Versatile plant genome engineering using anti-CRISPR-Cas12a systems.

Publisher: Science China Press, co-published with Springer Country of Publication: China NLM ID: 101529880 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1869-1889 (Electronic) Linking ISSN: 16747305 NLM ISO Abbreviation: Sci China Life Sci Subsets: MEDLINE

Original Publication: Beijing : Science China Press, co-published with Springer

CRISPR-Cas Systems* ; Gene Editing*/methods ; Genome, Plant* ; Plants, Genetically Modified*/genetics ; Oryza*/genetics ; CRISPR-Associated Proteins*/genetics ; CRISPR-Associated Proteins*/metabolism; Protoplasts/metabolism ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Genetic Engineering/methods ; Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Endodeoxyribonucleases

CRISPR-Cas12a genome engineering systems have been widely used in plant research and crop breeding. To date, the performance and use of anti-CRISPR-Cas12a systems have not been fully established in plants. Here, we conduct in silico analysis to identify putative anti-CRISPR systems for Cas12a. These putative anti-CRISPR proteins, along with known anti-CRISPR proteins, are assessed for their ability to inhibit Cas12a cleavage activity in vivo and in planta. Among all anti-CRISPR proteins tested, AcrVA1 shows robust inhibition of Mb2Cas12a and LbCas12a in E. coli. Further tests show that AcrVA1 inhibits LbCas12a mediated genome editing in rice protoplasts and stable transgenic lines. Impressively, co-expression of AcrVA1 mitigates off-target effects by CRISPR-LbCas12a, as revealed by whole genome sequencing. In addition, transgenic plants expressing AcrVA1 exhibit different levels of inhibition to LbCas12a mediated genome editing, representing a novel way of fine-tuning genome editing efficiency. By controlling temporal and spatial expression of AcrVA1, we show that inducible and tissue specific genome editing can be achieved in plants. Furthermore, we demonstrate that AcrVA1 also inhibits LbCas12a-based CRISPR activation (CRISPRa) and based on this principle we build logic gates to turn on and off target genes in plant cells. Together, we have established an efficient anti-CRISPR-Cas12a system in plants and demonstrate its versatile applications in mitigating off-target effects, fine-tuning genome editing efficiency, achieving spatial-temporal control of genome editing, and generating synthetic logic gates for controlling target gene expression in plant cells.
Competing Interests: Compliance and ethics. The authors declare no competing interests.
(© 2024. Science China Press.)

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Keywords: AcrVA1; CRISPRa; Cas12a; anti-CRISPR; fine-tuning genome editing; inducible and tissue specific genome editing; off-target effects; synthetic logic circuit

0 (CRISPR-Associated Proteins)
EC 3.1.- (Cas12a protein)
0 (Bacterial Proteins)
EC 3.1.- (Endodeoxyribonucleases)

Date Created: 20240819 Date Completed: 20241217 Latest Revision: 20241217

20250114

10.1007/s11427-024-2704-7

39158766