CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA

Li, Shi‐Yuan; Cheng, Qiuxiang; Liu, Jia-Kun; Nie, Xiaoqun; Zhao, Guoping; Wang, Jing‐Zhi

doi:10.1038/s41422-018-0022-x

Cited by 668 publications

(586 citation statements)

References 16 publications

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“…We could not detect homologous off‐targets in any of the plants under analysis. Moreover, we do not find evidences in any of the analysed lines of enhanced genome‐wide mutagenesis to occur as a result of Cas12a activation as suggested previously (Chen et al ., ; Li et al ., ). Just the opposite, WGS data showed that all analysed lines had variations with the reference genome and with their own relatives in the range of those observed in Cas12a‐free plants.…”

Section: Discussionmentioning

confidence: 97%

“…However, recent studies have reported large DNA rearrangements near the target loci, which remain undetected with traditional target-specific PCR analysis (Adikusuma et al, 2018;Kosicki et al, 2018). Also, indiscriminate single-strand DNA (ssDNA) DNase activity of Cas12a upon activation by target-specific cleavage has been observed in vitro (Chen et al, 2018;Li et al, 2018). These observations have led to the suggestion that increased mutagenesis rates could occur elsewhere in the genome, particularly in areas with transient ssDNA formation, such as active DNA replication and transcriptional sites.…”

Section: Modifications In the Crrna Dr Loop Affect Rgen Activity Butmentioning

confidence: 99%

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Assessment of Cas12a‐mediated gene editing efficiency in plants

Bernabé‐Orts

Casas‐Rodrigo

Minguet

et al. 2019

Plant Biotechnology Journal

101

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Summary The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA‐guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB‐assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target‐dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on‐target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a‐free segregating T2 lines to assess possible off‐target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of −10 to −2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off‐target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.

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Section: Discussionmentioning

confidence: 97%

Section: Modifications In the Crrna Dr Loop Affect Rgen Activity Butmentioning

confidence: 99%

Assessment of Cas12a‐mediated gene editing efficiency in plants

Bernabé‐Orts

Casas‐Rodrigo

Minguet

et al. 2019

Plant Biotechnology Journal

101

View full text Add to dashboard Cite

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“…Several Cas12a orthologs, including AsCas12a, Lachnospiraceae bacterium Cas12a (LbCas12a) and Francisella novicida Cas12a (FnCas12a), have been found to have indiscriminate single‐stranded (ss) DNase activity upon binding to its target DNA sequence(s) (Table and Figure ) . Although the exact mechanism has not been fully understood, studies have suggested that while the cis‐ cleavage by Cas12a requires recognition of PAM and a specific target sequence, the non‐target DNA degradation through the trans ‐cleavage occurs in a PAM‐ and sequence‐independent manner . It is noteworthy that the trans ‐cleavage activity by Cas12a has been re‐purposed for highly sensitive detection of specific nucleic acid sequence(s) …”

Section: Type V: the Crispr‐cas12 Systemsmentioning

confidence: 99%

The rapidly advancing Class 2 CRISPR‐Cas technologies: A customizable toolbox for molecular manipulations

Wang

Zhang

Feng

2020

J Cellular Molecular Medi

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The CRISPR‐Cas technologies derived from bacterial and archaeal adaptive immune systems have emerged as a series of groundbreaking nucleic acid‐guided gene editing tools, ultimately standing out among several engineered nucleases because of their high efficiency, sequence‐specific targeting, ease of programming and versatility. Facilitated by the advancement across multiple disciplines such as bioinformatics, structural biology and high‐throughput sequencing, the discoveries and engineering of various innovative CRISPR‐Cas systems are rapidly expanding the CRISPR toolbox. This is revolutionizing not only genome editing but also various other types of nucleic acid‐guided manipulations such as transcriptional control and genomic imaging. Meanwhile, the adaptation of various CRISPR strategies in multiple settings has realized numerous previously non‐existing applications, ranging from the introduction of sophisticated approaches in basic research to impactful agricultural and therapeutic applications. Here, we summarize the recent advances of CRISPR technologies and strategies, as well as their impactful applications.

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“…Additionally, force clamps did not aggregate after Cas12a digestion ( Figures S9-S13), suggesting their short poly-dT ends remained. [21] Other constraints, including dsDNA damages with excessive enzyme concentration or reaction time (Figures S55, S56) and enzyme accessibility to short, recessed ssDNA ( Figures S57, S58), need to be considered when applying this method. Because Cas12a digestion does not require toehold or protospacer adjacent motifs on ssDNA substrates, it is compatible with DNA structures built from virtually any sequences and uniquely suitable for post-processing of single-stranded DNA origami.…”

Section: Angewandte Chemiementioning

confidence: 99%

DNA Origami Post‐Processing by CRISPR‐Cas12a

et al. 2020

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Customizable nanostructures built through the DNA‐origami technique hold tremendous promise in nanomaterial fabrication and biotechnology. Despite the cutting‐edge tools for DNA‐origami design and preparation, it remains challenging to separate structural components of an architecture built from—thus held together by—a continuous scaffold strand, which in turn limits the modularity and function of the DNA‐origami devices. To address this challenge, here we present an enzymatic method to clean up and reconfigure DNA‐origami structures. We target single‐stranded (ss) regions of DNA‐origami structures and remove them with CRISPR‐Cas12a, a hyper‐active ssDNA endonuclease without sequence specificity. We demonstrate the utility of this facile, selective post‐processing method on DNA structures with various geometrical and mechanical properties, realizing intricate structures and structural transformations that were previously difficult to engineer. Given the biocompatibility of Cas12a‐like enzymes, this versatile tool may be programmed in the future to operate functional nanodevices in cells.

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CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA

Cited by 668 publications

References 16 publications

Assessment of Cas12a‐mediated gene editing efficiency in plants

Assessment of Cas12a‐mediated gene editing efficiency in plants

The rapidly advancing Class 2 CRISPR‐Cas technologies: A customizable toolbox for molecular manipulations

DNA Origami Post‐Processing by CRISPR‐Cas12a

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