Applications of engineered Streptococcus canis Cas9 variants on single-base PAM targets

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) (CRISPR-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 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 an engineered Streptococcus canis Cas9 (ScCas9) protein comprising SEQ ID No:
• 27, wherein said engineered ScCas9 protein further comprises:
• (i) the ScCas9 PID domain of SEQ ID No:
• 27 substituted with the PID domain from Streptococcus pyogenes Cas9 (SpCas9)-NG, and/or
• (ii) the substitution of amino acid positions 365-379 of SEQ ID NO:
• 27 with amino acids ADKKLRKRSGKLATE (SEQ ID No:
• 4), and/or
• (iii) a threonine-to-lysine substitution at position 1227 of SEQ ID No:
• 27, and,
• wherein components (a) and (b) are located on the same or different vectors of the system, whereby the guide RNA targets the target sequence and one or more of the proteins 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.