Versatile Nucleotides Substitution in Plant Using an Improved Prime Editing System

Abstract

In addition, for BEs, the target bases are limited to an editing window of at least three nucleotides (nt) away from the 5 0 end of the protospacer adjacent motif (PAM), and undesired base substitutions are common, especially when several Cs or As coexist in the editing window (Chen et al., 2019). Although CRISPR/Cas-mediated homologous recombination provides an alternative method for precise targeted gene replacement or gene insertion in plants, it is limited by the low efficiency (Miki et al., 2018). Most recently, a ''search-and-replace'' genome editing technology called prime editing was developed in human cells, which can mediate all 12 possible base-to-base conversions using a reverse transcriptase (RT) paired with CRISPR-Cas9 nickase (Cas9n) (H840A) and a prime editing guide RNA (pegRNA), without DSBs or donor DNA (Anzalone et al., 2019).

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

“…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. These results indicate that the reported primeediting tools require significant optimization before they are adopted for use by plant researchers.…”

“…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.…”