Abstract
Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.
Original language | English (US) |
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Pages (from-to) | 505-529 |
Number of pages | 25 |
Journal | Annual Review of Biophysics |
Volume | 46 |
DOIs | |
State | Published - May 22 2017 |
Externally published | Yes |
Keywords
- CRISPR
- Cas9
- Genome engineering
- Mechanism
- Off-target
- Structure
ASJC Scopus subject areas
- Biophysics
- Structural Biology
- Bioengineering
- Biochemistry
- Cell Biology