Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize

Abstract

Prime editing is a novel and universal CRISPR/Cas-derived precision genome-editing technology that has been recently developed. However, low efficiency of prime editing has been shown in transgenic rice lines. We hypothesize that enhancing pegRNA expression could improve prime-editing efficiency. In this report, we describe two strategies for enhancing pegRNA expression. We construct a prime editing vector harboring two pegRNA variants for W542L and S621I double mutations in ZmALS1 and ZmALS2. Compared with previous reports in rice, we achieve much higher prime-editing efficiency in maize. Our results are inspiring and provide a direction for the optimization of plant prime editors.

Keywords: ALS; CRISPR/Cas9; Herbicide resistance; Maize; PE2; Prime editing; pegRNA.

Fig. 1

Efficiency of prime editing in maize. a Sequences and their relationships of the two ALS genes, the pegRNA targets, the pegRNA RT templates, the pegRNA primer-binding sites (PBS), and the sgRNA targets for nicking the non-edited strands used for the generation of 4 maize prime-editing vectors. The PAMs, nicking sites, RT template and PBS lengths and introduced mutations are indicated. b T-DNA structures of the 4 PE vectors. The pZ1WS and pZ1WS-Csy4 vectors were designed for editing two targets. Each double-PE vector included two expression cassettes for each of the two pegRNAs or sgRNAs. Two strategies were used to express peg1&2: one was based on the Csy4 RNA processing system, and the other was based on tRNA and HDV ribozyme RNA-processing systems integrated with two drivers, polymerase II (35S enhancer-CmYLCV) and III (shortened U6-26) promoters. The structures of the pegRNA and sgRNA expression cassettes in pegR1-sgR1 and pegR2-sgR2 are the same as those in pZ1PE3/3b. The expression of two sgRNAs in sgR1-sgR2 is driven by OsU3p and TaU3p. c Prime-editing efficiencies based on direct sequencing of the PCR products. The values in the parentheses are based on NGS results. The transgenic lines transformed with pZ1WS-Csy4 were not obtained. d Sequencing chromatograms of the PCR fragments from prime-edited plants transformed with pZ1WS. Double peaks represent heterozygous or chimeric mutations, and an asterisk indicates a mutation induced by PE. The first mutation in #15 is the unwanted mutation introduced by the pegRNA scaffold

Fig. 2

Desired edits and unwanted byproducts in the two transformations with pZ1WS. a Desired edits and unwanted byproducts at the two target sites in the first transformation. Only partial sgRNA sequences are shown. The homologous sgRNA-rtT sequences shared by all the aligned sequences are shaded in yellow, and the mutated nucleotides are indicated by red letters. The number of cloned PCR fragments harboring the same edits is indicated in parentheses, and a vertical line indicates the same type of byproduct. For convenience, the byproducts derived from the two mechanisms were assorted into pegRNA scaffold-derived byproduct category. b Summary of the desired edits and byproducts from the first transformation according to sequences of the cloned PCR fragments. c Prime-editing efficiency achieved in the second transformation. Strong and weak, T0 lines harboring strong and weak peak signals for the edits, respectively, in the sequencing chromatograms. Both, total no. of lines harboring strong and weak edits. d Sequencing chromatograms from a prime-edited line harboring W542L edits and obtained in the second transformation. Double peaks represent heterozygous or chimeric mutations, and an asterisk indicates a mutation induced by PE. The first mutation in the S621 target is the pegRNA scaffold-derived byproducts. e Desired edits and unwanted byproducts at the two target sites in the second transformation. f Summary of the desired edits and byproducts in the second transformation