Massachusetts Institute of Technology

Whitehead Institute for Biomedical Research

Noah Michael Jakimo

Joseph M Jacobson

Pranam Chatterjee

David Sabatini

Naama Kanarek

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.

Applications of engineeredCas9 variants on single-base PAM targets

The present invention relates to a cell based genomic Recorded Accumulative Memory (geRAM) system (also referred to herein as Genomically Encoded Memory (GEM)) for recoding data (i.e., changes in nucleic acid sequences in cellular DNA in response to physical and/or chemical signal(s)) from the cellular environment.

Cell-based genomic recorded accumulative memory

The invention provides compositions and methods for repeatable directed endonucleases (RDEs) and methods for repeatedly, and specifically cleaving DNA offset from the RDE's DNA recognition sequence on the target nucleic acid rather than within the DNA recognition sequence. Conservation of the recognition sequence of the target nucleic acid enables for re-localization of an RDE back to the DNA recognition sequence for further cleavage. The RDEs and methods of the invention are useful in applications including, but not limited to, recording data into a genome, timing the order of biochemical pathway events, efficient genome engineering and encoding lagged cellular death.

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Boston, MA, United States of America

Noah Michael Jakimo