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ENGINEERING AND OPTIMIZATION OF IMPROVED SYSTEMS, METHODS AND ENZYME COMPOSITIONS FOR SEQUENCE MANIPULATION

  • US 20140335620A1
  • Filed: 06/02/2014
  • Published: 11/13/2014
  • Est. Priority Date: 12/12/2012
  • Status: Active Grant
First Claim
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1. A method of altering expression of at least one gene product comprising introducing into a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding the gene product an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system comprising one or more vectors comprising:

  • a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, andb) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Staphylococcus aureus Cas9 protein,wherein the CRISPR-Cas system further comprises one or more nuclear localization signal(s) (NLS(s)), and components (a) and (b) are located on same or different vectors of the system,whereby the guide RNA targets the target sequence and the Cas9 protein cleaves the DNA molecule;

    the method further comprising inserting DNA into a cleaved strand of the DNA molecule;

    whereby expression of the at least one gene product is altered; and

    , wherein the Cas9 protein and the guide RNA do not naturally occur together.

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