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 T 0 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.

Prime editing is achievable in the tetraploid potato, but needs improvement

“…Very recently, several reports have shown that prime editing can be applied to cereal plants Lin et al, 2020;Tang et al, 2020;Xu et al, 2020a;Xu et al, 2020b;Butt et al;Hua et al). All these studies showed that the efficiency of PPEs greatly varies between target sites and is sensitive to several parameters such as both the PBS and RT sequence.…”

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

“…Recently, a novel and universal precision genome-editing technology named prime editing was developed and tested in mammalian cells [1]. More recently, seven groups reported applications of prime editors (PE) in rice and wheat, and six groups achieved prime-edited rice lines [2][3][4][5][6][7][8]. Excluding the data based on enriching strategies or that are unsuitable for comparison, a total of 164 prime-edited rice lines transformed with 39 prime editors targeting 11 endogenous rice genes were generated in these previous reports [2,3,5,6,8].…”

“…More recently, seven groups reported applications of prime editors (PE) in rice and wheat, and six groups achieved prime-edited rice lines [2][3][4][5][6][7][8]. Excluding the data based on enriching strategies or that are unsuitable for comparison, a total of 164 prime-edited rice lines transformed with 39 prime editors targeting 11 endogenous rice genes were generated in these previous reports [2,3,5,6,8]. The highest editing efficiencies achieved by the five groups ranged from 2.22% to 31.3% [2,3,5,6,8].…”

“…Excluding the data based on enriching strategies or that are unsuitable for comparison, a total of 164 prime-edited rice lines transformed with 39 prime editors targeting 11 endogenous rice genes were generated in these previous reports [2,3,5,6,8]. The highest editing efficiencies achieved by the five groups ranged from 2.22% to 31.3% [2,3,5,6,8]. Of the total 164 primeedited rice lines, only one line, generated by Xu et al [6], harbors homozygous mutations; all the other lines mainly harbor chimeric mutations.…”

SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition

“…Recently, the SpCas9(H840A) nickase-guided Moloney murine leukemia virus reverse transcriptase (M-MLV RT) has been developed in both rice and wheat, facilitating the introduction of all 12 base-to-base conversions, small insertion as well as deletions in a precise and targeted manner through reverse transcription of the pegRNA [14][15][16][17].…”