Genome editing in archaea

Abstract

Methods of RNA-guided DNA endonuclease-mediated genome editing in Bacteria and Archaea are provided.

1 . A genetic engineering cassette comprising a first inducible promoter operably linked to one or more sgRNA sequences, a homologous recombination editing template comprising two homology arms with a deletion portion, a substitution portion comprising a single nucleotide polymorphism, or an insertion portion between the two homology arms, wherein the two homology arms have homology to a an archaeon nucleic acid molecule. 
     
     
         2 . The genetic engineering cassette of  claim 1 , wherein the deletion portion, the substitution portion, or the insertion portion between the two homology arms further comprises a restriction endonuclease site. 
     
     
         3 . The genetic engineering cassette of  claim 1 , wherein the first inducible promoter is a methanogen promoter, and wherein the archaeon nucleic acid molecule is a methanogen nucleic acid molecule. 
     
     
         4 . A vector comprising the genetic engineering cassette of  claim 1 , and further comprising a nucleic acid molecule encoding Type II Cas9 protein protein operably linked to a second inducible promoter. 
     
     
         5 . The vector of  claim 4 , wherein the second inducible promoter is a methanogen promoter. 
     
     
         6 . The vector of  claim 4 , further comprising a methanogen origin of replication. 
     
     
         7 . The vector of  claim 4 , further comprising a selection marker, a counter-selection marker, or both a selection marker and a counter-selection marker. 
     
     
         8 .- 15 . (canceled) 
     
     
         16 . The genetic engineering cassette of  claim 1 , wherein the homologous recombination editing template comprises two homology arms with a deletion portion. 
     
     
         17 . The genetic engineering cassette of  claim 1 , wherein the homologous recombination editing template comprises two homology arms with an insertion portion. 
     
     
         18 . The genetic engineering cassette of  claim 1 , wherein the homologous recombination editing template comprises two homology arms a substitution portion comprising a single nucleotide polymorphism. 
     
     
         19 . The genetic engineering cassette of  claim 1 , wherein the archaeon is a methanogen. 
     
     
         20 . The genetic engineering cassette of  claim 19 , wherein the archaeon is  Methanosarcina.    
     
     
         21 . The genetic engineering cassette of  claim 1 , wherein two or more sgRNA sequences are present in the genetic engineering cassette. 
     
     
         22 . A vector comprising the genetic engineering cassette of  claim 1 . 
     
     
         23 . A host cell comprising the vector of  claim 4 . 
     
     
         23 . A host cell comprising the vector of  claim 22 .