Methods for genomic integration
View Patent ↗Provided herein are methods of integrating one or more exogenous nucleic acids into one or more selected target sites of a host cell genome. In certain embodiments, the methods comprise contacting the host cell genome with one or more integration polynucleotides comprising an exogenous nucleic acid to be integrated into a genomic target site, a nuclease capable of causing a break at the genomic target site, and a linear nucleic acid capable of homologous recombination with itself or with one or more additional linear nucleic acids contacted with the population of cells, whereupon said homologous recombination results in formation of a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker. In some embodiments, the methods further comprise selecting a host cell that expresses the selectable marker.
1. A composition comprising:
(a) a site-specific nuclease, or a nucleic acid comprising a coding sequence for a site-specific nuclease; and
(b) a first linear nucleic acid and one or more additional linear nucleic acid, wherein the first and second linear nucleic acids are capable of homologously recombining with each other in a host cell, whereupon said homologous recombination results in formation of a circular nucleic acid comprising a coding sequence for a selectable marker.
2. The composition of claim 1 , wherein each linear nucleic acid comprises a partial, interrupted and/or non-contiguous coding sequence for the selectable marker, wherein the selectable marker cannot be expressed from each linear nucleic acid in a host cell, whereupon said formation of the circular nucleic acid results in formation of a complete coding sequence of the selectable marker, wherein the selectable marker can be expressed from the circular nucleic acid in a host cell.
3. The composition of claim 1 , wherein the circular nucleic acid further comprises a coding sequence for a site-specific nuclease.
4. The composition of claim 1 , wherein the site-specific nuclease is an RNA-guided DNA endonuclease.
5. The composition of claim 4 , wherein the RNA-guided DNA endonuclease is a Cas9 endonuclease.
6. The composition of claim 5 , wherein the circular nucleic acid further comprises a deoxyribonucleic acid that encodes a ribonucleic acid comprising a crRNA activity and a deoxyribonucleic acid that encodes a ribonucleic acid comprising a tracrRNA activity.
7. The composition of claim 4 , further comprising:
i. a ribonucleic acid comprising a crRNA activity and a ribonucleic acid comprising a tracrRNA activity; or
ii. a deoxyribonucleic acid that encodes a ribonucleic acid comprising a crRNA activity and a deoxyribonucleic acid that encodes a ribonucleic acid comprising a tracrRNA activity.
8. The composition of claim 7 , wherein the deoxyribonucleic acid that encodes the crRNA activity and the tracrRNA activity encodes said activities on a single contiguous RNA molecule.
9. The composition of claim 1 , wherein the site-specific nuclease is selected from the group consisting of an endonuclease, a zinc finger nuclease, a TAL-effector DNA binding domain-nuclease fusion protein (TALEN), a transposase, and a site-specific recombinase.
10. The composition of claim 9 , wherein the zinc finger nuclease is a fusion protein comprising the cleavage domain of a TypellS restriction endonuclease fused to an engineered zinc finger binding domain.
11. The composition of claim 10 , wherein the TypellS restriction endonuclease is selected from the group consisting of HO endonuclease and Fok I endonuclease.
12. The composition of claim 10 , wherein the zinc finger binding domain comprises 3, 5 or 6 zinc fingers.
13. The composition of claim 9 , wherein the endonuclease is a homing endonuclease selected from the group consisting of: an LAGLIDADG (SEQ ID NO: 1) homing endonuclease, an HNH homing endonuclease, a His-Cys box homing endonuclease, a GIY-YIG homing endonuclease, and a cyanobacterial homing endonuclease.
14. The composition of claim 9 , wherein the endonuclease is selected from the group consisting of: H-DreI, I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, Pi-PspI, F-SceI, F-SceII, F-SuvI, F-CphI, F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NclIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp68031, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TevI, I-TevII, I-TevIII, i-UarAP, i-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MgaI, PI-MtuI, PI-MtuHIP PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, or PI-TliII.
15. The composition of claim 9 , wherein the endonuclease is modified to specifically bind an endogenous genomic sequence, wherein the modified endonuclease no longer binds to its wild type endonuclease recognition sequence.
16. The composition of claim 15 , wherein the modified endonuclease is derived from a homing endonuclease selected from the group consisting of: an LAGLIDADG (SEQ ID NO: 1)homing endonuclease, an HNH homing endonuclease, a His-Cys box homing endonuclease, a GIY-YIG homing endonuclease, and a cyanobacterial homing endonuclease.
17. The composition of claim 15 , wherein the modified endonuclease is derived from an endonuclease selected from the group consisting of: H-Drel, I-Scel, I-Scell, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAIIP, I-Crel, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, Pi-PspI, F-SceI, F-SceII, F-SuvI, F-CphI, F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LIaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I-NgrIP, I-NitI, I-Njal, I-Nsp236IP, I-PakI, I-PboIP,I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP,I-PobIP, I-PorI,I-PorIIP,I-PbpIP, I-SpBetaIP, I-ScaI,I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomCP,I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp68031, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TdeIP,I-TevI,I-TevII, I-TevIII, i-UarAP, i-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MgaI, PI-MtuI, PI-MtuHIP PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, or PI-TliII.