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

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aBIOTECH 2024年第2期5卷 151-168页

作者： Debasmita Panda subhasis karmakar Manaswini Dash Swagat Kumar Tripathy Priya Das Sagar Banerjee Yiping Qi Sanghamitra Samantaray Pradipta Kumar Mohapatra Mirza J.Baig Kutubuddin A.Molla ICAR National Rice Research Institute CuttackOdisha 753006India Department of Botany Ravenshaw UniversityCuttackOdisha 753003India Department of Plant Science and Landscape Architecture University of MarylandCollege ParkMD 20742USA

The CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic ***-predicted guide RNAs(gRNAs)often fail to induce efficient cleavage at target *** target loci are inaccessible due to complex chromatin ***,there is no suitable tool available to predict the architecture of genomic target sites and their ***,significant time and resources are spent on performing editing experiments with inefficient *** in vitro-cleavage assay could provide a rough assessment of gRNA efficiency,it largely excludes the interference of native genomic *** in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic ***,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 *** have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and *** introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice,Arabidopsis,and chickpea *** dual sgRNAs,our CRISPRdeletion strategy offers straightforward detection of genome editing success by simple agarose gel *** sequencing of PCR products confirmed the editing efficiency of specific ***,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 *** high-efficiency protocol for protoplast isolation and transfection enables rapid(less than 7 days)validation of sgRNA cleavage efficiency before proceeding with stable *** isolation and transfection method can als

关键词： Protoplast Genome editing Transgene-free edited plants Rice Chickpea

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