ArticleInternational journal of molecular sciences2021
High-Throughput Profiling of Cas12a Orthologues and Engineered Variants for Enhanced Genome Editing Activity.
Article in International journal of molecular sciences, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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Who cites it
7 citing papers in PubMed.
- Synergistic engineering of Casδ nuclease for robust genome editing.Journal of integrative plant biology · 2026Article
- Wheat's Up with CRISPR-Cas-Current Advances, Obstacles and Perspectives.International journal of molecular sciences · 2026Review
- Benchmarking the PAM compatibility of Cas12a variants for high-throughput yeast genetic variant engineering.Applied and environmental microbiology · 2025Article
- Engineering of a high-fidelity Cas12a nuclease variant capable of allele-specific editing.PLoS biology · 2024Article
- Enhancing the quality of staple food crops through CRISPR/Cas-mediated site-directed mutagenesis.Planta · 2023Review
- Optimization of Nuclear Localization Signal Composition Improves CRISPR-Cas12a Editing Rates in Human Primary Cells.GEN biotechnology · 2022Article
- Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity.BMC biology · 2022Article
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Authors and funding
5 authors.
Funding
Abstract
CRISPR/Cas12a (formerly Cpf1), an RNA-guided endonuclease of the Class II Type V-A CRISPR system, provides a promising tool for genome engineering. Over 10 Cas12a orthologues have been identified and employed for gene editing in human cells. However, the functional diversity among emerging Cas12a orthologues remains poorly explored. Here, we report a high-throughput comparative profiling of editing activities across 16 Cas12a orthologues in human cells by constructing genome-integrated, self-cleaving, paired crRNA-target libraries containing >40,000 guide RNAs. Three Cas12a candidates exhibited promising potential owing to their compact structures and editing efficiency comparable with those of AsCas12a and LbCas12a, which are well characterized. We generated three arginine substitution variants (3Rv) via structure-guided protein engineering: BsCas12a-3Rv (K155R/N512R/K518R), PrCas12a-3Rv (E162R/N519R/K525R), and Mb3Cas12a-3Rv (D180R/N581R/K587R). All three Cas12a variants showed enhanced editing activities and expanded targeting ranges (NTTV, NTCV, and TRTV) compared with the wild-type Cas12a effectors. The base preference analysis among the three Cas12a variants revealed that PrCas12a-3Rv shows the highest activity at target sites with canonical PAM TTTV and non-canonical PAM TTCV, while Mb3Cas12a-3Rv exhibits recognition features distinct from the others by accommodating for more nucleotide A at position -3 for PAM TATV and at position -4 for PAM ATCV. Thus, the expanded Cas12a toolbox and an improved understanding of Cas12a activities should facilitate their use in genome engineering.
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