Abstract
Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.
Highlights
Substantial efforts are being made to optimize the CRISPR/Cas[9] system for precision crop breeding
We showed that transient expression of CRISPR/Cas[9] DNA or RNA (TECCDNA or TECCRNA) in wheat resulted in efficient genome editing with significantly reduced transgene integration[3]
As the first step in our work, we tested if CRISPR/Cas[9] RNPs may cleave targeted genomic sites and induce mutations in wheat protoplasts using the gw2-sgRNA that we previously found to be highly active on the TaGW2 gene[3]
Summary
Substantial efforts are being made to optimize the CRISPR/Cas[9] system for precision crop breeding. Because no foreign DNA is used in CRISPR/Cas[9] RNP mediated genome editing, the mutants obtained are completely transgene free This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized. At present, the biosecurity of genome-edited plants is an important public concern[7] In response to this concern, substantial efforts are being made to optimize CRISPR/Cas[9] mediated genome editing with the aim of avoiding transgene integration and off-target mutations. Woo et al.[8] demonstrated that the use of preassembled CRISPR/Cas[9] ribonucleoproteins (RNPs) completely avoided transgene integration, and greatly decreased off-target mutations They delivered the RNPs into lettuce protoplasts and obtained transgene-free mutant plants. We aimed to develop a CRISPR/Cas[9] ribonucleoprotein mediated genome editing method for efficient and specific genome editing of major monocot crops using hexaploid bread wheat (Triticum aestivum L., AABBDD, 2n 1⁄4 6x 1⁄4 42) as experimental material
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