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

Abstract

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.

Epigenome engineering: new technologies for precision medicine

“…It is worth noting that Cas12a orthologues (AsCas12a more so than LbCas12a) exhibit higher DNA binding specificity than SpCas9, evaluated by the B reaks L abeling I n S itu and S equencing (BLISS-seq), which is a sensitive method for detecting genome-wide off-target activity ( 149 ). Additionally, the novel CeCas12a orthologue from Coprococcus eutactus , with its more stringent non-canonical PAM recognition, provides another potentially more specific toolkit for epigenome engineering ( 231 ). Lastly, another h igh- f idelity version of Cas12a is the en hanced en As d Cas12a- HF 1 variant from Acidaminococcus sp .…”

CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

“…Cas12a editing has been widely utilized in many crops (see Table 1 ) including rice ( Endo A. et al, 2016 ; Begemann et al, 2017 ; Hu et al, 2017 ; Tang et al, 2017 , 2018 ; Wang et al, 2017a , b ; Yin et al, 2017 ; Li L. et al, 2018 ; Li et al, 2019a ; Jun et al, 2019 ; Malzahn et al, 2019 ; Banakar et al, 2020 ; Chen et al, 2020 ; Schindele and Puchta, 2020 ), wheat ( Liu et al, 2020 ), maize ( Lee K. et al, 2019 ), soybean ( Kim et al, 2017 ), cotton ( Li B. et al, 2019 ), tomato ( van Vu et al, 2020 ), citrus ( Jia et al, 2019 ), tobacco ( Endo A. et al, 2016 ; Endo and Toki, 2019 ), and the model plant Arabidopsis ( Wolter and Puchta, 2019 ; Schindele and Puchta, 2020 ). At present, three Cas12a genome editing systems AsCas12a, FnCas12a, and LbCas12a have been demonstrated in plants ( Zhong et al, 2018 ) with varied efficiency.…”

“…After engineering five independent nucleotide changes into Cas12a that had been shown in previous studies to modulate PAM-site selectivity and enzyme cutting efficiency, the impLbCas12a enzyme was able to cut at a TNTN consensus sequence with increased activity ( Tóth et al, 2020 ). In addition to engineering relaxed specificities, Chen and colleagues identified two Cas12a variants ( CeCas12a and BeCas12a ) with a more stringent PAM site requirement in order to minimize off target events ( Chen et al, 2020 ). This may have applications in engineering synthetic circuits when tight control of target sites is necessary.…”

Types I and V Anti-CRISPR Proteins: From Phage Defense to Eukaryotic Synthetic Gene Circuits

“…As shown in Teng et al (2019), type V-A editing efficiency is enhanced by optimizing the loop region formed by the repeat sequence in the crRNA. Moreover a novel Cas12a-CeCas12a from Coprococcus eutactus-was reported to recognize PAM sequences more stringently than the other Cas12as currently used, with a consequent considerable reduction in the rate of off-target editing (Teng et al, 2019;Chen et al, 2020).…”

“…This was due to the fact that AcrIF1 and AcrIF2 prevented the Csy complex from binding the phzM promoter, whereas AcrIF3 binds and inactivates Cas3 nuclease. The Csy Guo et al, 2017;Tang et al, 2017;Tu et al, 2017;Teng et al, 2019;Chen et al, 2020 AcrIF2 90 Cas7.6f and Cas8f…”