US2016281072A1PendingUtilityA1

Crispr-cas systems and methods for altering expression of gene products

66
Assignee: BROAD INST INCPriority: Dec 12, 2012Filed: May 20, 2016Published: Sep 29, 2016
Est. expiryDec 12, 2032(~6.4 yrs left)· nominal 20-yr term from priority
Inventors:Feng Zhang
C12N 9/52C12N 9/22C12N 15/63C12N 15/907C12N 2800/30C12N 15/1082C12N 2310/10C12N 15/102A61K 38/43C12N 2800/80C12N 15/111C12N 15/85C12N 9/96C12N 15/902C12N 9/14C12Q 1/68A61K 38/46A61K 38/47C12N 2800/90C12N 2310/20C12N 15/00C12N 9/226
66
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Claims

Abstract

The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A non-naturally occurring or engineered composition comprising a delivery system operably configured to deliver an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) complex to a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM), the system comprising:
 I. one or more regulatory elements operably linked to one or more CRISPR-Cas complex polynucleotide sequences comprising   (a) a guide sequence capable of hybridizing to the target sequence in the eukaryotic cell,   (b) a tracr mate sequence, and   (c) a tracr sequence, and   II. a second regulatory element operably linked to an enzyme-coding sequence encoding a Type II CRISPR enzyme,   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, 
 the guide sequence comprises more than about 10 nucleotides in length, and 
 the tracr sequence comprises more than about 30 nucleotides in length, and 
 when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence and PAM recognition, and 
 the tracr sequence exhibits at least 50% sequence complementarity along the length of the tracr mate, and 
 the CRISPR enzyme and the guide RNA do not naturally occur together. 
   
     
     
         2 . The composition according to  claim 1 , wherein the CRISPR-Cas complex polynucleotide sequences comprises a chimeric guide RNA comprising the guide sequence, the tracr sequence, and a tracr mate sequence. 
     
     
         3 . The composition according to  claim 1 , wherein the expression of two or more gene products is altered. 
     
     
         4 . The composition according to  claim 1 , wherein the eukaryotic cell is a mammalian cell. 
     
     
         5 . The composition according to  claim 1  wherein the eukaryotic cell is a human cell. 
     
     
         6 . The composition according to  claim 1 , wherein the expression of one or more gene products is decreased. 
     
     
         7 . The composition according to  claim 1 , wherein the tracr sequence comprises more than about 40 nucleotides in length. 
     
     
         8 . The composition according to  claim 1 , wherein the tracr sequence comprises more than about 50 nucleotides in length 
     
     
         9 . The composition according to  claim 1 , wherein the guide sequence comprises more than about 75 nucleotides in length. 
     
     
         10 . The composition according to  claim 8 , wherein the guide sequence comprises more than about 75 nucleotides in length. 
     
     
         11 . The composition according to  claim 1 , wherein the CRISPR enzyme comprises one or more mutations. 
     
     
         12 . The composition according to  claim 11 , wherein the one or more mutations comprise one or more mutations of D10 with reference to  Streptococcus pyogenes  Cas9 (“SpCas9”), H840 SpCas9, N854 SpCas9 or N863 SpCas9. 
     
     
         13 . The composition according to  claim 12  wherein the one or more mutations comprise D10A SpCas9, H840A SpCas9, N854A SpCas9 or N863A SpCas9. 
     
     
         14 . The composition according to  claim 11  wherein the one or more mutations comprise two mutations. 
     
     
         15 . The composition according to  claim 14  wherein the mutations comprise D10A SpCas9 and H840A SpCas9, or corresponding residues of other CRISPR enzymes. 
     
     
         16 . The composition according to  claim 11 , wherein the one or more mutations is in a catalytically active domain of the CRISPR enzyme comprising RuvCI, RuvCII or RuvCIII. 
     
     
         17 . The composition according to  claim 1 , wherein the CRISPR complex includes one or more nuclear localization signals (NLS(s)). 
     
     
         18 . The composition according to  claim 1 , wherein the CRISPR enzyme comprises a fusion of a Type II Cas9 protein and one or more effector domains. 
     
     
         19 . The composition according to  claim 18 , wherein the one or more effector domains comprises one or more NLS(s). 
     
     
         20 . The composition according to  claim 19 , wherein the one or more effector domains comprises a transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA demethylase domain, histone acetylase domain, histone deacetylases domain, nuclease domain, repressor domain, activator domain, nuclear-localization signal domain, transcription-protein recruiting domain, cellular uptake activity associated domain, nucleic acid binding domain or antibody presentation domain. 
     
     
         21 . The composition according to  claim 1 , which is a multiplexed composition comprising multiple guide sequences capable of hybridizing to multiple target sequences. 
     
     
         22 . The composition according to  claim 2 , which is a multiplexed composition comprising multiple guide sequences capable of hybridizing to multiple target sequences. 
     
     
         23 . The composition according to  claim 1 , wherein the delivery system comprises a vector system comprising one or more vectors, and wherein components I and II are located on the same or different vectors of the system; or, wherein the delivery system comprises a nanoparticle, liposome, exosome, yeast system, microvesicle, or gene gun. 
     
     
         24 . The composition according to  claim 23 , wherein the delivery system comprises a vector system comprising one or more vectors and components I and II are located on the same vector. 
     
     
         25 . The composition according to  claim 23 , wherein the delivery system comprises a vector system comprising one or more vectors and the one or more vectors comprise one or more viral vectors, and the one or more viral vectors comprise one or more retrovirus, lentivirus, adenovirus, adeno-associated virus or herpes simplex virus vectors. 
     
     
         26 . A eukaryotic cell comprising the composition or CRISPR complex of  claim 1 . 
     
     
         27 . A cell culture from the cell of  claim 26 . 
     
     
         28 . A eukaryotic cell translation product from the cell of  claim 26 . 
     
     
         29 . A eukaryotic cell translation product from a cell of the culture of  claim 27 . 
     
     
         30 . The composition of  claim 1  wherein the PAM has a nucleotide sequence comprising NNAGAAW.

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