RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2

Bravo, Jack P.K.; Hallmark, Thomson; Naegle, Bronson; Beisel, Chase L.; Jackson, Ryan N.; Taylor, David W.

doi:10.1038/s41586-022-05560-w

Cited by 39 publications

(35 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These results demonstrate that RNA targeting by SuCas12a2 causes dsDNA damage of the bacterial chromosome that in turn induces the SOS response and abortive infection in bacteria, reflecting a distinct mechanism of immunity that relies on indiscriminate dsDNase activity. Consistent with this observation, recent cryo-electron microscopy structures revealed that Cas12a2 binds to and cuts dsDNA through a mechanism that is completely distinct from all other CRISPR-associated nucleases, and structure-guided mutants with impaired in vitro collateral dsDNase but not ssRNase and ssDNase activities abolished in vivo defence activity against plasmids 19 .…”

Section: Sos Response and Dormancy By Cas12a2mentioning

confidence: 62%

“…The flexible PFS recognition and a tolerance for guide-target mismatches indicate that SuCas12a2 exhibits promiscuous target recognition and appears to lack a canonical seed that is hypersensitive to guide-target mismatches 34,35 . However, the necessary pairing with the 3′ end of the guide is consistent with this end being pre-ordered in the structure of the crRNA-Cas12a2 binary complex 19 and possibly initiating base pairing with the target. Furthermore, the promiscuity further enabled SuCas12a2 to recognize target mutations that disrupt targeting by Cas12a (Extended Data Fig.…”

Section: Cas12a2 Exhibits Targeting Flexibilitymentioning

confidence: 69%

“…between self and non-self targets. Recent cryo-electron microscopy structures of Cas12a2 at stages of RNA targeting and collateral dsDNA capture are already fulfilling this need 19 .…”

Section: Discussionmentioning

confidence: 99%

“…2). Considering their original classification combined with our phylogenetic analyses as well as recent structural results 19 , we named these distinct type V nucleases Cas12a2.…”

Section: Articlementioning

confidence: 99%

See 3 more Smart Citations

Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA

Dmytrenko

Neumann

Hallmark

et al. 2023

Nature

Self Cite

View full text Add to dashboard Cite

Bacterial abortive-infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate1,2. Several RNA-targeting CRISPR–Cas systems (that is, types III and VI) cause abortive-infection phenotypes by activating indiscriminate nucleases3–5. However, a CRISPR-mediated abortive mechanism that leverages indiscriminate DNase activity of an RNA-guided single-effector nuclease has yet to be observed. Here we report that RNA targeting by the type V single-effector nuclease Cas12a2 drives abortive infection through non-specific cleavage of double-stranded DNA (dsDNA). After recognizing an RNA target with an activating protospacer-flanking sequence, Cas12a2 efficiently degrades single-stranded RNA (ssRNA), single-stranded DNA (ssDNA) and dsDNA. Within cells, the activation of Cas12a2 induces an SOS DNA-damage response and impairs growth, preventing the dissemination of the invader. Finally, we harnessed the collateral activity of Cas12a2 for direct RNA detection, demonstrating that Cas12a2 can be repurposed as an RNA-guided RNA-targeting tool. These findings expand the known defensive abilities of CRISPR–Cas systems and create additional opportunities for CRISPR technologies.

show abstract

Section: Sos Response and Dormancy By Cas12a2mentioning

confidence: 62%

Section: Cas12a2 Exhibits Targeting Flexibilitymentioning

confidence: 69%

“…between self and non-self targets. Recent cryo-electron microscopy structures of Cas12a2 at stages of RNA targeting and collateral dsDNA capture are already fulfilling this need 19 .…”

Section: Discussionmentioning

confidence: 99%

“…2). Considering their original classification combined with our phylogenetic analyses as well as recent structural results 19 , we named these distinct type V nucleases Cas12a2.…”

Section: Articlementioning

confidence: 99%

See 2 more Smart Citations

Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA

Dmytrenko

Neumann

Hallmark

et al. 2023

Nature

Self Cite

View full text Add to dashboard Cite

show abstract

“…A reverse-transcription step is inconvenient because it adds to the time, cost, error, and complexity of the assay. The other alternative is to use an RNA-targeting enzyme such as Cas12a2 15 , Cas12g 24 , or Cas13a-d [25][26][27] ; however, these systems can only detect RNA.…”

Section: Introductionmentioning

confidence: 99%

Programmable RNA detection with CRISPR-Cas12a

Rananaware

Vesco

Shoemaker

et al. 2023

Preprint

View full text Add to dashboard Cite

CRISPR is a prominent bioengineering tool and the type V CRISPR-associated protein complex, Cas12a, is widely used in diagnostic platforms due to its innate ability to cleave DNA substrates. Here we demonstrate that Cas12a can also be programmed to directly detect RNA substrates without the need for reverse transcription or strand displacement. We discovered that while the PAM-proximal seed region of the crRNA exclusively recognizes DNA for initiating trans-cleavage, the PAM-distal region or 3'-end of the crRNA can tolerate both RNA and DNA substrates. Utilizing this property, we developed a method named Split Activators for Highly Accessible RNA Analysis (SAHARA) to detect RNA sequences at the PAM-distal region of the crRNA by merely supplying a short ssDNA or a PAM containing dsDNA to the seed region. Notably, SAHARA is Mg2+ concentration- and pH-dependent, and it was observed to work robustly at room temperature with multiple orthologs of Cas12a. SAHARA also displayed a significant improvement in the specificity for target recognition as compared to the wild-type CRISPR-Cas12a, at certain positions along the crRNA. By employing SAHARA we achieved amplification-free detection of picomolar concentrations of miRNA-155 and hepatitis C virus RNA. Finally, SAHARA can use a PAM-proximal DNA as a switch to control the trans-cleavage activity of Cas12a for the detection of both DNA and RNA targets. With this, multicomplex arrays can be made to detect distinct DNA and RNA targets with pooled crRNA/Cas12a complexes. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.

show abstract

Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR‐Cas12a

Zhang,

Li,

Guo

et al. 2024

Angewandte Chemie

View full text Add to dashboard Cite

The CRISPR‐Cas12a system has emerged as a powerful tool for nucleic acid‐based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA‐enabled trans‐cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by fully complementary RNA targets, although LbCas12a exhibits weaker trans‐cleavage activity than AsCas12a on single‐stranded DNA and RNA substrates. Remarkably, we find that the RNA‐activated Cas12a exhibits higher specificity compared to DNA activation. Based on these findings, we develop the “Universal Nuclease for Identification of Virus Empowered by RNA‐sensing” (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM‐less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay, demonstrating random target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a‐based nucleic acid detection and further enhance the understanding of CRISPR‐Cas biochemistry.

show abstract

RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2

Cited by 39 publications

References 56 publications

Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA

Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA

Programmable RNA detection with CRISPR-Cas12a

Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR‐Cas12a

Contact Info

Product

Resources

About