Methods for genomic modification
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, and a nuclease capable of causing a double-strand break near or within the genomic target site.
1. A host cell comprising:
(a) a plurality of (n) exogenous donor nucleic acids to be integrated into the host cell genome, wherein:
n is at least 2, x is an integer from 1 to n, and for each integer x, each exogenous donor nucleic acid (ES) x comprises a first homology region (HR1) x and a second homology region (HR2) x , wherein (HR1) x and (HR2) x are capable of initiating host cell mediated homologous recombination of (ES) x at a target site (TS) x selected from a plurality of (n) target sites of said host cell genome; and
(b) for each said target site (TS) x , a nuclease (N) x capable of cleaving at (TS) x , whereupon said cleaving results in homologous recombination of (ES) x at (TS),
wherein the host cell comprises one or more heterologous nucleotide sequences encoding one or more enzymes of a biosynthetic pathway.
2. The host cell of claim 1 , wherein (HR1) x is homologous to a 5′ region of (TS) x , and (HR2) x is homologous to a 3′ region of (TS) x .
3. The host cell of claim 2 , wherein (N) x is capable of cleaving at a region positioned between said 5′ and 3′ regions of (TS) x .
4. The host cell of claim 1 , wherein a single nuclease is capable of cleaving each (TS) x .
5. The host cell of claim 1 , wherein n=3, 4, 5, 6, 7, 8, 9 or 10.
6. The host cell of claim 1 , wherein (N) x is capable of cleaving an endogenous genomic sequence within (TS) x .
7. The host cell of claim 1 , wherein (N) x is capable of cleaving an exogenous sequence within (TS) x , wherein x is 1 or any integer from 1 to n.
8. The host cell of claim 7 , wherein the exogenous sequence is a recognition sequence for a homing endonuclease.
9. The host cell of claim 8 , wherein the homing endonuclease is F-cphI.
10. The host cell of claim 7 , wherein (ES) x further comprises a nucleic acid of interest (D) x positioned 3′ of (HR1) x and 5′ of (HR2) x .
11. The host cell of claim 10 , wherein (D) x is selected from the group consisting of a protein-coding sequence, reporter gene, fluorescent marker coding sequence, promoter, enhancer, terminator, transcriptional activator, transcriptional repressor, transcriptional activator binding site, transcriptional repressor binding site, intron, exon, poly-A tail, multiple cloning site, nuclear localization signal, mRNA stabilization signal, integration loci, epitope tag coding sequence, and degradation signal.
12. The host cell of claim 1 , wherein (ES) x is linear.
13. The host cell of claim 1 , wherein the one or more heterologous nucleotide sequences encoding one or more enzymes of a biosynthetic pathway are genomically integrated.
14. The host cell of claim 1 , wherein each said exogenous nucleic acid (ES) x comprises a nucleic acid of interest (D) x positioned 3′ of (HR1) x and 5′ of (HR2) x , encoding an enzyme of a biosynthetic pathway.
15. The host cell of claim 1 , wherein (N) x is provided as an expression vector comprising a nucleic acid sequence encoding (N) x .
16. The host cell of claim 1 , wherein (N) x is transformed into the host cell as a purified protein.
17. The host cell of claim 1 , wherein (N) x is transformed into the host cell as a purified RNA.
18. The host cell of claim 1 , wherein (N) x 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.
19. The host cell of claim 18 , 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.
20. The host cell of claim 19 , wherein the TypeIIS restriction endonuclease is selected from the group consisting of HO endonuclease and Fok I endonuclease.
21. The host cell of claim 19 , wherein the zinc finger binding domain comprises 3, 5 or 6 zinc fingers.
22. The host cell of claim 18 , 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.
23. The host cell of claim 18 , 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, and PI-TliII.
24. The host cell of claim 18 , wherein the endonuclease is modified to specifically bind an endogenous host cell genomic sequence, wherein the modified endonuclease no longer binds to its wild type endonuclease recognition sequence.
25. The host cell of claim 24 , 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.
26. The host cell of claim 24 , 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-TeeI, 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, and PI-TliII.
27. The host cell of claim 1 , wherein the host cell is selected from the group consisting of a fungal cell, a bacterial cell, a plant cell, and an animal cell.
28. The host cell of claim 1 , wherein the host cell is a yeast cell.
29. The yeast cell of claim 28 , wherein the yeast cell is a Saccharomyces cerevisiae cell.
30. A host cell comprising:
(a) a plurality of (n) libraries, wherein n is at least 2, x is any integer from 1 to n, and for each integer x, each library (L) x comprises a plurality of exogenous donor nucleic acids to be integrated into the host cell genome, wherein a selected exogenous donor nucleic acid comprises, in a 5′ to 3′ orientation, a first homology region (HR1) x , any nucleic acid of interest selected from a group (D) x , and a second homology region (HR2) x , wherein (HR1) x and (HR2) x are capable of initiating host cell mediated homologous recombination of said selected exogenous nucleic acid at a target site (TS) x of said host cell genome; and
(b) for each said target site (TS) x , a nuclease (N) x capable of cleaving at (TS) x , whereupon said cleaving results in homologous recombination of said selected exogenous nucleic acid at (TS) x .
31. The host cell of claim 30 , wherein (D) x is selected from the group consisting of a protein-coding sequence, reporter gene, fluorescent marker coding sequence, promoter, enhancer, terminator, transcriptional activator, transcriptional repressor, transcriptional activator binding site, transcriptional repressor binding site, intron, exon, poly-A tail, multiple cloning site, nuclear localization signal, mRNA stabilization signal, integration loci, epitope tag coding sequence, and degradation signal.
32. The host cell of claim 30 or 31 , wherein the host cell comprises at least 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , or more than 10 6 unique nucleic acids of interest (D) x in each library (L) x .
33. The host cell of claim 30 , wherein each exogenous nucleic acid (ES) x does not comprise nucleic acid encoding a selectable marker.
34. The host cell of claim 30 , wherein (HR1) x is homologous to a 5′ region of (TS) x , and (HR2) x is homologous to a 3′ region of (TS) x .
35. The host cell of claim 34 , wherein (N) x is capable of cleaving at a region positioned between said 5′ and 3′ regions of (TS) x .
36. The host cell of claim 30 , wherein a single nuclease is capable of cleaving each (TS) x .
37. The host cell of claim 30 , wherein (N) x is capable of cleaving an endogenous genomic sequence within (TS) x .
38. The host cell of claim 30 , wherein (N) x is capable of cleaving an exogenous sequence within (TS) x , wherein x is 1 or any integer from 1 to n.
39. The host cell of claim 38 , wherein the exogenous sequence is a recognition sequence for a homing endonuclease.
40. The host cell of claim 30 , wherein (N) x 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.
41. The host cell of claim 40 , wherein the endonuclease is modified to specifically bind an endogenous host cell genomic sequence, wherein the modified endonuclease no longer binds to its wild type endonuclease recognition sequence.
42. The host cell of claim 30 , wherein the host cell is selected from the group consisting of a fungal cell, a bacterial cell, a plant cell, and an animal cell.
43. The host cell of claim 30 , wherein the host cell is a yeast cell.
44. The yeast cell of claim 43 , wherein the yeast cell is a Saccharomyces cerevisiae cell.