Enhanced mammalian genome editing by new Cas12a orthologs with optimized crRNA scaffolds

Teng, Fei; Li, Jing; Cui, Tongtong; Xu, Kai; Guo, Lu; Gao, Qingqin; Feng, Guihai; Chen, Chuanyuan; Han, Dali; Zhou, Qi; Li, Wei

doi:10.1186/s13059-019-1620-8

Cited by 77 publications

(68 citation statements)

References 26 publications

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“…For example, position 1 tolerated A or G but not C, whereas active constructs were observed with all nucleotides at position 12 and 14 in the loop. Interestingly, this observation in the 12th position of the loop agrees with alignment of direct repeat sequences across Cas12a orthologs 33 . All recovered active sequences maintained basepairing in the stem, but basepairing alone was insufficient for activity, as the nucleotide sequence proved important.…”

Section: Development Of Variant Direct Repeat Sequencessupporting

confidence: 80%

Optimization of AsCas12a for combinatorial genetic screens in human cells

Sanson

DeWeirdt

Sangree

et al. 2019

Preprint

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Cas12a enzymes have attractive properties for scalable delivery of multiplexed perturbations, yet widespread usage has lagged behind Cas9-based strategies. Here we describe the optimization of Cas12a from Acidaminococcus (AsCas12a) for use in pooled genetic screens in human cells. By assaying the activity of thousands of guides, we confirm on-target design rules and extend them to an enhanced activity variant, enAsCas12a. We also develop the first comprehensive set of off-target rules for Cas12a, and demonstrate that we can predict and exclude promiscuous guides. Finally, to enable efficient higher-order multiplexing via lentiviral delivery, we screen thousands of direct repeat variants and identify 38 that outperform the wildtype sequence. We validate this optimized AsCas12a toolkit by targeting 12 synthetic lethal gene pairs with up to 400 guide pairs each, and demonstrate effective triple knockout via flow cytometry. These results establish AsCas12a as a robust system for combinatorial applications of CRISPR technology.

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Section: Development Of Variant Direct Repeat Sequencessupporting

confidence: 80%

Optimization of AsCas12a for combinatorial genetic screens in human cells

Sanson

DeWeirdt

Sangree

et al. 2019

Preprint

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“…LbaCas12a appears to be the ortholog of choice for high trans activity applications. However, recent studies have expanded the known diversity and classification of Cas12 systems and have introduced mutant Cas12 proteins with altered PAM specificities (43)(44)(45)(46)(47). There is the possibility that there may be other orthologs with very high or very low trans activity and future studies will be needed to determine why orthologs vary in magnitude of trans activity.…”

Section: Discussionmentioning

confidence: 99%

Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA

Fuchs

Curcuru

Mabuchi

et al. 2019

Preprint

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CRISPR-Cas12a (Cpf1) are RNA-guided nuclease effectors of acquired immune response that act in their native organisms by cleaving targeted DNA sequences. Like CRISPR-Cas9 RNA-guided DNA targeting enzymes, Cas12a orthologs have been repurposed for genome editing in non-native organisms and for DNA manipulation in vitro. Recent studies have shown that activation of Cas12a via guide RNA-target DNA pairing causes multiple turnover, non-specific ssDNA degradation in trans, after single turnover ontarget cleavage in cis. We find that the non-specific trans nuclease activity affects RNA and dsDNA in addition to ssDNA, an activity made more evident by adjustment of reaction buffer composition. The magnitude of the trans nuclease activity varies depending on features of the guide RNA being used, specifically target sequence composition and length. We also find that the magnitude of trans nuclease activity varies between the three most well-studied Cas12a orthologs and that the Cas12a from Lachnospiraceae bacterium ND2006 appears to be the most active.

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“…Previous studies suggested that the crRNA carrying nucleotide substitutions in the direct repeats had variable effects on the cleavage activity of Cas12a nucleases [15,31]. To investigate the effects of mutations in the loop region, we employed 8 crRNA loop regions (AsCas12a, 5-UCUU-3; LbCas12a, 5-UAAGU-3; FnCas12a/BfCas12a/CeCas12a, 5-UGUU-3; PbCas12a/PeCas12a/LiCas12a, 5-UUUU-3; Lb2Cas12a/PmCas12a/PcCas12a, 5-UAUU; MbCas12a, 5- Figure S1B).…”

Section: Gene Editing With Cecas12a and Bfcas12a In Human Cellsmentioning

confidence: 99%

A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing

et al. 2020

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Background: AsCas12a and LbCas12a nucleases are reported to be promising tools for genome engineering with protospacer adjacent motif (PAM) TTTV as the optimal. However, the C-containing PAM (CTTV, TCTV, TTCV, etc.) recognition by Cas12a might induce extra off-target edits at these non-canonical PAM sites. Results: Here, we identify a novel Cas12a nuclease CeCas12a from Coprococcus eutactus, which is a programmable nuclease with genome-editing efficiencies comparable to AsCas12a and LbCas12a in human cells. Moreover, CeCas12a is revealed to be more stringent for PAM recognition in vitro and in vivo followed by very low off-target editing rates in cells. Notably, CeCas12a renders less off-target edits located at C-containing PAM at multiple sites compared to LbCas12a and AsCas12a, as assessed by targeted sequencing methods. Conclusions: Our study shows that CeCas12a nuclease is active in human cells and the stringency of PAM recognition could be an important factor shaping off-target editing in gene editing. Thus, CeCas12a provides a promising candidate with distinctive characteristics for research and therapeutic applications.

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Enhanced mammalian genome editing by new Cas12a orthologs with optimized crRNA scaffolds

Cited by 77 publications

References 26 publications

Optimization of AsCas12a for combinatorial genetic screens in human cells

Optimization of AsCas12a for combinatorial genetic screens in human cells

Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA

A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing

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