Engineering of systems, methods and optimized guide compositions with new architectures for sequence manipulation

Abstract

The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are structural information on the Cas protein of the CRISPR-Cas system, use of this information in generating modified components of the CRISPR complex, vectors and vector systems which encode one or more components or modified components of a CRISPR complex, as well as methods for the design and use of such vectors and components. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system. In particular the present invention comprehends optimized functional CRISPR-Cas enzyme systems. In particular the present invention comprehends engineered new guide architectures to be used in optimized CRISPR-Cas enzyme systems.

1 - 93 . (canceled) 
     
     
         94 . A non-naturally occurring or engineered CRISPR-Cas system guide RNA, or a chimeric single guide RNA molecule (sgRNA), capable of effecting the manipulation of a target nucleic acid within a prokaryotic or eukaryotic cell when in complex within the cell with a CRISPR enzyme comprising a CRISPR enzyme, or a non-naturally occurring or engineered composition comprising a CRISPR-Cas system comprising said guide RNA or sgRNA, the guide RNA or sgRNA comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell wherein architecture of the guide RNA or sgRNA is modified. 
     
     
         95 . The guide RNA or sgRNA or composition of  claim 94 , wherein the modification of architecture is replacing a poly U tract (22-25 with respect to the position numbering of +85 sgRNA architecture) with poly C. 
     
     
         96 . The guide RNA or sgRNA or composition of  claim 94 , wherein the modification of architecture is replacing a poly A tract (46-49 with respect to the position numbering of +85 sgRNA architecture) with poly G. 
     
     
         97 . The guide RNA or sgRNA or composition of  claim 94 , wherein the modification of architecture is replacing the repeat:anti-repeat duplex A28 and A41, A42 with C28, U28, or G28, or U41 and U42 with respect to the position numbering of +85 sgRNA architecture. 
     
     
         98 . The guide RNA or sgRNA or composition of  claim 94 , wherein the modification of architecture is replacing bases in stemloop 2 with respect to the position numbering of +85 sgRNA architecture. 
     
     
         99 . The guide RNA or sgRNA or composition of  claim 98 , wherein “acuu” and “aagu” bases in stemloop2 are replaced with complimentary GC-rich regions of 4 nucleotides. 
     
     
         100 . The guide RNA or sgRNA or composition of  claim 99 , wherein the complimentary GC-rich regions of 4 nucleotides are “cgcc” and “gcgg”. 
     
     
         101 . The guide RNA or sgRNA or composition of  claim 94 , wherein the modification is selected from the following, corresponding with +85 sgRNA for SpCas9: mutation of a proximal duplex; truncation of a proximal duplex; removing a bulge duplex: removal of a stem loop 1; truncation of a linker; replacement or lengthening of the stemloop 2; mutation or truncation of stemloop 3; a G43C mutation; a U44G mutation; a U44C mutation; a U50C mutation: a U59A mutation; a G89U mutation; or a U90A mutation. 
     
     
         102 . The guide RNA or sgRNA or composition of  claim 94 , wherein at least one loop of the sgRNA is further modified by the insertion of distinct RNA sequence(s), that binds to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains. 
     
     
         103 . The guide RNA or sgRNA or composition of  claim 102 , wherein the inserted RNA sequence is an aptamer. 
     
     
         104 . A non-naturally occurring or engineered composition comprising the guide RNA or sgRNA of  claim 94  and a CRISPR enzyme or a Cas9. 
     
     
         105 . The non-naturally occurring or engineered composition of  claim 104 , wherein the CRISPR enzyme is SaCas9 or SpCas9. 
     
     
         106 . The composition of  claim 104 , wherein the CRISPR enzyme is associated with one or more functional domains. 
     
     
         107 . The composition of  claim 106 , wherein the one or more functional domains comprises a transcriptional activation domain, a transcriptional repressor domain or a nuclease domain. 
     
     
         108 . The guide RNA or sgRNA or composition of  claim 94 , wherein the cell is a eukaryotic cell, or a mammalian cell, or a human cell or a mouse cell. 
     
     
         109 . A polynucleotide encoding the guide RNA or sgRNA of  claim 94 . 
     
     
         110 . A vector comprising the polynucleotide of  claim 109  operably linked to a suitable promoter. 
     
     
         111 . The vector of  claim 110 , which further comprises a polynucleotide encoding a CRISPR enzyme or a Cas9 or SaCas9 or SpCas9. 
     
     
         112 . A vector system, comprising one vector according to  claim 110  and a second vector comprising a polynucleotide encoding a CRISPR enzyme, or a Cas9 or SaCas9 or SpCas9. 
     
     
         113 . A transformant organism or model or progeny thereof transformed with any one of the vectors of any one of  claims 111  to  112  to thereby express the guide RNA or sgRNA. 
     
     
         114 . The transformant organism or model or progeny thereof of  claim 113 , which further expresses the CRISPR enzyme. 
     
     
         115 . A method of modifying a genomic locus of interest to alter gene expression in a cell by introducing into the cell the guide RNA or sgRNA or composition, polynucleotide or vectors of any one of  claims 94 - 103  and a CRISPR enzyme, optionally a Cas9 as defined in any one of  claims 104 - 107 .