Enhanced editing efficiency in Arabidopsis with a LbCas12a variant harboring D156R and E795L mutations

Cas12a(Cpf1),a Class 2 Type V CRISPR/Cas nuclease,has several unique attributes for genome editing and may provide a valuable alternative to Cas9.However,a low editing efficiency due to temperature sensitivity and insufficient cleavage activity of the Cas12a nuclease are major obstacles to its broad application.In this report,we generated two variants,ttAsCas12 Ultra and ttLbCas12a Ultra harboring three(E174R,M537R,and F870L)or two(D156R and E795L)mutations,respectively,by combining the mutations from the temperature-tolerant variants ttAsCas12a(E174R)and ttLbCas12a(D156R),and those from the highly active variants AsCas12a Ultra(M537R and F870L)and LbCas12a Ultra(E795L).We compared editing efficiencies of the five resulting Cas12a variants(LbCas12a,ttLbCas12a,ttLbCas12a Ultra,AsCas12a Ultra,and ttAsCas12 Ultra)at six target sites of four genes in Arabidopsis(Arabidopsis thaliana).The variant ttLbCas12a Ultra,harboring the D156R and E795L mutations,exhibited the highest editing efficiency of all variants tested in Arabidopsis and can be used to generate homozygous or biallelic mutants in a single generation in Arabidopsis plants grown at 22 C.In addition,optimization of ttLbCas12a Ultra,by varying nuclear localization signal sequences and codon usage,further greatly improved editing efficiency.Collectively,our results indicate that ttLbCas12a Ultra is a valuable alternative to Cas9 for editing genes or promoters in Arabidopsis.

Ethylene Response Factor TERF1 Enhances Glucose Sensitivity in Tobacco through Activating the Expression of Sugar-related Genes

Ethylene response factor （ERF） proteins are important plant-specific transcription factors. Increasing evidence shows that ERF proteins regulate plant pathogen resistance, abiotic stress response and plant development through interaction with different stress responsive pathways. Previously, we revealed that overexpression of TERF1 in tobacco activates a cluster gene expression through interacting with GCC box and dehydration responsive element （DRE）, resulting in enhanced sensitivity to abscisic acid （ABA） and tolerance to drought, and dark green leaves of mature plants, indicating that TERF1 participates in the integration of ethylene and osmotic responses. Here we further report that overexpression of TERF1 confers sugar response in tobacco. Analysis of the novel isolated tomato TERF1 promoter provides information indicating that there are many cis-acting elements, including sugar responsive elements （SURE） and W box, suggesting that TERF1 might be sugar inducible. This prediction is confirmed by results of reverse transcription-polymerase chain reaction amplification, indicating that transcripts of TERF1 are accumulated in tomato seedlings after application of glucose. Further investigation indicates that the expression of TERF1 in tobacco enhances sensitivity to glucose during seed germination, root and seedling development, showing a decrease of the fresh weight and root elongation under glucose treatment. Detailed investigations provide evidence that TERF1 interacts with the sugar responsive cis-acting element SURE and activates the expression of sugar response genes, establishing the transcriptional regulation of TERF1 in sugar response. Therefore, our results deepen our understanding of the glucose response mediated by the ERF protein TERF1 in tobacco.

Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS

Dear Editor,Prime editing is a novel and universal CRISPR-Cas-derived precise genome-editing technology and has great potentials for applications in basic plant research and crop molecular breeding(Anzalone et al.,2019).Although low efficiency has restrained the original prime editors(PEs)from being used as a routine tool for precise genome editing in plants,an iterative update of the PEs is removing this obstacle(Lin et al.,2021;Xu et al.,2022).Recently,the Liu group reported three optimization strategies for improving prime editing efficiency(Chen et al.,2021;Nelson et al.,2022).The first strategy is based on engineered prime editing guide RNAs(epegRNAs),which were generated by incorporating structured RNA motifs to the 3′terminus of pegRNAs.This strategy enhances pegRNA stability and prevents degradation of the 3′extension(Nelson et al.,2022).The second strategy is based on the optimized PE2 protein(PEmax),which harbors a SpCas9 variant with increased nuclease activity,an additional nuclear localization signal(NLS)sequences,and a new linker between nCas9 and reverse transcriptase(Chen et al.,2021).The third strategy is based on inhibition of DNA mismatch repair(MMR)in cells(Chen et al.,2021).In this work,we tested the optimized PEs generated with these three strategies in rice,demonstrating that the optimized PEs greatly improved prime editing efficiency in rice.We named the two optimized PEs ePE3max and ePE5max:the former is comprised of the PEmax protein,an epegRNA with evopreQ1 appended to its 3′end,and a nicking sgRNA;the latter is comprised of the ePE3max system and a dominant negative OsMLH1 variant for inhibiting MMR.Using the two optimized PEs,we efficiently generated homozygous and heterozygous T173I,A174V,and P177S(TAP-IVS)mutation in EPSPS in rice,which lays a solid foundation for rice non-transgenic glyphosate-resistance breeding.