Improvingmultiplex genome editing in rice by‐combo‐mediatedactivation
Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine
SUMMARY The recently developed CRISPR‐Combo technology enables simultaneous targeted mutagenesis and transcriptional activation in plants. However, its reliance on SpCas9 limits its use at AT‐rich genomic loci, such as promoter regions commonly targeted for transcription activation. To overcome this limitation, we explored the usage of Cas12b and iSpyMacCas9 in the CRISPR‐Combo architecture for simultaneous genome editing and gene activation. We tested these expanded CRISPR‐Combo systems for hormone‐free regeneration of rice plants by transcriptional activation of a morphogenic gene,, while knocking out the genes of interest. The Cas12b‐Combo system induced mildupregulation (~3‐fold), which did not affect the genome editing efficiency. By contrast, iSpyMacCas9‐Combo achieved approximately 12‐foldtranscriptional activation, supporting hormone‐free regeneration at a high rate (42%). As a result, iSpyMacCas9‐Combo conferred higher genome editing efficiency, including improved multiplex editing, than the standard iSpyMacCas9 system, either with or without hormones during rice regeneration. Hence, our data prove iSpyMacCas9‐Combo to be a more efficient system for genome editing in rice, especially at low‐efficiency target sites, when coupled withtranscriptional activation. These findings establish iSpyMacCas9‐Combo as a useful addition to the CRISPR‐Combo toolkit, expanding its genomic targeting scope and enabling more efficient genome editing by activation of an appropriate endog
Abstract
SUMMARY The recently developed CRISPR‐Combo technology enables simultaneous targeted mutagenesis and transcriptional activation in plants. However, its reliance on SpCas9 limits its use at AT‐rich genomic loci, such as promoter regions commonly targeted for transcription activation. To overcome this limitation, we explored the usage of Cas12b and iSpyMacCas9 in the CRISPR‐Combo architecture for simultaneous genome editing and gene activation. We tested these expanded CRISPR‐Combo systems for hormone‐free regeneration of rice plants by transcriptional activation of a morphogenic gene,, while knocking out the genes of interest. The Cas12b‐Combo system induced mildupregulation (~3‐fold), which did not affect the genome editing efficiency. By contrast, iSpyMacCas9‐Combo achieved approximately 12‐foldtranscriptional activation, supporting hormone‐free regeneration at a high rate (42%). As a result, iSpyMacCas9‐Combo conferred higher genome editing efficiency, including improved multiplex editing, than the standard iSpyMacCas9 system, either with or without hormones during rice regeneration. Hence, our data prove iSpyMacCas9‐Combo to be a more efficient system for genome editing in rice, especially at low‐efficiency target sites, when coupled withtranscriptional activation. These findings establish iSpyMacCas9‐Combo as a useful addition to the CRISPR‐Combo toolkit, expanding its genomic targeting scope and enabling more efficient genome editing by activation of an appropriate endogenous gene such asin rice. Significance Statement This study establishes iSpyMacCas9‐Combo as an expanded CRISPR‐Combo platform that enables efficient simultaneous genome editing and transcriptional activation in rice, particularly at AT‐rich or low‐efficiency target sites. By activating endogenous, iSpyMacCas9‐Combo supports hormone‐free regeneration and improves multiplex genome editing efficiency, broadening the utility of CRISPR‐Combo for plant genome engineering. graphical