摘要
With the widespread use of clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease(Cas) technologies in plants, large-scale genome editing is increasingly needed. Here, we developed a geminivirus-mediated surrogate system, called Wheat Dwarf Virus-Gate(WDV-surrogate), to facilitate high-throughput genome editing.WDV-Gate has two parts: one is the recipient callus from a transgenic rice line expressing Cas9 and a mutated hygromycin-resistant gene(HygM) for surrogate selection;the other is a WDV-based construct expressing two single guide RNAs(sgRNAs) targeting HygM and a gene of interest, respectively. We evaluated WDV-Gate on six rice loci by producing a total of 874 T_0 plants. Compared with the conventional method, the WDV-Gate system, which was characterized by a transient and high level of sgRNA expression, significantly increased editing frequency(66.8% vs. 90.1%), plantlet regeneration efficiency(2.31-fold increase), and numbers of homozygous-edited plants(36.3%vs. 70.7%). Large-scale editing using pooled sg RNAs targeting the SLR1 gene resulted in a high editing frequency of 94.4%, further demonstrating its feasibility. We also tested WDVGate on sequence knock-in for protein tagging.By co-delivering a chemically modified donor DNA with the WDV-Gate plasmid, 3xFLAG peptides were successfully fused to three loci with an efficiency of up to 13%. Thus, by combining transiently expressed sgRNAs and a surrogate selection system, WDV-Gate could be useful for high-throughput gene knock-out and sequence knock-in.
基金
supported by the National Key R&D Program of China (2021YFD1201300 to Y.L., 2021YFA1300404 to J.-K.Z.)
the National Natural Science Foundation of China (32070396 to Y.L., 32188102 to J.-K.Z.)
the China Postdoctoral Science Foundation (BX20220098 to Y.T.)。
