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The chemistry of Cas9 and its CRISPR colleagues

Abstract

RNA-guided binding and cleavage of nucleic acids by CRISPR–Cas systems is a defining feature of bacterial and archaeal adaptive immunity against viruses and plasmids. As a result of their programmable ability to cut specific DNA and RNA sequences, Cas9 and related single-subunit effector proteins from CRISPR–Cas systems have been widely adopted for research and therapeutic genome engineering applications. In this Review, we discuss the chemistry of macromolecules involved in the multistep interference pathway used by CRISPR–Cas systems that mediate accurate nucleic acid target recognition and cutting. Although this Review mainly focuses on DNA interference by Cas9, we briefly explore nucleic acid targeting by the single-effector proteins Cas12 and Cas13 to emphasize the conserved themes of precision DNA and RNA cleavage within class 2 CRISPR–Cas systems. We further highlight the unique mechanisms of surveillance complex formation, substrate recognition and target cleavage in molecular detail across diverse single-subunit CRISPR–Cas interference proteins.

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Figure 1: Overview of CRISPR–Cas adaptive immunity and interference genes from class 2 systems.
Figure 2: TracrRNA-dependent and self-processing pathways for crRNA biogenesis and RNP complex formation.
Figure 3: PAM recognition and sequence-specific target identification.
Figure 4: Model for unidirectional DNA unwinding and R-loop formation in Cas9 and Cas12 interference proteins.
Figure 5: Nuclease domain structure and metal-dependent nucleic acid target cleavage.

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Acknowledgements

The authors thank M. L. Hochstrasser, L. B. Harrington and A. V. Wright for critical reading and valuable input on the manuscript. J.S.C. is a National Science Foundation Graduate Research Fellow and J.A.D. is a Howard Hughes Medical Institute Investigator.

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The authors contributed equally to this manuscript.

Corresponding author

Correspondence to Jennifer A. Doudna.

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Competing interests

J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine and Intellia Therapeutics; a scientific adviser to Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics and Driver; and executive director of the Innovative Genomics Institute at the University of California Berkeley (UC Berkeley) and the University of California San Francisco (UCSF). J.S.C. and J.A.D. are inventors on UC Berkeley and Howard Hughes Medical Institute patents for clustered regularly interspaced short palindromic repeats (CRISPR) technologies.

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Glossary

Mobile genetic elements

DNA sequences that are capable of moving around a genome, including transposons, plasmids and bacteriophages.

Protospacers

Spacer precursors that are captured from foreign DNA and that are complementary to the CRISPR RNA (crRNA) spacer sequence.

Protospacer adjacent motif sequence

(PAM sequence). A short sequence adjacent to the protospacer within foreign DNA. Recognition of the PAM sequence by effector Cas proteins triggers target interference.

Endonucleolytic cleavage

Achieved by Cas proteins that hydrolyse internal phosphodiester bonds within a nucleotide chain.

Guide RNA

An RNA molecule that includes the CRISPR RNA (crRNA) and directs the Cas interference protein to a target site that is complementary to the spacer sequence.

Interference

The final stage of CRISPR immunity that involves RNA-directed cleavage of target nucleic acids by Cas proteins.

Nuclease

An enzyme that catalyses the cleavage of phosphodiester bonds between nucleic acids.

Helicase

An enzyme that unwinds double-stranded DNA using the energy from ATP hydrolysis.

RuvC nuclease domain

Contains an RNase H-like fold and cleaves single-stranded DNA through a two metal mechanism.

HEPN domain

(Higher eukaryotes and prokaryotes nucleotide-binding RNase domain). Contains conserved motifs and functions as an RNase or a non-catalytic RNA-binding domain.

Ribonuclease

An enzyme that hydrolyses the phosphodiester bonds of an RNA backbone.

Exonuclease

An enzyme that hydrolyses phosphodiester bonds, one at a time, from the ends of a nucleic acid chain.

Single-guide RNA

A chimeric RNA molecule in which the CRISPR RNA (crRNA), which contains a sequence complementary to the target DNA, is covalently linked to a trans-activating crRNA (tracrRNA).

Scissile phosphate

The phosphate within the nucleic acid backbone that is cleaved by a nuclease.

DNA melting

The process of DNA strand separation that does not require an external energy source such as ATP.

RNA strand invasion

A process in which the guide RNA segment interrogates a double-stranded DNA (dsDNA) target to initiate DNA unwinding and to form an RNA–DNA heteroduplex.

R-loop

An RNA–DNA heteroduplex and a displaced DNA strand, which is the end result of RNA strand invasion by Cas ribonucleoprotein (RNP) complexes.

Kinetic inhibition

A model in the context of RNA strand invasion that explains how impeding the rate of R-loop formation reduces Cas9 cleavage activity.

Kinetic Monte Carlo analyses

Reveal the time evolution of a given process (that is, stability of the R-loop over time with mismatched substrates or single-guide RNA (sgRNA) variants).

HNH nuclease domain

Contains a histidine–asparagine–histidine (HNH) motif and is the nuclease within Cas9 that hydrolyses the target DNA strand through a one metal ion mechanism.

Apoprotein

The inactive, unbound state of the protein.

Molecular dynamics simulation

(MD simulation). Computer simulations that capture the time evolution of atomic and/or molecular systems by numerically solving Newton's equations of motion.

Cas9-digested whole-genome sequencing

An in vitro method for detecting Cas9 cleavage sites within genomic DNA using whole-genome sequencing.

cis-cleavage

Occurs when the Cas13–CRISPR RNA (crRNA) complex binds a complementary single-stranded RNA (ssRNA) target, which activates the external higher eukaryotes and prokaryotes nucleotide-binding RNase (HEPN) domain catalytic pocket to cleave the bound ssRNA target.

trans-cleavage

Occurs when the Cas13–CRISPR RNA (crRNA) complex binds a complementary single-stranded RNA (ssRNA) target, which activates the external higher eukaryotes and prokaryotes nucleotide-binding RNase (HEPN) domain catalytic pocket to cleave nonspecific ssRNAs in solution.

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Chen, J., Doudna, J. The chemistry of Cas9 and its CRISPR colleagues. Nat Rev Chem 1, 0078 (2017). https://doi.org/10.1038/s41570-017-0078

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