	US 12,203,123 B2
	Recombinant type I CRISPR-Cas system and uses thereof for screening for variant cells
Rodolphe Barrangou, Raleigh, NC (US); and Claudio Hidalgo Cantabrana, Cary, NC (US)
Assigned to North Carolina State University, Raleigh, NC (US)
Appl. No. 17/280,454
Filed by NORTH CAROLINA STATE UNIVERSITY, Raleigh, NC (US)
PCT Filed Sep. 25, 2019, PCT No. PCT/US2019/052878 § 371(c)(1), (2) Date Mar. 26, 2021, PCT Pub. No. WO2020/072253, PCT Pub. Date Apr. 9, 2020.
Claims priority of provisional application 62/739,686, filed on Oct. 1, 2018.
Prior Publication US 2022/0177943 A1, Jun. 9, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/04 (2006.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01); C12N 15/74 (2006.01); C12N 15/90 (2006.01)

CPC C12Q 1/04 (2013.01) [C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 15/746 (2013.01); C12N 15/902 (2013.01); C12N 2310/20 (2017.05)]

20 Claims

1. A method of screening for a variant cell of a population of Lactobacillus or Bifidobacterium bacterial cells, the method comprising

(a) introducing into the population of Lactobacillus or Bifidobacterium bacterial cells

(i) 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 a target DNA in the population of Lactobacillus or Bifidobacterium bacterial cells, wherein 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 target sequence;

(ii) 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

(iii) a Cas3 polypeptide, optionally as a ribonucleoprotein particle (RNP) or a

polynucleotide encoding a Cas3 polypeptide;

wherein the recombinant nucleic acid construct comprising a CRISPR array, the recombinant nucleic acid construct encoding a Cascade complex, and when present the polynucleotide encoding a Cas3 polypeptide, each comprise a polynucleotide encoding a polypeptide conferring resistance to a selection marker; and

(b) selecting from the population of cells produced in (a) one or more cells comprising resistance to the selection marker(s), thereby selecting from the population of cells one or more variant cells that are not killed and do not comprise the target sequence.