Engineering Prime Editing System for Concurrent Gene Knockout and Point Mutation Induction (KAPSi) in Rice

doi:10.1016/j.rsci.2026.02.008

Abstract

Liu Xiaoshuang, Ma Chong, Wang Dongmei, Xu Youshen, Qin Ruiying, Li Juan, Xu Rongfang, Wei Pengcheng. Engineering Prime Editing System for Concurrent Gene Knockout and Point Mutation Induction (KAPSi) in Rice[J]. Rice Science, 2026, 33(3): 277-281.

Figures/Tables 1

Fig. 1. Design of KAPSi strategies for concurrent knockout and point mutation in rice. A, Schematic illustration of working mechanisms of five KAPSi strategies. The upper panels depict the point mutation modules, the lower panels show the knockout modules, and the middle panels indicate the distinct forms of DNA nicks by each strategy. Each strategy integrates a point mutation unit with a knockout unit through a different nuclease configuration: C4ePE2-P employs two Csy4-processed prime editing guide RNA (pegRNA) modules; C4ePE2-Nick combines point mutation editing with paired nick-mediated knockout; C4ePE2-Cas12a combines enhanced PE2 (ePE2) with a Cas12a-crRNA knockout module; C4ePE2-Cas9 separates point mutation and knockout via aptamer-guided recruitment of either ePE2 or SpCas9; and C4ePE2-Full uses SpCas9 to introduce double-strand break (DSB)-mediated knockout. NC, Viral nucleocapsid protein; M-MLV, Moloney-murine leukemia virus reverse transcriptase; MCP, MS2 coat protein; N22P, N-terminal 22-amino-acid peptide; boxB, boxB RNA hairpin; crRNA, CRISPR RNA; InDel, Insertion/deletion. B and C, Precise point mutation efficiencies (B) amd knockout editing efficiencies (C) of KAPSi at GS2-GW2-T, BRI1-SD8-T, and D10-PIN1b-T target pairs. Efficiencies were calculated as the percentage of edited reads among total reads. Violin plots display data points from the three genomic loci as independent measurements. Statistical significance was assessed using a two-tailed Mann-Whitney test comparing each strategy against C4ePE2-P (ns, Not significant; **, P < 0.01). D, Precise point mutation profiles of T0 transgenic rice plants generated using KAPSi. Genotyping was performed by high-throughput tracking of mutations (Hi-TOM) sequencing, with 48 independent T0 lines analyzed per target (15% read-frequency threshold). The stacked bars indicate the proportions of homozygous (Ho) and heterozygous (He) mutants. E, Knockout profiles of T0 transgenic rice plants generated using KAPSi. The bars show the proportions of homozygous/biallelic knockout mutants (HO/Bi), biallelic mutants (Bi), and chimeric plants (Chi). F, Co-editing and homozygous rates in T0 transgenic plants at GS2-GW2-T, BRI1-SD8-T, and D10-PIN1b-T target pairs using KAPSi. G, Grain phenotypes of T1 plants with distinct editing types [co-edited (OsGS2AA/osgw2), only precise point mutation (OsGS2AA), or only knockout (osgw2)] generated by C4ePE2-P at the GS2-GW2-T target pair. WT, Wild type. H, Quantification of grain-size traits in T1 plants carrying different editing types. Measurements include grain width, grain length, and 1000-grain weight. Data are mean ± SD (n = 12). Statistical significance was assessed using a two-tailed t-test (ns, Not significant; **, P < 0.01).

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