DNA interference states of the hypercompact CRISPR–CasΦ effector

Pausch, Patrick; Soczek, Katarzyna M; Herbst, D.A.; Tsuchida, Connor A.; Al-Shayeb, Basem; Banfield, Jillian F.; Nogales, Eva; Doudna, Jennifer A.

doi:10.1038/s41594-021-00632-3

Cited by 55 publications

(32 citation statements)

References 60 publications

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“…Collectively these data show that Cas12a2 mediates Abi through a unique mechanism of dsDNA cleavage, distinct from Abi-mediated indiscriminate RNA cleavage seen in other RNA targeting CRISPR-Cas systems 10,32 . The catalytic mechanism of Cas12a2 is consistent with that of other RuvC endonucleases 21,30,33,34 , where protein-induced structural tension of the DNA facilitates proper scissile phosphate coordination. However, within the available Cas9 and Cas12a2 structures, the NCS aromatic clamps provide a unique strategy to cleave duplexed nucleic acids.…”

Section: Resultssupporting

confidence: 70%

“…This groove is of sufficient size to accommodate both ss-and ds-nucleic acids. While other Cas12 proteins undergo conformational changes upon crRNA hybridization (up to ∼25Å in for Cas12a 20 and Cas12j 21 , but more typically up to ∼10 Å 22 ). The conformational rearrangements we observe for Cas12a2 are considerably larger, highlighting the distinct activation mechanism of Cas12a2 ( Supplemental movie 1 ).…”

Section: Resultsmentioning

confidence: 99%

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Large-scale structural rearrangements unleash indiscriminate nuclease activity of CRISPR-Cas12a2

Bravo

Hallmark

Naegle

et al. 2022

Preprint

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Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided degradation of non-specific single-stranded (ss)RNA, ssDNA and double-stranded (ds)DNA upon recognition of a complementary RNA target, culminating in abortive infection (Dmytrenko 2022). Here, we report structures of Cas12a2 in binary, ternary, and quaternary complexes to reveal a complete activation pathway. Our structures reveal that Cas12a2 is autoinhibited until binding a cognate RNA target, which exposes the RuvC active site within a large, positively charged cleft. Double-stranded DNA substrates are captured through duplex distortion and local melting, stabilized by pairs of "aromatic clamp" residues that are crucial for dsDNA degradation and in vivo immune system function. Our work provides a structural basis for this unprecedented mechanism of abortive infection to achieve population-level immunity, which can be leveraged to create rational mutants that degrade a spectrum of collateral substrates.

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Section: Resultssupporting

confidence: 70%

Section: Resultsmentioning

confidence: 99%

Large-scale structural rearrangements unleash indiscriminate nuclease activity of CRISPR-Cas12a2

Bravo

Hallmark

Naegle

et al. 2022

Preprint

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“…Cas12i is an attractive type V CRISPR-Cas nuclease for genome editing because of its compact size that fits in AAV vector with short 43-mer gRNA, absence of tracrRNA, ability to process pre-crRNA, and high specificity. With perhaps the exception of the recently discovered type V-J Cas12j 34 , 35 , no other CRISPR-Cas nuclease possesses this unique combination of favorable properties (Supplementary Table 1 ). The robust activity and high specificity shown by the first engineered version, ABR-001, establishes Cas12i2 as a versatile genome editing platform with broad capabilities.…”

Section: Discussionmentioning

confidence: 99%

Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing

McGaw¹,

Garrity²,

Munoz³

et al. 2022

Nat Commun

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The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.

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“…Interestingly, CampyICE1 lacks the cas1 and cas2 genes [ 56 ], a feature which has also been noted for the hypercompact Cas12j (CasΦ) system found on certain bacteriophages [ 57 ]. The Cas12j system closely resembles the CampyICE1 Cas9 system described here, as they share a limited CRISPR spacer repertoire [ 58 ]. The lack of Cas1 and Cas2 components could mean that the CampyICE1 system is incapable of acquiring new spacers, which is supported by the relative lack of spacer diversity in the 214 genomes containing CampyICE1.…”

Section: Discussionmentioning

confidence: 99%

A Campylobacter integrative and conjugative element with a CRISPR-Cas9 system targeting competing plasmids: a history of plasmid warfare?

2021

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Microbial genomes are highly adaptable, with mobile genetic elements (MGEs) such as integrative conjugative elements (ICEs) mediating the dissemination of new genetic information throughout bacterial populations. This is countered by defence mechanisms such as CRISPR-Cas systems, which limit invading MGEs by sequence-specific targeting. Here we report the distribution of the pVir, pTet and PCC42 plasmids and a new 70–129 kb ICE (CampyICE1) in the foodborne bacterial pathogens Campylobacter jejuni and Campylobacter coli . CampyICE1 contains a degenerated Type II-C CRISPR system consisting of a sole Cas9 protein, which is distinct from the previously described Cas9 proteins from C. jejuni and C. coli . CampyICE1 is conserved in structure and gene order, containing blocks of genes predicted to be involved in recombination, regulation and conjugation. CampyICE1 was detected in 134/5829 (2.3 %) C . jejuni genomes and 92/1347 (6.8 %) C . coli genomes. Similar ICEs were detected in a number of non-jejuni/coli Campylobacter species, although these lacked a CRISPR-Cas system. CampyICE1 carries three separate short CRISPR spacer arrays containing a combination of 108 unique spacers and 16 spacer-variant families. A total of 69 spacers and 10 spacer-variant families (63.7 %) were predicted to target Campylobacter plasmids. The presence of a functional CampyICE1 Cas9 protein and matching anti-plasmid spacers was associated with the absence of the pVir, pTet and pCC42 plasmids (188/214 genomes, 87.9 %), suggesting that the CampyICE1-encoded CRISPR-Cas has contributed to the exclusion of competing plasmids. In conclusion, the characteristics of the CRISPR-Cas9 system on CampyICE1 suggests a history of plasmid warfare in Campylobacter .

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DNA interference states of the hypercompact CRISPR–CasΦ effector

Cited by 55 publications

References 60 publications

Large-scale structural rearrangements unleash indiscriminate nuclease activity of CRISPR-Cas12a2

Large-scale structural rearrangements unleash indiscriminate nuclease activity of CRISPR-Cas12a2

Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing

A Campylobacter integrative and conjugative element with a CRISPR-Cas9 system targeting competing plasmids: a history of plasmid warfare?

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