Genome editing in rice using CRISPR/Cas12i3

Abstract

The CRISPR/Cas type V-I is a family of programmable nuclease systems that prefers a T-rich protospacer adjacent motif (PAM) and is guided by a short crRNA. In this study, the genome-editing application of Cas12i3, a type V-I family endonuclease, was characterized in rice. We developed a CRIPSR/Cas12i3-based Multiplex direct repeats (DR)-spacer Array Genome Editing (iMAGE) system that was efficient in editing various genes in rice. Interestingly, iMAGE produced chromosomal structural variations with a higher frequency than CRISPR/Cas9. In addition, we developed base editors using deactivated Cas12i3 and generated herbicide-resistant rice plants using the base editors. These CRIPSR/Cas12i3-based genome editing systems will facilitate precision molecular breeding in plants.

Keywords: Base editors; CRISPR/Cas12i3; DNA structural variations; Multiplex DR-spacer Array.

Figure 1

Genome editing in rice using CRISPR/Cas12i3. (a) Diagrams of the genome editing construct, and crRNAs and pre‐crRNA arrays. Target sequences of the OsYSA, OsNAL, OsMIR396e and OsPYL6 genes are underlined, and PAM sequences are highlighted in red. (b) Percentages of T0 plants with mutations in the target sequences. (c) Examples of OsYSA gene mutations generated by CRISPR/Cas12i3. (d) Statistics of homozygous, bi‐allelic, heterozygous, or chimeric mutations in the OsYSA, OsNAL, OsMIR396e, and OsPYL6 target genes. (e) Types and frequencies of T0 generation mutations in the OsYSA, OsNAL, OsMIR396e, and OsPYL6 target genes. d: deletion; i: insertion; s: substitution. (f) Representative Sanger sequencing chromatograms of OsYSA, OsNAL, OsMIR396e and OsPYL6 gene mutations. Homo: homozygous; Het: heterozygous.

Figure 2

The multiplex crRNA array improves mutation frequencies. (a) A schematic diagram of the target sites in the OsNRAMP5 gene. Shown are the five crRNAs (B1, A2, A3, B4 and A5) in the CRISPR/Cas12i3 single target vector and the five‐crRNAs array:A (A1–A5) and five crRNA array:B (B1–B5) in the multiplex crRNA array vector, named iMAGE‐array:A and iMAGE‐array:B respectively. (b) Percentages of T0 transgenic plants with mutations in the OsNramp5 gene caused by iMAGE‐Array:A or iMAGE‐Array:B. (c) Percentage of T0 plants with mutations in the target gene. (d) The rates of mutations caused by iMAGE‐array vector and single sgRNA vector.

Figure 3

Generation of HPPD gene duplication by the multiplex crRNA array system. (a) Diagram of duplication of the HPPD gene by a multiplex crRNA array system. The two crRNAs targeting the OsUbi and OsHPPD genes were assembled into the multiplex crRNA array vector iMAGE‐pUbi:HPPD. (b) Gel bands indicating duplication events in rice calli. (c) T0 plants with duplication alleles displayed tolerance to the bipyrazone herbicide under regeneration conditions. (d) T0 plants with homozygous duplication alleles were identified, and the corresponding Sanger sequencing chromatogram is shown. PAM sequences are highlighted in red. (e) The frequencies of duplication events between the OsHPPD and OsUbi2 genes generated by CRISPR/Cas9 and CRISPR/Cas12i3.

Figure 4

dCas12i3‐based base editors are active in rice. (a) A native agarose gel image showing that mutations of the RuvC catalytic residues of Cas12i3 (D619A, E844A, D1017A and 3 M) prevent double‐stranded DNA cleavage. (b) Schematic representations of the dCas12i3 cytidine base editor (iBE) and dCas12i3 adenine base editor (iABE) vectors. (c) Base editing efficiencies of iBE ⁶¹⁹ , iBE ⁸⁴⁴ , iBE ¹⁰¹⁷ and iBE ^3M in rice calli. (d) Base editing efficiencies of iABE ⁶¹⁹ , iABE ⁸⁴⁴ , iABE ¹⁰¹⁷ and iABE ^3M in rice calli. (e) Diagram of the GFP reporter system. (f) The transgenic rice calli with GFP signals. Scale bars represent 500 μm. (g) Summary of the base editing frequency at each adenine in the spacer region for the indicated 20 crRNAs. (h) Sanger sequencing chromatograms of the OsACCase gene mutations generated by iABE ⁸⁴⁴ . (i) T0 plants with base‐editing in the OsACCase gene showed tolerance to the sethoxydim herbicide. (+) with sethoxydim herbicide spray; (−) without sethoxydim herbicide spray.