Development of Plant Prime-Editing Systems for Precise Genome Editing

Abstract

Prime-editing systems have the capability to perform efficient and precise genome editing in human cells. In this study, we first developed a plant prime editor 2 (pPE2) system and test its activity by generating a targeted mutation on an HPT^-ATG reporter in rice. Our results showed that the pPE2 system could induce programmable editing at different genome sites. In transgenic T₀ plants, pPE2-generated mutants occurred with 0%-31.3% frequency, suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures. To optimize editing efficiency, guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells. However, at the genomic sites tested in this study, pPE3 systems generated only comparable or even lower editing frequencies. Furthemore, we developed a surrogate pPE2 system by incorporating the HPT^-ATG reporter to enrich the prime-edited cells. The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system, presumably due to the enhanced screening efficiency of edited cells. Taken together, our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.

Keywords: CRISPR; precise editing; prime editing; rice; surrogate system.

Figure 1

pPE2-Induced Editing at the HPT^-ATG Reporter in Rice. (A) Schematic illustration of the expression cassette of pPE2. The maize Ubiquitin 1 promoter and the terminator of CaMV 35S were used for expression of the pPE2 fusion protein. NLS, nuclear location signal. (B) pegRNAs designed for editing on HPT^-ATG. Upper: schematic illustration of the HPT^-ATG reporter. Lower: the target sequence for pegRNA design. The artificial target sequence is indicated in lowercase letters. The uppercase letters indicate the coding sequence for HPT. The expected mutation in the underlined start codon is labeled in red. The region corresponding to the protospacer, PBS, and RT template is shown by brackets. PAM is labeled in orange. The editing position was counted from the nick site on the edited strand. (C) The events harboring editing at the HPT^-ATG reporter was selected by hygromycin. After Agrobacterium-mediated transformation, calli were selected under 50 mg/l hygromycin for 4 weeks. (D) pPE2-mediated precise editing at the HPT^-ATG reporter in rice. Calli were selected by 50 mg/l hygromycin for 4 weeks. The newly emerged yellowish compact calli were recognized as resistant events. Samples for genotyping were randomly selected from the resistant events with extra calli after regeneration. To regenerate plants, we transferred 1- to 5-well growth calli of each resistant event, as a single event, to regeneration medium with 25 mg/l hygromycin for another 4–5 weeks. All resistance events were used for regeneration. (E) Representative Sanger sequencing chromatograph of editing at the target site of HPT^-ATG. The red arrow indicates the expected base conversion in a T₀ heterozygous mutant.

Figure 2

Precise Genome Editing with the pPE2 System in Transgenic Plants. (A) The genome sequence for designing pegRNA. The length of the PBS sequence and RT template is indicated by the brackets. The reading frame around the editing target is underlined. At Wx target, intron sequence is indicated by lowercase letters. (B) Representative sequencing chromatograph of targeted genome editing. The editing is labeled by red arrows. For pePDS1, the insertion was determined by clone sequencing of the target region. For peACC1, PCR products were directly applied for Sanger sequencing. No editing was found at OsWx site by the pPE2 system. (C) Prime editing by pPE2 at the OsPDS site. The position is counted from the nick site. (D) Prime-editing-induced W2125C mutant of the OsACC1 gene confers herbicide resistance. Wild-type line (WT; left) and edited line (right) were incubated in rooting medium supplied with 5 μM haloxyfop-R-methyl for 10 days.

Figure 3

The Surrogate pPE2 System Eases the Screening of Prime-Edited Plants. (A) Schematic illustration of pPE2, pPE3/3b, and surrogate pPE2 system. The wheat TaU3 promoter was used for the expression of gRNA or peHPT1, and the pegRNA of the genomic target was driven by the rice OsU3 promoter. (B) Editing efficiency of pePDS2 and peWX1. After 2 weeks of selection, ~100 newly emerged resistant calli were selected together as one sample. The target region was then amplified to evaluate editing frequency by sequencing 32 randomly selected T-A clones. SD was generated from three biological repetitions. Significance was determined by two-tailed t-test (∗p < 0.05).