Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a

Swarts, Daan C.; Oost, John van der; Jinek, Martin

doi:10.1016/j.molcel.2017.03.016

Cited by 482 publications

(713 citation statements)

References 61 publications

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“…Thus, these observations suggested that the Cpf1 Nuc domain plays a role in guiding the target strand into the RuvC active site, rather than catalyzing the cleavage of the target strand. Supporting this notion, a recent study indicated that Francisella novicida Cpf1 cleaves the target and non-target DNA strands, using the same RuvC domain active site (Swarts et al, 2017). …”

Section: Resultsmentioning

confidence: 96%

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

et al. 2017

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Summary The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA), and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic genome editing. Cpf1 requires a specific nucleotide sequence, called a protospacer adjacent motif (PAM), for the target recognition. Besides the canonical TTTV PAM, Cpf1 recognizes suboptimal C-containing PAMs. Here, we report four crystal structures of LbCpf1 in complex with the crRNA and its target DNA, containing either TTTA, TCTA, TCCA or CCCA as the PAM. These structures revealed that, depending on the PAM sequences, LbCpf1 undergoes conformational changes to form altered interactions with the PAM-containing DNA duplexes, thereby achieving the relaxed PAM recognition. Collectively, the present structures advance our mechanistic understanding of the PAM-dependent crRNA-guided DNA cleavage by the Cpf1 family nucleases.

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Section: Resultsmentioning

confidence: 96%

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

et al. 2017

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“…In the first stage, adaptation of a complex of Cas1, Cas2, and in some cases additional Cas proteins mediates excision of fragments from the target DNA (these fragments are known as protospacers), followed by their insertion into the CRISPR array, typically at the leader end of the array (3,12). In the second stage, the CRISPR array is transcribed, and the primary transcript, the pre-CRISPR RNA, is processed into mature CRISPR RNAs (crRNAs) either by a distinct complex of Cas proteins (in type I and type III class 1 CRISPR-Cas systems), by the effector protein (in type VA and type VI systems of class 2), or by the bacterial RNase III with aid from the trans-acting CRISPR (tracr) RNA (in type II and type V-B systems of class 2) (13)(14)(15)(16)(17)(18). The mature crRNA consists of a unique spacer of 25 to 65 bp in length (depending on the CRISPR-Cas type and subtype) flanked by portions of the adjacent repeats.…”

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confidence: 99%

The CRISPR Spacer Space Is Dominated by Sequences from Species-Specific Mobilomes

Shmakov

Sitnik

Makarova

et al. 2017

mBio

202

195

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Clustered regularly interspaced short palindromic repeats and CRISPRassociated protein (CRISPR-Cas) systems store the memory of past encounters with foreign DNA in unique spacers that are inserted between direct repeats in CRISPR arrays. For only a small fraction of the spacers, homologous sequences, called protospacers, are detectable in viral, plasmid, and microbial genomes. The rest of the spacers remain the CRISPR "dark matter." We performed a comprehensive analysis of the spacers from all CRISPR-cas loci identified in bacterial and archaeal genomes, and we found that, depending on the CRISPR-Cas subtype and the prokaryotic phylum, protospacers were detectable for 1% to about 19% of the spacers (~7% global average). Among the detected protospacers, the majority, typically 80 to 90%, originated from viral genomes, including proviruses, and among the rest, the most common source was genes that are integrated into microbial chromosomes but are involved in plasmid conjugation or replication. Thus, almost all spacers with identifiable protospacers target mobile genetic elements (MGE). The GC content, as well as dinucleotide and tetranucleotide compositions, of microbial genomes, their spacer complements, and the cognate viral genomes showed a nearly perfect correlation and were almost identical. Given the near absence of self-targeting spacers, these findings are most compatible with the possibility that the spacers, including the dark matter, are derived almost completely from the species-specific microbial mobilomes. IMPORTANCEThe principal function of CRISPR-Cas systems is thought to be protection of bacteria and archaea against viruses and other parasitic genetic elements. The CRISPR defense function is mediated by sequences from parasitic elements, known as spacers, that are inserted into CRISPR arrays and then transcribed and employed as guides to identify and inactivate the cognate parasitic genomes. However, only a small fraction of the CRISPR spacers match any sequences in the current databases, and of these, only a minority correspond to known parasitic elements. We show that nearly all spacers with matches originate from viral or plasmid genomes that are either free or have been integrated into the host genome. We further demonstrate that spacers with no matches have the same properties as those of identifiable origins, strongly suggesting that all spacers originate from mobile elements.KEYWORDS CRISPR-Cas, bacteriophages, mobilome, oligonucleotide composition, spacer acquisition C RISPR-Cas (clustered regularly interspaced palindromic repeats and CRISPRassociated proteins) systems are adaptive (acquired) immune systems of archaea and bacteria that store memory of past encounters with foreign DNA in unique spacer Citation Shmakov SA, Sitnik V, Makarova KS, Wolf YI, Severinov KV, Koonin EV. 2017. The CRISPR spacer space is dominated by sequences from species-specific mobilomes.

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“…Based on the similarity of domain organization, effectors associated with these five sub-types have been classified as variants of the Cas12 family. Only Cas12a (formerly Cpf1) and Cas12b (formerly C2c1) have been extensively characterized both structurally and functionally (Dong et al, 2016; Gao et al, 2016; Liu et al, 2017a; Shmakov et al, 2015; Stella et al, 2017; Swarts et al, 2017b; Wu et al, 2017; Yamano et al, 2016; Yang et al, 2016; Zetsche et al, 2015). Two additional Type V effectors, Cas12d (formerly CasY) and the compact 980 amino acid Cas12e (formerly CasX), have demonstrated RNA-dependent DNA interference activity in in vivo studies (Burstein et al, 2017), while activity of Cas12c (formerly C2c3) has not been established (Shmakov et al, 2015).…”

Section: Type V Crispr-cas Systemsmentioning

confidence: 99%

“…Formation of the RNA:DNA heteroduplex is stabilized through backbone interactions within the cleft formed between the REC and NUC lobes. Interestingly, although the Cas12a crRNA guide region is 24 nt, 4 nt longer than Cas12b, RNA:DNA heteroduplex formation is interrupted after 20 base pairs by the insertion of an aromatic sidechain, resulting in a duplex that is the same length as in Cas12b (Swarts et al, 2017b; Yamano et al, 2016) (Figure 1F–G, 3D, F). …”

Section: Type V Crispr-cas Systemsmentioning

confidence: 99%

“…In Cas9, the PAM-proximal region of the non-target strand is bound within a tight channel through the NUC lobe, which places the strand within the RuvC active site and positions the scissile phosphate for cleavage (Jiang et al, 2016) (Figure 3B). Notably, in Cas12a and Cas12b crystal structures, much of the non-target strand is disordered beyond the first few PAM-proximal nucleotides, suggesting that the central region of the strand does not interact stably with the protein (Swarts et al, 2017b; Yang et al, 2016) (Figure 3D, F). Instead, the non-target strand may be stabilized through interactions on the PAM-distal end, near the cleavage site.…”

Section: Type V Crispr-cas Systemsmentioning

confidence: 99%

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

et al. 2017

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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.

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Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a

Cited by 482 publications

References 61 publications

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

The CRISPR Spacer Space Is Dominated by Sequences from Species-Specific Mobilomes

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

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