Crispr editing to embed nucleic acid landing pads into genomes of live cells

Abstract

The present disclosure relates to compositions, methods, modules and automated integrated instrumentation for multiplex delivery of “landing pad” edits into the genomes of a population of live cells. The landing pads then may be leveraged to insert very large DNA sequences into the genomes of the population of live cells.

1 - 30 . (canceled) 
     
     
         31 . A method for insertion of a DNA payload into a cell genome, the method comprising:
 (a) introducing into the cell:
 (i) an editing vector, wherein the editing vector comprises one or more editing cassettes comprising:
 (A) at least one landing pad; and 
 (B) a guide RNA (gRNA) recognizing a target sequence in the cell genome and a repair template comprising 5′ and 3′ homology arms flanking the at least one landing pad; and 
 
 (ii) one or more vectors carrying one or more large DNA payloads; and 
 (iii) one or more vectors comprising a coding sequence for an integrase; and providing the cell conditions to allow the editing vector to edit the cell and the integrase to insert the one or more large DNA payloads into the at least one landing pad. 
   
     
     
         32 . The method of  claim 31 , wherein the one or more editing cassettes each comprises one or more primer binding sites. 
     
     
         33 . The method of  claim 31 , wherein the at least one landing pad comprises an integrase recognition sequence. 
     
     
         34 . The method of  claim 31 , wherein the at least one landing pad is more than 65 nucleotides in length. 
     
     
         35 . The method of  claim 31 , wherein the gRNA has more than 85% homology to the target sequence. 
     
     
         36 . The method of  claim 31 , wherein the gRNA has homology to the target sequence wherein 20 or more nucleotides overlap with and are complementary to the target sequence. 
     
     
         37 . The method of  claim 31 , wherein the editing vector comprises a coding sequence for a nucleic acid-guided nuclease. 
     
     
         38 . The method of  claim 31 , wherein a separate vector comprising a coding sequence for a nucleic acid-guided nuclease is introduced into the cell at the same time as the editing vector. 
     
     
         39 . The method of  claim 31 , wherein the integrase is selected from the group consisting of a bacteriophage lambda integrase, a HK2022 integrase, a phiC31 integrase, and a R4Tp901 integrase. 
     
     
         40 . The method of  claim 31 , wherein the coding sequence for the integrase is under the control of an inducible promoter. 
     
     
         41 . The method of  claim 31 , wherein the one or more vectors carrying one or more large DNA payloads and the one or more vectors comprising the coding sequence for the integrase are the same vector. 
     
     
         42 . The method of  claim 31 , wherein the one or more large DNA payloads are between 100 nucleotides and 100,000 nucleotides in length. 
     
     
         43 . The method of  claim 31 , wherein the cell is a microbial cell or a mammalian cell. 
     
     
         44 . The method of  claim 31 , wherein the editing vector further comprises a selectable marker. 
     
     
         45 . The method of  claim 31 , wherein the one or more vectors carrying one or more large DNA payloads further comprises a selectable marker that is different from the selectable marker in the editing vector. 
     
     
         46 . The method of  claim 31 , wherein the method further comprises screening for a desired phenotype or genotype in the cell, wherein the screening step comprises polymerase chain reaction (PCR) analysis with appropriate primer sets; a metabolic test; measurement of transcript level; a phenotypic assay; detection of a protein product using an antibody specific to the protein product; or DNA sequencing of the integrated large DNA payload. 
     
     
         47 . A system for insertion of DNA payloads into a cell genome comprising one or more vectors comprising:
 (a) at least one landing pad;   (b) a guide RNA (gRNA) recognizing a target sequence in the cell genome and a repair template comprising 5′ and 3′ homology arms flanking each landing pad;   (c) a coding sequence for a nucleic acid-guided nuclease;   (d) one or more large DNA payloads; and   (e) a coding sequence for an integrase.   
     
     
         48 . The system of  claim 47 , wherein the at least one landing pad encodes an integrase recognition sequence. 
     
     
         49 . The system of  claim 47 , wherein the gRNA has more than 95% homology to the target sequence. 
     
     
         50 . The system of  claim 47 , wherein the one or more vectors further comprises at least one selectable marker.