Back to results
US 2022/0177943 A1Published application
Recombinant type i crispr-cas system and uses thereof for screening for variant cells
AssigneeNorth Carolina State University
InventorsRodolphe Barrangou, Claudio Hidalgo Cantabrana
Life of this patent
Estimating the term…
Abstract
This invention relates to recombinant Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) arrays and recombinant nucleic acid constructs encoding Type I-E CASCADE complexes, plasmids, retroviruses and bacteriophage comprising the same, and methods of use thereof for screening for variant cells of an organism.
Claims24
A method of screening for a variant cell of an organism, the method comprising
- (a) introducing into a population of cells from (or of) an organism
- (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 cells from the organism, wherein the target sequence is located immediately adjacent (3′) to a protospacer adjacent motif (PAM);
- (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 (e.g., 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.
11 more elements
A method of screening for variant bacterial cells comprising an endogenous Type I-E CRISPR-Cas system, the method comprising
- (a) introducing into a population of bacterial cells 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 bacterial cells,
- wherein the target sequence is located immediately adjacent (3′) to a protospacer adjacent motif (PAM);
- and
- wherein the recombinant nucleic acid construct comprising a CRISPR array comprises a polynucleotide encoding a polypeptide conferring resistance to a selection marker;
- and
- (b) selecting from the population of bacterial cells produced in (a) one or more bacterial cells comprising resistance to the selection marker(s),
- wherein the two or more repeat sequences comprise any one of the nucleotide sequences of SEQ ID NOs:1 to 68, in any combination, thereby selecting from the population of bacterial cells one or more variant bacterial cells that do not comprise the target sequence and are not killed.
5 more elements
(canceled)
6-11. (canceled)
(canceled)
15-21. (canceled)
Dependencies are inferred from the claim text.