CRISPR ribonucleoprotein-mediated genetic engineering in plants

Abstract

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.

Multiplex Site-Directed Gene Editing Using Polyethylene Glycol-Mediated Delivery of CRISPR gRNA:Cas9 Ribonucleoprotein (RNP) Complexes to Carrot Protoplasts

“…However, it should be emphasized that different methods can be used to estimate mutation efficiency making direct comparisons ambiguous. Nevertheless, the mutation efficiency obtained in this work reached 71%, which is one of the highest reported to date [ 34 ]. Such high efficiency was partly due to the simultaneous use of RNPs cleaving two independent targets and was more than double that of using a single RNP.…”

“…Carrot protoplasts start to recover their cell wall no earlier than three days after the initiation of culture, and the first cell divisions are observed within the following days [ 36 ]. Pre-assembled RNP complexes are delivered to cells and are not synthesized de novo, so their number in the cell decreases over time due to their natural degradation and separation to daughter cells at each subsequent cell division [ 34 , 37 ]. Moreover, editing efficiency reaches a plateau in prolonged culture [ 38 ].…”

A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs

“…Successful CRISPR-Cas9-gRNA-mediated genome editing in plant cells was first demonstrated in protoplasts isolated from Arabidopsis, tobacco, rice and wheat with varying degrees of efficiency for various targets in different plants ( Li et al, 2013 ; Shan et al, 2013 ). Despite widespread applications of CRISPR technologies via NHEJ-mediated mutagenesis in basic plant research and crop improvement ( Zhu et al, 2020 ), the underlying mechanisms for variable editing efficiency and limited precise editing through HDR remain unresolved challenges for future advances ( Chechik et al, 2020 ; Zhang et al, 2021 ). Recent CRISPR-Cas9 innovations have significantly expanded the range of genetic variants by PEs ( Anzalone et al, 2019 ; Lin et al, 2020 , Jin et al, 2021 ) and enabled targeted insertion and replacement ( Lu et al, 2020 ).…”

Utilizing CRISPR-Cas in Tropical Crop Improvement: A Decision Process for Fitting Genome Engineering to Your Species

“…However, the integration of transgenes is non-specific and sometimes unstable ( Jaganathan et al, 2018 ). Transfection techniques are possible because the gene editing reagents can be assembled as RNP complexes or RNA molecules in vitro ( Zhang Y. et al, 2021 ) and subsequently delivered to embryogenic calli via particle bombardment or introduced in isolated protoplasts via transfection ( Liang et al, 2018b ). The advantage of using the transfection approach is that the edited events are transgene-free, which may or may not influence regulatory processes required for commercial release of gene edited crops.…”