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DELIVERY, ENGINEERING AND OPTIMIZATION OF TANDEM GUIDE SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION

  • US 20160186213A1
  • Filed: 12/17/2015
  • Published: 06/30/2016
  • Est. Priority Date: 06/17/2013
  • Status: Active Grant
First Claim
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1. A method of modifying a genomic locus of interest in a cell comprisingdelivering a non-naturally occurring or engineered composition comprising a tandem guideRNA construct comprising:

  • I. a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises;

    (a) a first guide sequence capable of hybridizing to the first target sequence,(b) a first tracr mate sequence,(c) a first tracr sequence,(d) a second guide sequence capable of hybridizing to the second target sequence,(e) a second tracr mate sequence, and(f) a second tracr sequence, andII. a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences (NLS)s,wherein the polynucleotide sequence comprises a linker sequence between the first tracr sequence and the second guide sequence, whereby the first guide sequence and the second guide sequence are in tandem,wherein when transcribed, the first and the second tracr mate sequence hybridize to the first and second tracr sequence respectively and the first and the second guide sequence directs sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively,wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridized to the first target sequence, and (2) the first tracr mate sequence that is hybridized to the first tracr sequence,wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridized to the second target sequence, and (2) the second tracr mate sequence that is hybridized to the second tracr sequence,wherein the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA, andwherein the first guide sequence directs cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directs cleavage of other strand near the second target sequence inducing a double strand break, thereby modifying the genomic locus.

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