Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants

Abstract

Background Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants. Results To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis in E. coli and identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency in T0 plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites. Conclusions Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.

Lachnospiraceae are emerging industrial biocatalysts and biotherapeutics

“…Lachnospiraceae proteins that have been applied as molecular tools in other organisms are strong candidates to advance genome engineering in Lachnospiraceae . For example, LbCas12a, which was isolated from Lachnospiraceae bacterium ND 2006 ( Tak et al, 2017 ), has been applied for genome editing in eukaryotes including fungi ( Chen T. et al, 2023 ), plants ( Kim et al, 2021 ), flies ( Port et al, 2020 ), and human cells ( Zhang et al, 2023 ). Another Cas12a variant, Lb2Cas12a, derived from Lachnospiraceae bacterium MA 2020, has been developed to have enhanced editing activity and broadened protospacer adjacent motif (PAM) recognition in human cells ( Tran et al, 2021 ).…”

Hs1Cas12a and Ev1Cas12a confer efficient genome editing in plants

“…Previous studies showed Cas12a orthologs and engineered Cas12a had low off-target effects ( Zhang et al, 2021b ; Zhang et al, 2023 ). To further verify this feature of Cas12a, we analyzed off-target effects of both Ev1Cas12a and Hs1Cas12a in poplar T 0 plants.…”

“…So far, multiple Cas12a nucleases have been demonstrated in plants, including AsCas12a, LbCas12a ( Tang et al, 2017 ), FnCas12a ( Zhong et al, 2018 ), ErCas12a (also known as MAD7) ( Lin et al, 2021 ), and Mb2Cas12a ( Zhang et al, 2021b ). Moreover, researchers have improved genome editing efficiency of Cas12a through protein engineering, such as LbCas12a-D156R/ttLbCas12a ( Schindele and Puchta, 2020 ), AsCas12a ultra ( Zhang et al, 2021a ), intron-containing ttLbCas12a ( Schindele et al, 2023 ), LbCas12a-RV and LbCas12a-RRV ( Zhang et al, 2023 ). Nevertheless, the temperature sensitivity of Cas12a represents a constraint that affects Cas12-mediated genome editing ( Malzahn et al, 2019 ; Zhang et al, 2021b ).…”

Optimizing ErCas12a for efficient gene editing in Arabidopsis thaliana

“…In the case of K172R, additional contacts with the backbone of the DNA in the PAM‐proximal region as well as additional hydrogen bonds with the base at the 3′ end of the PAM might form. The resulting enhanced editing efficiency is in line with results from other studies, which showed that non‐negatively charged amino acids within DNA‐interacting protein domains increase editing activities of different Cas12a orthologues, probably due to improved DNA binding (Zhang et al ., 2021b , 2023 ). However, it should be kept in mind that this structure only reflects a probable model of Er Cas12a and that the crRNA/DNA duplex was only visually inserted into the model, based on the analogous As Cas12a.…”

“…Can the cutting efficiency of imErCas12a be further improved? This will most probably be the case as the example of LbCas12a demonstrates: Recently, besides the already known activity enhancing mutation D156R (Schindele and Puchta, 2020 ), two other mutation could be identified by saturation mutagenesis, that in combination resulted in the variant LbCas12a‐RRV showing a further increase of editing efficiency (Zhang et al ., 2023 ). On the other hand, the integration of introns in the ORF of ttLbCas12a lead to drastic enhancement of gene editing and gene targeting frequencies (Schindele et al ., 2023 ).…”