Streptococcus Canis Cas9 as a Genome Engineering Platform with Novel PAM Specificity

Abstract

A Streptococcus canis Cas9 (ScCas9) ortholog and its engineered variants, possessing novel PAM specificity, is an addition to the family of CRISPR-Cas9 systems. ScCas9 endonuclease is used in complex with guide RNA, consisting of identical non-target-specific sequence to that of the guide RNA SpCas9, for specific recognition and activity on a DNA target immediately upstream of either an “NNGT” or “NNNGT” PAM sequence. A novel DNA-interacting loop domain within ScCas9, and other Cas9 orthologs, such as those from Streptococcus gordonii and Streptococcus angionosis facilitates a divergent PAM sequence from the “NGG” PAM of SpCas9.

1 . An isolated  Streptococcus canis  Cas9 (ScCas9) protein or transgene expression thereof. 
     
     
         2 . The protein of  claim 1 , comprising at least one of the mutations K857A, K1012A, R1069A, N507A, R671A, Q705A, Q935A, N702A, M704A, Q705A, and H708A. 
     
     
         3 . A CRISPR-associated DNA endonuclease with PAM interacting domain (PID) amino acid sequences that are at least 80% identical to that of the isolated  Streptococcus canis  Cas9 (ScCas9) protein. 
     
     
         4 . The CRISPR-associated DNA endonuclease of  claim 3 , having a PAM specificity of “NNGT” or “NNNGT”. 
     
     
         5 . The CRISPR-associated DNA endonuclease of  claim 3 , comprising a 10 amino acid loop insertion of “IKHRKRTTKL” [SEQ ID No. 4]. 
     
     
         6 . The CRISPR-associated DNA endonuclease of  claim 3 , comprising a 2 amino acid insertion of “KQ” two positions upstream of the first critical arginine (R) residue for PAM binding. 
     
     
         7 . An isolated, engineered Streptococcus pyogenes Cas9 (SpCas9),  Streptococcus thermophilus  Cas9, or Cpf1 protein with a PID as either the PID amino acid composition of the isolated  Streptococcus canis  Cas9 (ScCas9) protein or of CRISPR-associated DNA endonucleases with PAM interacting domain (PID) amino acid sequences that are at least 80% identical to that of the isolated  Streptococcus canis  Cas9 (ScCas9) protein. 
     
     
         8 . The protein of  claim 7 , comprising at least one of the amino acid insertions “IKHRKRTTKL” [SEQ ID No. 4] or a 2 amino acid insertion of “KQ” two positions upstream of the first critical arginine (R) residue for PAM binding 
     
     
         9 . A DNA-interacting loop domain within ScCas9, or a Cas9 ortholog, that facilitates a divergent PAM sequence from the “NGG” PAM of SpCas9. 
     
     
         10 . The DNA-interacting loop domain of  claim 9 , wherein the Cas9 orthologs comprise  Streptococcus gordonii  or  Streptococcus angionosis.    
     
     
         11 . A method for altering expression of at least one gene product by employing  Streptococcus canis  Cas9 (ScCas9) endonucleases in complex with guide RNA, consisting of identical non-target-specific sequence to that of the guide RNA SpCas9, for specific recognition and activity on a DNA target immediately upstream of either an “NNGT” or “NNNGT” PAM sequence. 
     
     
         12 . 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 CRISPR-Cas system comprising one or more vectors comprising (a) a regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR system guide RNA that hybridizes with the target sequence, and (b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding one or more of the proteins in  claims 1 - 10 , wherein components (a) and (b) are located on same or different vectors of the system, whereby the guide RNA targets the target sequence and one or more of the proteins in  claims 1 - 10  cleave the DNA molecule, whereby expression of the at least one gene product is altered; and, wherein the proteins and the guide RNA do not naturally occur together.