US 12,264,330 B2
Recombinant type I CRISPR-Cas system and uses thereof for killing target cells
Rodolphe Barrangou, Raleigh, NC (US); and Claudio Hidalgo Cantabrana, Cary, NC (US)
Assigned to North Carolina State University, Raleigh, NC (US)
Appl. No. 17/280,493
Filed by NORTH CAROLINA STATE UNIVERSITY, Raleigh, NC (US)
PCT Filed Sep. 25, 2019, PCT No. PCT/US2019/052883
§ 371(c)(1), (2) Date Mar. 26, 2021,
PCT Pub. No. WO2020/072254, PCT Pub. Date Apr. 9, 2020.
Claims priority of provisional application 62/739,694, filed on Oct. 1, 2018.
Prior Publication US 2022/0170048 A1, Jun. 2, 2022
Int. Cl. C12N 15/90 (2006.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01)
CPC C12N 15/902 (2013.01) [C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 2310/20 (2017.05)] 28 Claims
 
1. A method of killing one or more cells in a population of Lactobacillus spp. or Bifidobacterium spp. bacterial cells, the method comprising introducing into the one or more cells of the population of Lactobacillus spp. or Bifidobacterium spp. bacterial cells:
(a) a recombinant nucleic acid construct comprising a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) array comprising two or more repeat sequences and one or more spacer nucleotide sequence(s),
wherein each of the one or more spacer sequences comprises a 3′ end and a 5′ end and is linked at its 5′ end and at its 3′ end to a repeat sequence, and each of the one or more spacer sequences is complementary to a target sequence (protospacer) in the genome of the Lactobacillus spp. or Bifidobacterium spp. bacterial cells of the population,
wherein the target sequence is a genomic sequence that is conserved among the one or more cells within the population of Lactobacillus spp. or Bifidobacterium spp. bacterial cells and the target sequence is located immediately adjacent (3′) to a protospacer adjacent motif (PAM), wherein the PAM comprises a nucleotide sequence of 5′-NAA-3′, 5′-AAA-3′ or 5′-AA-3′ that is immediately adjacent to and 5′ of the protospacer;
(b) a recombinant nucleic acid construct encoding a Type I-E CRISPR associated complex for antiviral defense complex (Cascade complex) comprising:
a Cse1 polypeptide encoded by the nucleotide sequence of SEQ ID NO: 82,
a Cse2 polypeptide encoded by the nucleotide sequence of SEQ ID NO: 83,
a Cas7 polypeptide encoded by the nucleotide sequence of SEQ ID NO: 84,
a Cas5 polypeptide encoded by the nucleotide sequence of SEQ ID NO: 85, and
a Cas6 polypeptide encoded by the nucleotide sequence of SEQ ID NO: 86; and
(c) a Cas3 polypeptide or a polynucleotide encoding a Cas3 polypeptide, thereby killing one or more cells in the population of Lactobacillus spp. or Bifidobacterium spp. bacterial cells that comprise the target sequence in their genome.