Development and Optimization of CRISPR Prime Editing System in Photoautotrophic Cells

Abstract

Prime editor (PE), a versatile editor that allows the insertion and deletion of arbitrary sequences, and all 12-point mutations without double-strand breaks (DSB) and a donor template, dramatically enhances research capabilities. PE combines nickase Cas9(H840A) and reverse transcriptase (RT), along with prime editing guide RNA (pegRNA). It has been reported in several plant species, but a weak editing efficiency has led to a decrease in applications. This study reports an optimized-prime editor (O-PE) for endogenous gene editing in Arabidopsis thaliana cells, with an average 1.15% editing efficiency, which is 16.4-fold higher than previously reported. Meanwhile, we observed an increase in indels when testing alternative reverse transcriptase and found out that nCas9(H840A) fused to non-functional reverse transcriptase was responsible for the increase. This work develops an efficient prime editor for plant cells and provides a blueprint for applying PE in other photoautotrophic cells, such as microalgae, that have a high industrial value.

Prime editing: Mechanism insight and recent applications in plants

“…Interestingly, the PE reaction and its consequences may be different from those reported in mammalian cells since introducing second nicks to PE2 did not improve the overall PE efficiency (Hua et al ., 2020; Jiang et al ., 2022b; Lin et al ., 2020; Tang et al ., 2020; Xu et al ., 2020a). The PE application progress in dicot plants was slow, possibly due to unexpectedly lower efficiency (Biswas et al ., 2022; Jiang et al ., 2022c; Lu et al ., 2021; Perroud et al ., 2022; Vu et al ., 2022b; Wang et al ., 2021a). The exact reasons have not been revealed besides the possibility of PBS‐spacer autoinhibition and the reduction of Cas9 activity when fused with RT or activated by pegRNAs (Vu et al ., 2022b).…”

“…Subsequently, they were the first to introduce paired pegRNAs and developed a pegRNA designer webpage for facilitating paired pegRNAs selection (Lin et al ., 2021). Though not all the strategies helped to improve PE performance, the pioneering PE works in plants have inspired many plant groups to study and apply PE for precision plant breeding in monocots (Butt et al ., 2020; Hua et al ., 2020; Jiang et al ., 2020, 2022b; Li et al ., 2020a, 2022a, 2022b, 2022e, 2023b; Liang et al ., 2023; Lin et al ., 2020, 2021; Molla et al ., 2021; Ni et al ., 2023; Qiao et al ., 2023; Tang et al ., 2020; Wang et al ., 2021a; Xu et al ., 2020a, 2021b, 2022; Zou et al ., 2022), dicots (Biswas et al ., 2022; Jiang et al ., 2022c; Lu et al ., 2021; Perroud et al ., 2022; Vu et al ., 2022b; Wang et al ., 2021a) and moss ( Physcomitrella patens) (Perroud et al ., 2022, 2023) (Table 1). Recently, using the PE2max and pU6 composite promoter driving epegRNAs expression (Figure 2c), PE efficiency reached up to 40% in rice, 21.5% in maize and 74.5% in wheat, which are sufficiently high for practical applications in plant breeding (Li et al ., 2022b; Ni et al ., 2023; Qiao et al ., 2023).…”

“…Several recent data show improvements in PE in different aspects of its reaction (Chen and Liu, 2023). However, PE applications in plants have shown inconsistency among different loci and plant species and still require much effort to improve efficiency, especially in dicot plants (Chen and Liu, 2023; Jiang et al ., 2022c; Lu et al ., 2021; Vu et al ., 2022b; Wang et al ., 2021a). Therefore, this work aims to dissect the PE mechanism, update the recent advances in PE engineering and discuss several major technical issues to be considered to improve PE performance in plants.…”