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CRISPR-Cas component systems, methods and compositions for sequence manipulation

  • US 9,822,372 B2
  • Filed: 01/07/2016
  • Issued: 11/21/2017
  • Est. Priority Date: 12/12/2012
  • Status: Active Grant
First Claim
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1. A method of selecting one or more prokaryotic cell(s) by introducing one or more mutations in one or more prokaryotic cell(s), the method comprising:

  • introducing one or more vectors into the prokaryotic cell(s);

    wherein the one or more vectors drive expression of;

    a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template for recombination into a targeted chromosomal locus comprising a target polynucleotide;

    and all of the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and a tracr sequence, and the editing template are produced in the prokaryotic cell(s), whereby a CRISPR complex forms;

    the CRISPR complex comprising the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to the tracr sequence;

    wherein the CRISPR complex binds to the target polynucleotide;

    wherein the editing template is introduced into the target polynucleotide through recombination, wherein the editing template comprises one or more mutations of the target polynucleotide that alter either a protospacer-adjacent motif (PAM) sequence or a protospacer sequence, and that abolishes CRISPR enzyme cleavage of the target polynucleotide;

    wherein in those cells that have not had the editing template introduced there is cleavage of the target polynucleotide by the CRISPR complex and cell death; and

    selecting one or more prokaryotic cell(s) in which the one or more mutations have been introduced.

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