A highly efficient in vivo plasmid editing tool based on CRISPR-Cas12a and phage λ Red recombineering.
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ABSTRACT:
SUBMITTER: Geng Y
PROVIDER: S-EPMC7172145 | biostudies-literature | 2019 Sep
REPOSITORIES: biostudies-literature
Publications
A highly efficient in vivo plasmid editing tool based on CRISPR-Cas12a and phage λ Red recombineering.
Geng Yiman Y Yan Haiqin H Li Pei P Ren Gaixian G Guo Xiaopeng X Yin Peiqi P Zhang Leiliang L Qian Zhaohui Z Zhao Zhendong Z Sun Yi-Cheng YC
Journal of genetics and genomics = Yi chuan xue bao 20190910 9
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Project description:The development of CRISPR-based gene-editing technologies has brought an unprecedented revolution in the field of genome engineering. Cas12a, a member of the Class 2 Type V CRISPR-associated endonuclease family distinct from Cas9, has been repurposed and developed into versatile gene-editing tools with distinct PAM recognition sites and multiplexed gene targeting capability. However, with current CRISPR/Cas12a technologies, it remains a challenge to perform efficient and precise genome editing of long sequences in mammalian cells. To address this limitation, we utilized phage recombination enzymes and developed an efficient CRISPR/Cas12a tool for multiplexed precision editing in mammalian cells. Through protein engineering, we were able to recruit phage recombination proteins to Cas12a to enhance its homology-directed repair efficiencies. Our phage-recombination-assisted Cas12a system achieved up to 3-fold improvements for kilobase-scale knock-ins in human cells without compromising the specificity of the enzyme. The performance of this system compares favorably against Cas9 references, the commonly used enzyme for gene-editing tasks, with improved specificity. Additionally, we demonstrated multi-target editing with similar improved activities thanks to the RNA-processing activity of the Cas12a system. This compact, multi-target editing tool has the potential to assist in understanding multi-gene interactions. In particular, it paves the way for a gene therapy method for human diseases that complements existing tools and is suitable for polygenic disorders and diseases requiring long-sequence corrections.
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Project description:[Figure: see text].
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