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ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH FUNCTIONAL DOMAINS

  • US 20140186958A1
  • Filed: 12/12/2013
  • Published: 07/03/2014
  • Est. Priority Date: 12/12/2012
  • Status: Active Grant
First Claim
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1. A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprisingI. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises(a) a guide sequence capable of hybridizing to a target sequence in a cell,(b) a tracr mate sequence, and(c) a tracr sequence, andII. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences,wherein (a), (b) and (c) are arranged in a 5′

  • to 3′

    orientation,wherein components I and II are located on the same or different vectors of the system,wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,wherein the CRISPR enzyme comprises two or more mutations, such that the enzyme has altered nuclease compared with the wild type enzyme, andwherein the enzyme-coding sequence further encodes one or more heterologous functional domains.

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