Modular universal plasmid design strategy for the assembly and editing of multiple dna constructs for multiple hosts

Abstract

Methods, compositions, and kits for high throughput DNA assembly reactions in vitro. Modular CRISPR DNA constructs comprising modular insert DNA parts flanked by cloning tag segments comprising pre-validated CRISPR protospacer/protospacer adjacent motif sequence combinations.

What is claimed is: 
     
         1 . A modular CRISPR DNA construct comprising a CRISPR multi-clonal site, said multi-clonal site comprising:
 a) a plurality of distinct cloning tags (cTAGs), said plurality of distinct cTAGs having been artificially introduced into the CRISPR DNA construct, wherein each cTAG comprises:
 i) a validated CRISPR landing site comprising a protospacer sequence operably linked to a protospacer adjacent motif (PAM); wherein the validated CRISPR landing site in at least three of the plurality of distinct cTAGs is different from any other CRISPR landing sites within the modular CRISPR DNA construct; and 
   b) a plurality of distinct DNA inserts;
 i) wherein each DNA insert is flanked by a pair of cTAGs; and 
   
       wherein the construct further comprises at least 5 cTAGs. 
     
     
         2 . The modular CRISPR DNA construct of  claim 1 , wherein said modular CRISPR DNA construct is circular. 
     
     
         3 . The modular CRISPR DNA construct of  claim 1 , wherein said modular CRISPR DNA construct is linear. 
     
     
         4 . The modular CRISPR DNA construct of  claim 1 , wherein said modular CRISPR DNA construct is integrated into an organism's genome. 
     
     
         5 . The modular CRISPR DNA construct of  claim 1 , wherein the validated CRISPR landing site is for a Cas9 endonuclease. 
     
     
         6 . The modular CRISPR DNA construct of  claim 1 , wherein the validated CRISPR landing site is for a Cpf1 endonuclease. 
     
     
         7 . The modular CRISPR DNA construct of  claim 1 , wherein at least one of said distinct cTAGs comprises a restriction endonuclease site at least 8 bases in length. 
     
     
         8 . A reaction mixture comprising:
 A) a modular CRISPR DNA construct comprising a plurality of distinct cloning tags (cTAGs), said plurality of distinct cTAGS having been artificially introduced into the CRISPR DNA construct, wherein each cTAG comprises:
 i) a validated CRISPR landing site(s), each comprising a protospacer sequence operably linked to a protospacer adjacent motif (PAM); and 
   B) a replacement DNA insert, wherein said replacement DNA insert is flanked by a first and second insert cTAG;
 i) wherein the first insert cTAG comprises the validated CRISPR landing site of one of the distinct cTAGs of the CRISPR DNA construct, and the second insert cTAG comprises the validated CRISPR landing site of another distinct cTAG of the CRISPR DNA construct; and 
   C) a first and second CRISPR complex targeting the first and second insert cTAGs, respectively, each CRISPR complex comprising:
 i) a CRISPR endonuclease, and 
 ii) a guide RNA capable of recruiting said CRISPR endonuclease to one of said targeted insert cTAGs. 
   
     
     
         9 . The reaction mixture of  claim 8 , wherein said modular CRISPR DNA construct is circular. 
     
     
         10 . The reaction mixture of  claim 8 , wherein said modular CRISPR DNA construct is linear. 
     
     
         11 . The reaction mixture of  claim 8 , wherein said modular CRISPR DNA construct is integrated into an organism's genome. 
     
     
         12 . The reaction mixture of  claim 8 , wherein the CRISPR endonuclease is Cas9. 
     
     
         13 . The reaction mixture of  claim 8 , wherein the CRISPR endonuclease is Cpf1. 
     
     
         14 . The reaction of  claim 8 , wherein at least one of said distinct cTAGs comprises a restriction endonuclease site at least 8 bases in length. 
     
     
         15 . The reaction of  claim 8 , wherein the guide RNA comprises a tracrRNA and a crRNA. 
     
     
         16 . A reaction mixture comprising:
 A) a plurality of different DNA inserts, wherein each DNA insert is flanked by a pair of artificially introduced cloning tags (cTAGs), each cTAG comprising:
 i) a validated CRISPR landing site(s), each comprising a protospacer sequence operably linked to a protospacer adjacent motif (PAM); and 
 ii) one or more CRISPR complexes targeting at least one of the cTAGs present in at least two of the plurality of different DNA inserts, each CRISPR complex comprising:
 a) a CRISPR endonuclease, and 
 b) a guide RNA capable of recruiting said CRISPR endonuclease to one of said targeted cTAGs. 
 
   
     
     
         17 . The reaction mixture of  claim 16 , wherein the CRISPR endonuclease is Cas9. 
     
     
         18 . The reaction mixture of  claim 16 , wherein the CRISPR endonuclease is Cpf1 . 
     
     
         19 . The reaction mixture of  claim 16 , wherein at least one of the cTAGs comprises a restriction endonuclease site at least 8 bases in length. 
     
     
         20 . The reaction mixture of  claim 16 , wherein the guide RNA comprises a tracrRNA and a crRNA.