CRISPR ribonucleoprotein-mediated genetic engineering in plants

CRISPR-derived biotechnologies have revolutionized the genetic engineering field and have been widely applied in basic plant research and crop improvement. Commonly used Agrobacterium- or particle bombardment-mediated transformation approaches for the delivery of plasmid-encoded CRISPR reagents can result in the integration of exogenous recombinant DNA and potential off-target mutagenesis. Editing efficiency is also highly dependent on the design of the expression cassette and its genomic insertion site. Genetic engineering using CRISPR ribonucleoproteins (RNPs) has become an attractive approach with many advantages: DNA/transgene-free editing, minimal off-target effects, and reduced toxicity due to the rapid degradation of RNPs and the ability to titrate their dosage while maintaining high editing efficiency. Although RNP-mediated genetic engineering has been demonstrated in many plant species, its editing efficiency remains modest, and its application in many species is limited by difficulties in plant regeneration and selection. In this review, we summarize current developments and challenges in RNP-mediated genetic engineering of plants and provide future research directions to broaden the use of this technology. CRISPR-derived genome engineering technologies have been widely applied in plant science. Delivery of CRISPR reagents as RNPs is a promising approach for the generation of transgene-free genome-edited crops. This review summarizes recent progress, discusses present challenges, and presents some future perspectives on this topic.