Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity

Abstract

Background The CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a). Results We created new AsCas12a variant termed “AsCas12a-Plus” with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAFV600E mutant, AsCas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAFV600E cancer cell killing. By introducing the corresponding substitutions into LbCas12a, we also generated LbCas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity. Conclusions Our discoveries provide alternative AsCas12a and LbCas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.

Improved genome editing by an engineered CRISPR-Cas12a

“…Unlike Cas9, the Cas12a nucleases are not as widely utilized for genome editing due to lower efficiency and specificity ( 33 , 34 ). To overcome these obstacles, several studies using either structure-guided protein engineering ( 26 , 35 ) or directed evolution ( 25 ) have generated highly active variants of Acidaminococcus Sp . Cas12a (AsCas12a).…”

Comparison of DNA targeting CRISPR editors in human cells

“…Tag-seq can parallelly profile the off-target cleavages induced by Cas-nuclease at various sites in a single experiment and thus is a convenient and cost-efficient method for comparing the specificity among different nucleases, which was performed and described as previously reported [ 13 , 27 ]. Briefly, for the HEK293T and MCF7, ~ 6.0 × 10 5 cells were transfected by PEI with 10 pmol Tag, 600 ng of Cas nuclease, and 600 ng pooled sgRNAs (21 guides) per well in a 12-well plate.…”

Comparison of DNA targeting CRISPR editors in human cells

“…Tag-seq can parallelly profile the off-target cleavages induced by Cas-nuclease at various sites in a single experiment and thus is a convenient and cost-efficient method for comparing the specificity among different nucleases, which was performed and described as previously reported 13, 27 . Briefly, for the HEK293T and MCF7, ∼6.0×10 5 cells were transfected by PEI with 10 pmol Tag, 600 ng of Cas nuclease, and 600 ng pooled sgRNAs (21 guides) per well in a 12-well plate.…”

“…Tag-seq can parallelly profile the off-target cleavages induced by Cas-nuclease at various sites in a single experiment and thus is a convenient and costefficient method for comparing the specificity among different nucleases, which was performed and described as previously reported 13,27 . Briefly, for the HEK293T and MCF7, ~6.0x10 The Tag-seq pipeline is available at https://github.com/zhoujj2013/Tag-seq and https://doi.org/10.5281/zenodo.4679460.…”