US 12,065,641 B2
NgAgo-based gene-editing method and the uses thereof
Kevin V. Solomon, Lafayette, IN (US); and Kok Zhi Lee, West Lafayette, IN (US)
Assigned to Purdue Research Foundation, West Lafayette, IN (US)
Filed by Purdue Research Foundation, West Lafayette, IN (US)
Filed on Mar. 15, 2019, as Appl. No. 16/354,415.
Claims priority of provisional application 62/643,814, filed on Mar. 16, 2018.
Prior Publication US 2019/0284547 A1, Sep. 19, 2019
Int. Cl. C12N 9/22 (2006.01); C12N 15/10 (2006.01); C12N 15/11 (2006.01)
CPC C12N 15/102 (2013.01) [C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 2800/80 (2013.01)] 17 Claims
 
1. A method for modifying a chromosomal or an extrachromosomal genetic material of a prokaryotic host cell, comprising:
providing a gene editing system comprising:
a donor DNA;
a plurality of designed DNA sequences each having about 15-30 nucleotides and 5′ phosphorylation, wherein said DNA sequences are complementary to a target nucleic acid sequence in a prokaryotic host cell;
a lambda red recombinase or other recombinase system, wherein the lambda red recombinase or other recombinase system is driven by an inducible promoter that is sufficient to induce homologous recombination; and
an NgAgo enzyme or a mutant thereof, a DNA expression cassette encoding the NgAgo enzyme or mutant thereof, or an mRNA encoding the NgAgo enzyme or mutant thereof, wherein said NgAgo enzyme or a mutant thereof is capable of interacting with said designed DNA sequences in the prokaryotic host cell and is capable of nicking the target nucleic acid sequence in the cell through the guidance of said designed DNA sequences;
introducing the gene editing system into the prokaryotic host cell;
wherein said NgAgo or a mutant thereof forms complexes with the designed DNA sequences, directing the complexes to bind to and nick the target nucleic acid sequence; and
wherein the plurality of designed DNA sequences are targeted to different regions of said target nucleic acid sequence in a site-specific manner.