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 method of selecting a host cell competent for homologous recombination, comprising:
(a) contacting one or more host cells with 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; and
(b) selecting a host cell that expresses the selectable marker.
2. A method for integrating an exogenous nucleic acid into a target site of a host cell genome, the method comprising:
(a) contacting one or more host cells with:
(i) an exogenous nucleic acid (ES) capable of recombining, via homologous recombination, at the target site (TS) of the host cell genome;
(ii) a nuclease (N) capable of generating a break at TS; and
(iii) a linear nucleic acid capable of homologous recombination with itself or with one or more additional linear nucleic acids contacted with the host cell, whereupon said homologous recombination results in formation of a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker;
and
(b) selecting a host cell that expresses the selectable marker.
3. The method of claim 2 , wherein the linear nucleic acid comprises two internal homology regions that are capable of homologously recombining with each other, whereupon homologous recombination of the internal homology regions results in formation of the circular extrachromosomal nucleic acid expressing the selectable marker.
4. The method of claim 2 , wherein the linear nucleic acid comprises a homology region that is capable of recombining with a homology region of an additional linear nucleic acid contacted with the host cell, whereupon homologous recombination of the two linear nucleic acids results in formation of the circular extrachromosomal nucleic acid expressing the selectable marker.
5. The method of any one of claims 1 to 4 , wherein the 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 the linear nucleic acid, whereupon said formation of the circular extrachromosomal nucleic acid results in formation of a complete coding sequence of the selectable marker, wherein the selectable marker can be expressed from the circular extrachromosomal nucleic acid.
6. The method of claim 2 , wherein the contacted host cell(s) are cultured for a period of at least about 12, 24, 36, 48, 72 or more than 72 hours prior to said selecting.
7. The method of claim 2 , wherein the contacted cells are cultured under culturing conditions that select against the survival of cells not expressing the selectable marker.
8. The method of claim 2 , wherein said selecting of step (b) comprises detecting the expression of the selectable marker via visual, colorimetric or fluorescent detection methods.
9. The method of claim 2 , further comprising the step of recovering a host cell wherein ES has homologously recombined at TS.
10. The method of claim 9 , wherein said recovering does not require integration of a selectable marker into the host cell genome.
11. The method of claim 9 , wherein said recovering occurs at a frequency of at least about one every 10, 9, 8, 7, 6, 5, 4, 3, or 2 contacted host cells, or clonal populations thereof, screened.
12. The method of claim 2 , further comprising the step of eliminating the circular extrachromasomal nucleic acid from the selected host cell.
13. The method of claim 2 , wherein a plurality of (n) exogenous nucleic acids is integrated into a plurality of (n) target sites of the host cell genome, wherein n is at least two, wherein step (a) comprises contacting the host cell with:
(i) said plurality of exogenous nucleic acids, wherein
x is an integer that varies from 1 to n, and for each integer x, each exogenous nucleic acid (ES) x is capable of recombining, via homologous recombination, at a target site (TS) x selected from said plurality of (n) target sites of said host cell genome;
(ii) for each said target site (TS) x , a nuclease (N) x capable of generating a break at (TS) x .
14. The method of claim 13 , wherein a single nuclease is capable of cleaving each (TS) x .
15. The method of claim 13 , wherein n=3, 4, 5, 6, 7, 8, 9 or 10.
16. The method of claim 2 , wherein ES comprises a first homology region (HR1) and a second homology region (HR2), wherein HR1 and HR2 are capable of recombining, via homologous recombination, with a third homology region (HR3) and a fourth homology region (HR4), respectively, wherein HR3 and HR4 are each at TS.
17. The method of claim 2 , wherein N is capable of generating a single stranded break or a double stranded break at TS.
18. The method of claim 2 , wherein ES further comprises a nucleic acid of interest D.
19. The method of claim 18 , wherein D is selected from the group consisting of a selectable marker, a promoter, a nucleic acid sequence encoding an epitope tag, a gene of interest, a reporter gene, and a nucleic acid sequence encoding a termination codon.
20. The method of claim 2 , wherein ES is linear.
21. The method of claim 2 , wherein the circular extrachromasomal nucleic acid further comprises a coding sequence for the nuclease.
22. The method of claim 2 , wherein the nuclease is an RNA-guided DNA endonuclease.
23. The method of claim 22 , wherein the RNA-guided DNA endonuclease is a Cas9 endonuclease.
24. The method of claim 22 , wherein the circular extrachromosomal nucleic acid further comprises a sequence that encodes a crRNA activity and a tracrRNA activity that enables site-specific recognition and cleavage of TS by the RNA-guided DNA endonuclease.
25. The method of claim 24 , wherein the crRNA activity and the tracrRNA activity are expressed as a single contiguous RNA molecule.
26. The method of claim 2 , wherein the 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.
27. The method of claim 26 , wherein the zinc finger nuclease is a fusion protein comprising the cleavage domain of a TypeIIS restriction endonuclease fused to an engineered zinc finger binding domain.
28. The method of claim 27 , wherein the TypeIIS restriction endonuclease is selected from the group consisting of HO endonuclease and Fok I endonuclease.
29. The method of claim 27 , wherein the zinc finger binding domain comprises 3, 5 or 6 zinc fingers.
30. The method of claim 26 , wherein the endonuclease is a homing endonuclease selected from the group consisting of: an LAGLIDADG homing endonuclease, an HNH homing endonuclease, a His-Cys box homing endonuclease, a GIY-YIG homing endonuclease, and a cyanobacterial homing endonuclease.
31. The method of claim 26 , 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.
32. The method of claim 26 , 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.
33. The method of claim 32 , wherein the modified endonuclease is derived from a homing endonuclease selected from the group consisting of: an LAGLIDADG homing endonuclease, an HNH homing endonuclease, a His-Cys box homing endonuclease, a GIY-YIG homing endonuclease, and a cyanobacterial homing endonuclease.
34. The method of claim 32 , wherein the modified endonuclease is derived from an endonuclease 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.
35. The method of claim 2 , wherein the host cell is a prokaryotic cell.
36. The method of claim 2 , wherein the host cell is a eukaryotic cell.
37. The method of claim 2 , wherein the host cell is selected from the group consisting of a fungal cell, a bacterial cell, a plant cell, an insect cell, an avian cell, a fish cell and a mammalian cell.
38. The method of claim 2 , wherein the host cell is a mammalian cell selected from the group consisting a rodent cell, a primate cell and a human cell.
39. The method of claim 2 , wherein the host cell is a human cell.
40. The method of claim 2 , wherein the host cell is a yeast cell.
41. The method of claim 40 , wherein the yeast is Saccharomyces cerevisiae.
42. A method for integrating an exogenous nucleic acid into a target site of a host cell genome, the method comprising:
(a) contacting one or more host cells with:
(i) an exogenous nucleic acid (ES) capable of recombining, via homologous recombination, at the target site (TS) of the host cell genome; and
(ii) a linear nucleic acid capable of homologous recombination with itself or with one or more additional linear nucleic acids contacted with the host cell, whereupon said homologous recombination results in formation of a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker and a coding sequence for a nuclease (N) capable of generating a break at TS;
and
(b) selecting a host cell that expresses the selectable marker.
43. A method for integrating one or more exogenous nucleic acids into one or more target sites of a host cell genome, the method comprising:
(a) contacting one or more host cells with:
(i) one or more exogenous donor nucleic acids (ES) capable of recombining, via homologous recombination, at one or more target sites (TS) of the host cell genome;
(ii) an RNA-guided endonuclease (RGEN);
(iii) one or more ribonucleic acids that enable site-specific recognition and cleavage of the one or more TS by the RGEN; and
(iv) a linear pre-recombination nucleic acid capable of homologous recombination with itself or with one or more additional linear pre-recombination nucleic acids contacted with the host cell, whereupon said homologous recombination results in formation of a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker;
and
(b) selecting a host cell that expresses the selectable marker, thereby selecting for a cell that has integrated the one or more exogenous nucleic acids into the one or more target sites of a host cell genome.