Methods for genomic modification

Abstract

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 method for simultaneously integrating a plurality of (n) exogenous nucleic acids into a plurality of (n) target sites of a host cell genome, wherein n is at least two, the method comprising:
 (a) simultaneously contacting a 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  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 said plurality of (n) target sites of said host cell genome; and 
 (ii) 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) x ; 
   and   (b) recovering a host cell wherein each exogenous nucleic acid (ES) x  has integrated at its selected target sequence (TS) x ,   wherein x is any integer from 1 to n wherein n is at least 2.   
     
     
         2 . The method 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 method of  claim 1 , wherein (N) x  is capable of cleaving at a region positioned between said 5′ and 3′ regions of (TS) x . 
     
     
         4 . The method of  claim 1 , wherein a single nuclease is capable of cleaving each (TS) x . 
     
     
         5 . The method of  claim 1 , wherein n=3, 4, 5, 6, 7, 8, 9 or 10. 
     
     
         6 . The method of  claim 1 , wherein said recovering does not require integration of a selectable marker. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein said recovering occurs at a frequency of about one every 10, 9, 8, 7, 6, 5, 4, 3, or 2 contacted host cells, or clonal populations thereof, screened. 
     
     
         9 . The method of  claim 1 , wherein said recovering comprises identifying said integrations by at least one method selected from the group consisting of PCR, Southern blot, restriction mapping, and DNA sequencing. 
     
     
         10 . The method of  claim 1 , wherein (N) x  is capable of cleaving an endogenous genomic sequence within (TS) x . 
     
     
         11 . The method of  claim 1 , wherein (N) x  is capable of cleaving an exogenous sequence within (TS) x  that is a recognition sequence for a homing endonuclease. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein (ES) x  further comprises a nucleic acid of interest (D) x  positioned 3′ of (HR1) x  and 5′ of (HR2) x . 
     
     
         15 . The method of  claim 14 , wherein (D) x  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, a nucleic acid sequence encoding a termination codon, and a nucleic acid sequence encoding an enzyme of a biosynthetic pathway. 
     
     
         16 . The method of  claim 1 , wherein (ES) x  is linear. 
     
     
         17 . The method of  claim 1 , wherein the host cell comprises one or more heterologous nucleotide sequences encoding one or more enzymes of a biosynthetic pathway. 
     
     
         18 . The method of  claim 17 , wherein the one or more heterologous nucleotide sequences encoding one or more enzymes of a biosynthetic pathway are genomically integrated. 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 15 , wherein (D) x  is a member of a library (L) x  comprising a plurality of nucleic acid molecules that encode variants of an enzyme of a biosynthetic pathway. 
     
     
         21 . The method of  claim 1 , wherein the host cell comprises one or more heterologous nucleotide sequences encoding one or more enzymes of a mevalonate (MEV) pathway for making isopentenyl pyrophosphate selected from the group consisting of: acetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase and mevalonate pyrophosphate decarboxylase. 
     
     
         22 . (canceled) 
     
     
         23 . The method of  claim 21 , wherein the host cell comprises a plurality of heterologous nucleic acids encoding all the enzymes of the MEV pathway. 
     
     
         24 . The method of  claim 21 , 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 a terpene synthase selected from the group consisting of: a monoterpene synthase, a diterpene synthase, a sesquiterpene synthase, a sesterterpene synthase, a triterpene synthase, a tetraterpene synthase, and a polyterpene synthase. 
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 1 , wherein (N) x  is provided as an expression vector comprising a nucleic acid sequence encoding (N) x . 
     
     
         27 . The method of  claim 1 , wherein (N) x  is transformed into the host cell as a purified protein. 
     
     
         28 . The method 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. 
     
     
         29 . The method of  claim 28 , 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. 
     
     
         30 . The method of  claim 29 , wherein the TypeIIS restriction endonuclease is selected from the group consisting of HO endonuclease and Fok I endonuclease. 
     
     
         31 . (canceled) 
     
     
         32 . The method of  claim 30 , 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. 
     
     
         33 . The method of  claim 30 , 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. 
     
     
         34 . The method of  claim 28 , 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. 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . The method 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. 
     
     
         38 . The method of  claim 1 , wherein the host cell is a yeast cell. 
     
     
         39 . (canceled) 
     
     
         40 . (canceled) 
     
     
         41 . A host cell generated by the method of  claim 1 . 
     
     
         42 - 99 . (canceled) 
     
     
         100 . A method for markerless integration of an exogenous nucleic acid into a target site of a yeast cell genome, the method comprising:
 (a) simultaneously contacting a yeast cell with:
 (i) an exogenous nucleic acid (ES) 1  comprising a first homology region (HR1) 1  and a second homology region (HR2) 1 , wherein (HR1) 1  and (HR2) 1  are capable of initiating host cell mediated homologous recombination at said target site (TS) 1 ; and 
 (ii) a nuclease (N) 1  capable of cleaving at (TS) 1 , whereupon said cleaving results in homologous recombination of (ES) 1  at (TS) 1 ; 
   and   (b) recovering a yeast cell having (ES) 1  integrated at (TS) 1 , wherein said recovering does not require integration of a selectable marker.   
     
     
         101 . (canceled) 
     
     
         102 . A method for simultaneously integrating a plurality of (n) exogenous nucleic acids into a plurality of (n) target sites of a host cell genome, wherein n is at least two, the method comprising:
 (a) simultaneously contacting a host cell with:
 (i) a plurality of libraries, wherein: 
 x is an integer that varies from 1 to n, and for each integer x, each library (L) x  comprises a plurality of exogenous nucleic acids, wherein a selected exogenous nucleic acid comprises, in a 5′ to 3′ orientation, a first homology region (HR1) x , any nucleic acid of interest selected from the 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 
 (ii) 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 ; 
   and   (b) recovering a host cell wherein each exogenous nucleic acid from each library (L) x  has integrated at each selected target sequence (TS) x ,   wherein x is any integer from 1 to n wherein n is at least 2.   
     
     
         103 . (canceled) 
     
     
         104 . (canceled)