Abstract
Delivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target editing effects, mitigation of random integration of non-native DNA fragments, independence of vector constructions, and less regulatory restrictions. Compared to the use in animal systems, RNP-mediated genome editing is still at the early development stage in plants. In this study, we established an efficient and simplified protoplast-based genome editing platform for CRISPR-Cas RNP delivery, and then evaluated the efficiency, specificity, and temperature sensitivity of six Cas9 and Cas12a proteins. Our results demonstrated that Cas9 and Cas12a RNP delivery resulted in genome editing frequencies (8.7–41.2%) at various temperature conditions, 22°C, 26°C, and 37°C, with no significant temperature sensitivity. LbCas12a often exhibited the highest activities, while AsCas12a demonstrated higher sequence specificity. The high activities of CRISPR-Cas RNPs at 22° and 26°C, the temperature preferred by plant transformation and tissue culture, led to high mutagenesis efficiencies (34.0–85.2%) in the protoplast-regenerated calli and plants with the heritable mutants recovered in the next generation. This RNP delivery approach was further extended to pennycress (Thlaspi arvense), soybean (Glycine max) and Setaria viridis with up to 70.2% mutagenesis frequency. Together, this study sheds light on the choice of RNP reagents to achieve efficient transgene-free genome editing in plants.
Highlights
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) systems were first discovered to cleave invading bacteriophage DNA as a prokaryotic adaptive immune system (Garneau et al, 2010; Horvath and Barrangou, 2010)
The use of CRISPR-Cas RNPs has been demonstrated in plant species, no systematic studies were conducted to provide the guideline for optimal selection of RNP reagents (Zhang et al, 2021b)
After optimizing the PEGmediated RNP delivery procedure, we sought to evaluate and compare the efficiency, specificity and temperature sensitivity of the three most widely used CRISPR-Cas system delivered as RNPs: SpCas9, AsCas12a and LbCas12a
Summary
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) systems were first discovered to cleave invading bacteriophage DNA as a prokaryotic adaptive immune system (Garneau et al, 2010; Horvath and Barrangou, 2010). CRISPR-Cas and a targeting gRNA are delivered as plasmid DNA either by Agrobacterium- or biolistic-mediated transformation methods (Atkins and Voytas, 2020) With these approaches, the plasmids usually need to integrate into the genome, and the expressed Cas protein and CRISPR RNA assemble to form functional RNPs for the targeted gene modifications. On the other hand, when the integrated cassettes are highly expressed, prolonged expression could lead to the increasing possibility of off-target mutations, and chimeric mutants (Chen et al, 2019) To overcome these challenges, several strategies have been developed to achieve DNA-free gene editing in plants. Our work indicated that RNP-mediated gene editing in plant protoplasts is a promising technique for quickly screening and optimizing the CRISPR-Cas systems as well as an effective platform for generating transgene-free gene edited plants
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