Dr. G. Brett Robb
New England Biolabs, Ipswich, MA
Friday, February 28, 2020 - 3:00pm
CCBR Red Room
Departmental Seminar
Abstract:
Cas9 nucleases are abundant in microbes. To explore this largely uncharacterized diversity
of orthologs for biotechnology and genome editing applications, we established a phylogeny-guided
bioinformatic approach to select Cas9s from sequenced organisms and sampled identifiable CRISPR
systems in metagenome sequence to select candidates. We developed and applied biochemical
screens for the rapid identification and characterization of the protospacer adjacent motif (PAM) and
guide RNA (gRNA) requirements of new Cas9 proteins. Using this approach, we characterized over
80 Cas9 orthologs with more than 50 distinct PAM sequence requirements. The set contained
nucleases that generate staggered-end breaks and nucleases that require longer spacers to function
robustly in vitro. In addition, it contained Cas9s that are active at narrow and broad temperature
ranges. To establish broad applicability for use as genome editing tools, cellular activity was
examined in both plant and human cells. Of those examined, several were capable of generating
targeted chromosomal mutations and some exhibited robust mutational activity approaching that of
Streptococcus pyogenes Cas9. Our results demonstrate that the diversity of Cas9 orthologs provides
a rich source of PAM recognition and other potentially desirable properties that may be mined to
expand the biotechnology toolbox with new programmable nucleases.
Host:
Dr. Julie Claycomb
Department of Molecular Genetics
Poster: