , wherein:
 the nucleic acid is a reporter gene, optionally wherein the reporter gene is a fluorescent protein;   the nucleic acid is a degradation tag for programmable knockdown of proteins in the presence of small molecules;   the nucleic acid is a T-cell receptor (TCR), a chimeric antigen receptor (CAR), an interleukin, a cytokine, or an immune checkpoint gene for integration into a T-cell or natural killer (NK) cell, optionally wherein the TCR, the CAR, the interleukin, the cytokine, or the immune checkpoint gene is incorporated into the target site of the T-cell or NK cell genome using a minicircle DNA;   the nucleic acid is a beta hemoglobin (HBB) gene and the cell is a hematopoietic stem cell (HSC), optionally wherein the HBB gene is incorporated into the target site in the HSC genome using a minicircle DNA and/or the nucleic acid is a gene responsible for beta thalassemia or sickle cell anemia;   the nucleic acid is a metabolic gene, optionally wherein the metabolic gene is involved in alpha-1 antitrypsin deficiency or ornithine transcarbamylase (OTC) deficiency and/or the metabolic gene is a gene involved in an inherited disease; or   the nucleic acid is a gene involved in an inherited disease or an inherited syndrome, optionally wherein the inherited disease is cystic fibrosis, familial hypercholesterolemia, adenosine deaminase (ADA) deficiency, X-linked SCID (X-SCID), Wiskott-Aldrich syndrome (WAS), hemochromatosis, Tay-Sachs, fragile X syndrome, Huntington's disease, Marfan syndrome, phenylketonuria, or muscular dystrophy.   
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 1 , wherein the cell is a dividing cell or a non-dividing cell, optionally wherein:
 the desired location in the cell genome is the locus of a mutated gene; and/or   the cell is a mammalian cell, a bacterial cell or a plant cell.   
     
     
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         42 . A vector comprising a nucleic acid encoding the polypeptide of  claim 63 . 
     
     
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         54 . A cell comprising:
 (a) the vector of  claim 42 ;   (b) a gRNA comprising a primer binding sequence, an integration sequence, and a guide sequence, wherein the gRNA can interact with the encoded nuclease comprising a nickase activity;   (c) a DNA minicircle comprising a nucleic acid and a sequence recognized by the encoded integrase, recombinase, or reverse transcriptase; and   (d) a nicking guide RNA (ngRNA) capable of binding the encoded nuclease comprising a nickase activity, wherein the ngRNA targets a sequence away from the gRNA.   
     
     
         55 . The cell of  claim 54 , wherein:
 the minicircle does not comprise a sequence of bacterial origin;   the integration enzyme is selected from the group consisting of Cre, Dre, Vika, Bxb1, φC31, RDF, FLP, φBT1, R1, R2, R3, R4, R5, TP901-1, A118, φFC1, φC1, MR11, TG1, q 370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, retrotransposases encoded by R2, L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1), Minos, and any mutants thereof;   the DNA binding nuclease comprising a nickase activity is selected from the group consisting of Cas9-D10A, Cas9-H840A and Cas12a;   the reverse transcriptase is a M-MLV reverse transcriptase, optionally wherein the reverse transcriptase is a modified M-MLV reverse transcriptase, optionally wherein the amino acid sequence of the M-MLV reverse transcriptase comprises one or more mutations selected from the group consisting of D200N, T306K, W313F, T330P, and L603W; and/or   the cell further comprises a ngRNA.   
     
     
         56 . (canceled) 
     
     
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         63 . A polypeptide comprising a DNA binding nuclease comprising a nickase activity C-terminally linked to a reverse transcriptase linked to an integration enzyme via a linker. 
     
     
         64 . The polypeptide of  claim 63 , wherein:
 the linker is cleavable or non-cleavable;   the integration enzyme is fused to an estrogen receptor;   the DNA binding nuclease comprising a nickase activity is selected from the group consisting of Cas9-D10A, Cas9-H840A, and Cas12a/b/c/d/e/f/g/h/i/j;   the reverse transcriptase is a M-MLV reverse transcriptase, a AMV-RT, a MarathonRT, or a XRT, optionally wherein the reverse transcriptase is a modified M-MLV relative to a wild-type M-MLV reverse transcriptase, optionally wherein the M-MLV reverse transcriptase domain comprises one or more of mutations selected from the group consisting of D200N, T306K, W313F, T330P, and L603W;   the integration enzyme is selected from group consisting of Cre, Dre, Vika, Bxb1, φC31, RDF, FLP, φBT1, R1, R2, R3, R4, R5, TP901-1, A118, φFC1, φC1, MR11, TG1, φ370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, retrotransposases encoded by R2, L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1), Minos, and any mutants thereof.   
     
     
         65 . (canceled) 
     
     
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         73 . A gRNA that specifically binds to a DNA binding nuclease comprising nickase activity, the gRNA comprising:
 (a) a primer binding site, which hybridizes to a nicked DNA strand;   (b) a recognition site for an integration enzyme; and   (c) a target recognition sequence recognizing a target site in a cell genome and hybridizing to a genomic strand complementary to the strand that is nicked by the DNA binding nuclease.   
     
     
         74 . The gRNA of  claim 73 , wherein:
 the DNA binding nuclease comprising a nickase activity is selected from the group consisting of Cas9-D10A, Cas9-H840A, and Cas12a/b/c/d/e/f/g/h/i/j;   the primer binding site hybridizes to the 3′ end of the nicked DNA strand;   the recognition site for the integration enzyme is selected from an attB site, an attP site, an attL site, an attR site, a lox71 site, and a FRT site; and/or   the recognition site for the integration enzyme is a Bxb1 site.   
     
     
         75 . (canceled) 
     
     
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         78 . A method of site-specific integration of two or more nucleic acids into a cell genome, the method comprising:
 (a) incorporating two integration sites at desired locations in the cell genome by introducing into the cell:
 i. a DNA binding nuclease linked to a reverse transcriptase domain, wherein the DNA binding nuclease comprises a nickase activity; and 
 ii. two guide RNAs (gRNAs), each comprising, a primer binding sequence, and is linked to a unique integration sequence, wherein the gRNA interacts with the DNA binding nuclease and targets the desired locations in the cell genome, wherein the DNA binding nuclease nicks a strand of the cell genome and the reverse transcriptase domain incorporates each of the integration sequence of the gRNA into the nicked site, thereby providing the integration site at the desired locations of the cell genome; and 
   (b) integrating the nucleic acid by introducing into the cell:
 i. two or more DNA or RNA comprising the nucleic acids, wherein each DNA is flanked by orthogonal integration sites; and 
 ii. an integration enzyme, wherein the integration enzyme incorporates the nucleic acids into the cell genome at the integration sites by integrase, recombinase, or reverse transcriptase of the sequence that is complementary or associated to the integration site, thereby introducing the nucleic acids into the desired locations of the cell genome of the cell. 
   
     
     
         79 . The method of  claim 78 , wherein each of the two different integration sites inserted into the cell genome are attB and/or attP sequences comprising different palindromic or non-palindromic central dinucleotide, optionally wherein:
 the integration enzyme enables each of the two or more DNA or RNA comprising the nucleic acids to directionally enable integration of the nucleic acids into a genome via recombination of a pair of orthogonal attB site sequence and an attP site sequence; and/or   the pair of an attB site sequence and an attP site sequence are selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, and SEQ ID NO: 35 and SEQ ID NO: 36.   
     
     
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         92 . The method of  claim 17 , wherein the attB site is about 40-46 basepair. 
     
     
         93 . The gRNA of  claim 74 , wherein the attB site is about 40-46 basepair.