Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3

Carabias, Arturo; Fuglsang, Anders; Temperini, Piero; Pape, Tillmann; Sofos, N.; Stella, Stefano; Erlendsson, Simon; Montoya, Guillermo

doi:10.1038/s41467-021-24707-3

Cited by 27 publications

(15 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The cleavage results showed that the NTD and TD strands were cleaved at 14 nt and 23 nt after the PAM duplex, respectively (Figure 5A ). Compared with the multiple cleavage sites reported to be generated by Cas12a, Cas12b, Cas12e, Cas12i and Cas12j on the NTD and/or TD strand ( 25 , 48 , 50 , 52 , 54 , 56 , 57 ), Cas12c1 appears to produce a single and uniform cleavage site on the NTD and TD strands. This feature of Cas12c1 would be highly useful for high-fidelity genome editing.…”

Section: Resultsmentioning

confidence: 89%

Structural insights into target DNA recognition and cleavage by the CRISPR-Cas12c1 system

Zhang

Lin

Perčulija

et al. 2022

Nucleic Acids Research

View full text Add to dashboard Cite

Cas12c is the recently characterized dual RNA-guided DNase effector of type V-C CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems. Due to minimal requirements for a protospacer adjacent motif (PAM), Cas12c is an attractive candidate for genome editing. Here we report the crystal structure of Cas12c1 in complex with single guide RNA (sgRNA) and target double-stranded DNA (dsDNA) containing the 5′-TG-3′ PAM. Supported by biochemical and mutation assays, this study reveals distinct structural features of Cas12c1 and the associated sgRNA, as well as the molecular basis for PAM recognition, target dsDNA unwinding, heteroduplex formation and recognition, and cleavage of non-target and target DNA strands. Cas12c1 recognizes the PAM through a mechanism that is interdependent on sequence identity and Cas12c1-induced conformational distortion of the PAM region. Another special feature of Cas12c1 is the cleavage of both non-target and target DNA strands at a single, uniform site with indistinguishable cleavage capacity and order. Location of the sgRNA seed region and minimal length of target DNA required for triggering Cas12c1 DNase activity were also determined. Our findings provide valuable information for developing the CRISPR-Cas12c1 system into an efficient, high-fidelity genome editing tool.

show abstract

Section: Resultsmentioning

confidence: 89%

Structural insights into target DNA recognition and cleavage by the CRISPR-Cas12c1 system

Zhang

Lin

Perčulija

et al. 2022

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…The miniaturization of enzymes usually comes at a cost of efficiency or specificity, because of the simplification of accessory regulatory protein domains. For example, Cas12js constitute a minimal protein scaffold that can perform RNA-guided DNA cleavage. , However, their efficiency is lower than those of other gene editors, and attempts to improve their efficiency have resulted in a parallel increase in RNA/DNA mismatch tolerance and reduced RNA/DNA specificity . A recent study with Cas9 suggests that the tolerance to RNA/DNA mismatches inversely correlates with the number of structural contacts with the RNA/DNA hybrid .…”

Section: Is200/is605 Transposons: the Evolutionary Origin Of Cas9 And...mentioning

confidence: 99%

“…The development of compact CRISPR enzymes is of current interest due to their advantage when they are packed into mature delivery vehicles, such as adenoviral-associated vectors (AAVs). ,− OMEGA nucleases are substantially smaller (∼400 amino acids on average) than type II (Cas9s, 900–1300 amino acids) and type V (Cas12s, 500–1300 amino acids) enzymes, and still smaller than the most compact CRISPR-Cas gene editors (Cas12fs, ∼500 amino acids, and Cas12js, ∼800 amino acids) . The miniaturization of enzymes usually comes at a cost of efficiency or specificity, because of the simplification of accessory regulatory protein domains.…”

Section: Is200/is605 Transposons: the Evolutionary Origin Of Cas9 And...mentioning

confidence: 99%

Transposons and CRISPR: Rewiring Gene Editing

2022

Self Cite

View full text Add to dashboard Cite

CRISPR-Cas is driving a gene editing revolution because of its simple reprogramming. However, off-target effects and dependence on the double-strand break repair pathways impose important limitations. Because homology-directed repair acts primarily in actively dividing cells, many of the current gene correction/replacement approaches are restricted to a minority of cell types. Furthermore, current approaches display low efficiency upon insertion of large DNA cargos (e.g., sequences containing multiple gene circuits with tunable functionalities). Recent research has revealed new links between CRISPR-Cas systems and transposons providing new scaffolds that might overcome some of these limitations. Here, we comment on two new transposonassociated RNA-guided mechanisms considering their potential as new gene editing solutions. Initially, we focus on a group of small RNA-guided endonucleases of the IS200/IS605 family of transposons, which likely evolved into class 2 CRISPR effector nucleases (Cas9s and Cas12s). We explore the diversity of these nucleases (named OMEGA, obligate mobile element-guided activity) and analyze their similarities with class 2 gene editors. OMEGA nucleases can perform gene editing in human cells and constitute promising candidates for the design of new compact RNA-guided platforms. Then, we address the co-option of the RNA-guided activity of different CRISPR effector nucleases by a specialized group of Tn7-like transposons to target transposon integration. We describe the various mechanisms used by these RNA-guided transposons for target site selection and integration. Finally, we assess the potential of these new systems to circumvent some of the current gene editing challenges.

show abstract

“…The remarkable ability of Cas12a to degrade ssDNA in trans upon activation has led to its rapid development in diagnostics as a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) (15); however, recent work in vitro suggests its kinetics were overestimated (22). Nonetheless, the success of these applications and a recent explosion in the functional and evolutionary diversity of the Cas12 (Type V) superfamily have led to a flurry of new detection tools with similar capabilities (23)(24)(25)(26)(27)(28). Although the collateral damage observed in vitro for these nucleases is well-suited for diagnostics, it raises some concerns about their use in gene therapy (29).…”

Section: Introductionmentioning

confidence: 99%

CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity

Marino

Pinilla‐Redondo

Bondy‐Denomy

2022

Preprint

View full text Add to dashboard Cite

CRISPR-Cas12a (Cpf1) is a bacterial RNA-guided nuclease that cuts double-stranded DNA (dsDNA) at sites specified by a CRISPR RNA (crRNA) guide. Additional activities have been ascribed to this enzyme in vitro: site-specific (cis) single-stranded DNA (ssDNA) cleavage and indiscriminate (trans) degradation of ssDNA, RNA, and dsDNA after activation by a complementary target. The ability of Cas12a to cleave nucleic acids indiscriminately has been harnessed for many applications, including diagnostics, but it remains unknown if it contributes to bacterial immunity. Here, we provide evidence that cleavage of ssDNA in cis or in trans by Cas12a is insufficient to impact immunity. Using LbCas12a expressed in either Pseudomonas aeruginosa or Escherichia coli, we observed that cleavage of dsDNA targets did not elicit cell death or dormancy, suggesting insignificant levels of collateral damage against host RNA or DNA. Canonical immunity against invasive dsDNA also had no impact on the replicative fitness of co-infecting ssDNA phage or plasmid in trans. Lastly, crRNAs complementary to invasive ssDNA did not provide protection, suggesting that ssDNA cleavage does not occur in vivo or is insignificant. Overall, these results suggest that CRISPR-Cas12a immunity predominantly occurs via canonical targeting of dsDNA, and that the other activities are in vitro irregularities.

show abstract

Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3

Cited by 27 publications

References 49 publications

Structural insights into target DNA recognition and cleavage by the CRISPR-Cas12c1 system

Structural insights into target DNA recognition and cleavage by the CRISPR-Cas12c1 system

Transposons and CRISPR: Rewiring Gene Editing

CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity

Contact Info

Product

Resources

About