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CRISPR-Cas12a System With Synergistic Phage Recombination Proteins for Multiplex Precision Editing in Human Cells.

ABSTRACT: 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.

SUBMITTER: Wang C

PROVIDER: S-EPMC9237396 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundEfficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking.ResultsUsing Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed.ConclusionsThis study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

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CRISPR-Cas12a System With Synergistic Phage Recombination Proteins for Multiplex Precision Editing in Human Cells.

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