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Review
. 2017 Oct 5;68(1):15-25.
doi: 10.1016/j.molcel.2017.09.007.

The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit

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Free PMC article
Review

The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit

Karthik Murugan et al. Mol Cell. .
Free PMC article

Abstract

CRISPR-Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR-Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR-Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR-Cas systems. This diversity has enabled further expansion of CRISPR-Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR-Cas systems.

Keywords: C2c1; C2c2; CRISPR-Cas; Cas12; Cas13; Cas9; Cpf1; RNA-guided DNA cleavage; RNA-guided RNA cleavage; genome editing.

Figures

Figure 1
Figure 1. Mechanisms of Class 2 effectors
(A) Schematic of CRISPR array. Type II and V-B operons also contain tracrRNA loci, which is sometimes expressed from the opposite strand. Types V-A and VI contain only the CRISPR array. (B,C) Processing mechanisms for crRNA. (B) Type II crRNA:tracrRNA duplexes are cleaved by RNase III. It is unknown whether RNase III also processes Type V-B dual-RNA. (C) Cas12a and Cas13 process pre-crRNAs to produce mature guide crRNA. (D–G) Targeting mechanisms of Class 2 effectors. PAM or PFS locations and sequences are shown in yellow. SpCas9 PAM sequence is shown in (D). (D–F) N: A, C, G or T. (G) H: A, C or U.
Figure 2
Figure 2. Domain arrangement of Class 2 effectors
Domains comprising recognition (REC) lobe and nuclease (NUC) lobes are labeled. PI: PAM interacting, WED: wedge domain, BH: bridge helix, Nuc: nuclease domain, HEPN: Higher Eukaryotes and Prokaryotes Nucleotide-binding, HEL: Helical.
Figure 3
Figure 3. Structures of Class 2 effector binary and ternary complexes
(A) SpCas9 with sgRNA (PDB 4ZT0) and (B) SpCas9-sgRNA bound to dsDNA (PDB 5F9R). (C) Francisella novicida Cas12a (FnCas12a) bound to crRNA (PDB 5NG6) and (D) FnCas12a-pre-crRNA bound to dsDNA (PDB 5NFV). (E) Alicyclobacillus acidoterrestris Cas12b (AacCas12b) bound to sgRNA (PDB 5U34) and (F) AacCas12b-sgRNA bound to dsDNA with extended non-target strand (PDB 5U33). (G) Leptotrichia shahii Cas13a (LshCas13a) bound to crRNA (PDB 5WTK) and (H) Leptotrichia buccalis Cas13a (LbuCas13a) and crRNA bound to target ssRNA (PDB 5XWP). T-DNA: Target DNA strand, NT-DNA: Non-target DNA strand, T-RNA: Target RNA strand.
Figure 4
Figure 4. PAM recognition and phosphate locking mechanisms in Type II and V effectors
Close-ups of PAM regions for (A) SpCas9 (PDB 5F9R) (B) FnCas12a (PDB 5NFV) (C) AacCas12b (PDB 5U33). (A) SpCas9 reads the G-rich PAM sequence through major groove interactions. (B,C) FnCas12a and AacCas12b read T-rich PAM sequences through sequence and shape-specific interactions in major and minor groove. All three effectors stabilize the rotated phosphate of the PAM-proximal deoxyribonucleotide using a phosphate lock loop. Residues from the effectors are colored by domain as in Figure 3.
Figure 5
Figure 5. Class 2 effector active sites
(A–B) Cas12b active sites with (A) target strand (PDB 5U30) or (B) non-target strand (PDB 5U33) accommodated in RuvC active site. (C) Solvent exposed Cas13a HEPN1/2 domain active site (PDB 5XWP).

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