Massachusetts Institute of Technology

Pranam Chatterjee

Joseph M Jacobson

Noah Michael Jakimo

EngineeredCas9 (ScCas9) variants include an ScCas9 protein with its PID being the PID amino acid composition ofCas9 (SpCas9-NG, an ScCas9 protein having a threonine-to-lysine substitution mutation at position 1227 in its amino acid sequence (Sc+), and an ScCas9 protein having a threonine-to-lysine substitution mutation at position 1227 and a substitution of residues ADKKLRKRSGKLATE [SEQ ID No. 4] in position 365-379 in the ScCas9 open reading frame (Sc++). Also included are CRISPR-associated DNA endonucleases with a PAM specificity of 5'-NG-3′ or 5′-NNG-3′ and a method of altering expression of a gene product by utilizing the engineered ScCas9 variants.

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

ACas9 (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 fromandfacilitates a divergent PAM sequence from the “NGG” PAM of SpCas9.

Cas9 as a genome engineering platform with novel PAM specificity

EngineeredCas9 (ScCas9) variants include an ScCas9 protein with its PID being the PID amino acid composition ofCas9 (SpCas9)-NG, an ScCas9 protein having a threonine-to-lysine substitution mutation at position 1227 in its amino acid sequence (Sc+), and an ScCas9 protein having a threonine-to-lysine substitution mutation at position 1227 and a substitution of residues ADKKLRKRSGKLATE [SEQ ID No. 4] in position 365-379 in the ScCas9 open reading frame (Sc++). Also included are CRISPR-associated DNA endonucleases with a PAM specificity of 5'-NG-3&#x2032; or 5&#x2032;-NNG-3&#x2032; and a method of altering expression of a gene product by utilizing the engineered ScCas9 variants.

Growing community of inventors

Cambridge, MA, United States of America

Pranam Chatterjee