Optimized protoplast isolation and transfection with a breakpoint:accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot

The CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic loci.Software-predicted guide RNAs(gRNAs)often fail to induce efficient cleavage at target loci.Many target loci are inaccessible due to complex chromatin structure.Currently,there is no suitable tool available to predict the architecture of genomic target sites and their accessibility.Hence,significant time and resources are spent on performing editing experiments with inefficient guides.Although in vitro-cleavage assay could provide a rough assessment of gRNA efficiency,it largely excludes the interference of native genomic context.Transient in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic context.Here,we developed a modified protocol that offers highly efficient protoplast isolation from rice,Arabidopsis,and chickpea,using a sucrose gradient,transfection using PEG(polyethylene glycol),and validation of single guide RNAs(sgRNAs)cleavage efficiency of CRISPR-Cas9.We have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and dicots.We introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice,Arabidopsis,and chickpea protoplasts.Using dual sgRNAs,our CRISPRdeletion strategy offers straightforward detection of genome editing success by simple agarose gel electrophoresis.Sanger sequencing of PCR products confirmed the editing efficiency of specific sgRNAs.Notably,we demonstrated that isolated protoplasts can be stored for up to 24/48 h with little loss of viability,allowing a pause between isolation and transfection.This high-efficiency protocol for protoplast isolation and transfection enables rapid(less than 7 days)validation of sgRNA cleavage efficiency before proceeding with stable transformation.The isolation and transfection method can also be utilized for rapid validation of editing strategies,evaluating diverse editing reagents,regenerating plants from transfected protoplasts,gene expression studies,protein localization and functional analysis,and other applications.

Single-nucleotide editing for zebra3 and wsl5 phenotypes in rice using CRISPR/Cas9-mediated adenine base editors

The CRISPR/Cas9-mediated base editing technology can efficiently generate point mutations in the genome without introducing a double-strand break(DSB)or supplying a DNA donor template for homology-directed repair(HDR).In this study,adenine base editors(ABEs)were used for rapid generation of precise point mutations in two distinct genes,OsWSL5,and OsZEBRA3(Z3),in both rice protoplasts and regenerated plants.The precisely engineered point mutations were stably inherited to subsequent generations.These single nucleotide alterations resulted in single amino acid changes and associated wsl5 and z3 phenotypes as evidenced by white stripe leaf and light green/dark green leaf pattern,respectively.Through selfing and genetic segregation,transgene-free,base edited wsl5 and z3 mutants were obtained in a short period of time.We noticed a novel mutation(V540A)in Z3 locus could also mimic the phenotype of Z3 mutation(S542P).Furthermore,we observed unexpected non-A/G or T/C mutations in the ABE editing window in a few of the edited plants.The ABE vectors and the method from this study could be used to simultaneously generate point mutations in multiple target genes in a single transformation and serve as a useful base editing tool for crop improvement as well as basic studies in plant biology.