Molecular Basis and Genome Editing Applications of a Compact Eubacterium ventriosum CRISPR-Cas9 System

CRISPR-Cas9 systems have been widely
harnessed for diverse
genome
editing applications because of their ease of use and high efficiency.
However, the large molecular sizes and strict PAM requirements of
commonly used CRISPR-Cas9 systems restrict their broad applications
in therapeutics. Here, we report the molecular basis and genome editing
applications of a novel compact type II-A Eubacterium
ventriosum CRISPR-Cas9 system (EvCas9) with 1107 residues
and distinct 5′-NNGDGN-3′ (where D represents A, T,
or G) PAM specificity. We determine the cryo-EM structure of EvCas9
in a complex with an sgRNA and a target DNA, revealing the detailed
PAM recognition and sgRNA and target DNA association mechanisms. Additionally,
we demonstrate the robust genome editing capacity of EvCas9 in bacteria
and human cells with superior fidelity compared to SaCas9 and SpCas9,
and we engineer it to be efficient base editors by fusing a cytidine
or adenosine deaminase. Collectively, our results facilitate further
understanding of CRISPR-Cas9 working mechanisms and expand the compact
CRISPR-Cas9 toolbox.