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Updated: Nov 18, 2025
Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens
Published on: June 6, 2020
Engineered FnCas12a with enhanced activity through directional evolution in human cells
Xiexie Liu1, Xiaoyu Liu1, Chenchen Zhou1
1State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China.
Abstract:
Clustered regularly interspaced short palindromic repeat-Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a. Furthermore, to widen the target selection spectrum, eaFnCas12a was engineered through site-directed mutagenesis. eaFnCas12a and one engineered variant (eaFnCas12a-RR), used for correcting human RS1 mutation responsible for X-linked retinoschisis, had a 3.28- to 4.04-fold improved activity compared with WT. Collectively, eaFnCas12a and its engineered variants can be used for genome-editing applications that requires high activity.
Insights
Researchers developed a novel screening system to enhance CRISPR-Cas12a gene editing. This system identified a new variant, eaFnCas12a, with significantly improved DNA cleavage activity for genome editing applications.
Area of Science:
- Molecular Biology
- Gene Editing Technologies
Background:
- CRISPR-Cas12a systems are powerful tools for genome manipulation.
- Limitations include low cleavage efficiency and strict protospacer adjacent motif (PAM) requirements, hindering therapeutic applications.
Purpose of the Study:
- To develop a system for identifying novel FnCas12a variants with enhanced DNA cleavage activity.
- To engineer variants with a broader target selection spectrum for improved genome editing.
Main Methods:
- A directional evolution and screening system was established in human cells.
- Site-directed mutagenesis was employed to engineer eaFnCas12a variants.
- Assessed cleavage activity of wild-type (WT) FnCas12a, eaFnCas12a, and engineered variants.
Main Results:
- Identified IV-79 (eaFnCas12a), a variant with superior DNA cleavage activity compared to WT FnCas12a.
- Engineered eaFnCas12a variants, including eaFnCas12a-RR, demonstrated 3.28- to 4.04-fold improved activity.
- Demonstrated efficacy in correcting the RS1 mutation linked to X-linked retinoschisis.
Conclusions:
- The developed screening system successfully identified highly active FnCas12a variants.
- eaFnCas12a and its engineered forms offer enhanced performance for genome editing.
- These variants hold promise for therapeutic applications requiring efficient gene correction.
