US 12,264,313 B2
Recombinant type I CRISPR-Cas system and uses thereof for genome modification and alteration of expression
Rodolphe Barrangou, Raleigh, NC (US); and Claudio Hidalgo Cantabrana, Cary, NC (US)
Assigned to North Carolina State University, Raleigh, NC (US)
Appl. No. 17/280,436
Filed by NORTH CAROLINA STATE UNIVERSITY, Raleigh, NC (US)
PCT Filed Sep. 25, 2019, PCT No. PCT/US2019/052864
§ 371(c)(1), (2) Date Mar. 26, 2021,
PCT Pub. No. WO2020/072250, PCT Pub. Date Apr. 9, 2020.
Claims priority of provisional application 62/739,669, filed on Oct. 1, 2018.
Prior Publication US 2022/0056433 A1, Feb. 24, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/10 (2006.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01)
CPC C12N 15/102 (2013.01) [C12N 9/22 (2013.01); C12N 15/111 (2013.01); C12N 2310/20 (2017.05)] 30 Claims
 
1. A method of modifying the genome of a Lactobacillus spp. or Bifidobacterium spp. bacterium, comprising introducing into the Lactobacillus spp. or Bifidobacterium spp. bacterium
(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 bacterium that 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 target sequence;
(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 encoded by the nucleotide sequence of SEQ ID NO:86;
(c) a Cas3 polypeptide or a polynucleotide encoding a Cas3 polypeptide; and
(d) a repair template, thereby modifying the genome of the Lactobacillus spp. or Bifidobacterium spp. bacterium.