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

Abstract

The ultimate goal of technology development in genome editing is to enable precisely targeted genomic changes in any cells or organisms. Here we describe protoplast systems for precise and efficient DNA sequence changes with preassembled Cas9 ribonucleoprotein (RNP) complexes in Arabidopsis thaliana, Nicotiana benthamiana, Brassica rapa, and Camelina sativa. Cas9 RNP-mediated gene disruption with dual gRNAs could reach ∼90% indels in Arabidopsis protoplasts. To facilitate facile testing of any Cas9 RNP designs, we developed two GFP reporter genes, which led to sensitive detection of nonhomologous end joining (NHEJ) and homology-directed repair (HDR), with editing efficiency up to 85 and 50%, respectively. When co-transfected with an optimal single-stranded oligodeoxynucleotide (ssODN) donor, precise editing of the AtALS gene via HDR reached 7% by RNPs. Significantly, precise mutagenesis mediated by preassembled primer editor (PE) RNPs led to 50% GFP reporter gene recovery in protoplasts and up to 4.6% editing frequency for the specific AtPDS mutation in the genome. The rapid, versatile and efficient gene editing by CRISPR RNP variants in protoplasts provides a valuable platform for development, evaluation and optimization of new designs and tools in gene and genomic manipulation and is applicable in diverse plant species.

Cytokinins: A Genetic Target for Increasing Yield Potential in the CRISPR Era

“…The integration position of transgenes can also influence their expression ( Matzke and Matzke, 1998 ). Furthermore, Cas protein expression based on DNA is toxic in some mammalian cell types and organisms ( Morgens et al, 2017 ; Foster et al, 2018 ; Jiang et al, 2021 ). To avoid the problems that come with DNA delivery, particle bombardment can be a alternative method to deliver mRNA encoding Cas proteins along with the gRNA(s) into the plant system ( Zhang et al, 2016 ).…”

“…It is also possible to use multiple types of CRISPR/Cas systems and orthogonal Cas proteins at the same time if there is no crosstalk between them, allowing for the achievement of multiple editing outcomes. CRISPR/Cas delivered via plasmid, on the other hand, causes cell or organism toxicity, which could be avoided by using RNP-mediated genome editing ( Shin et al, 2016 ; Foster et al, 2018 ; Jiang et al, 2021 ). Caenorhabditis elegans , human cells, mice and plants all have been shown to benefit from RNP-mediated genome editing ( Cho et al, 2013 ; Kim et al, 2014 ; Woo et al, 2015 ; Zuris et al, 2015 ; Foster et al, 2018 ; Li et al, 2020 ).…”

“…Even though RNP-mediated genome editing does not require transcription or translation, it can be completed as soon as cells are transfected with the RNP construct ( Kim et al, 2014 ). There is evidence of RNP-mediated genetic engineering in plant protoplasts, as well as RNP-edited plants ( Jiang et al, 2021 ). Therefore, RNP-based CRISPR technology has the potential to generate transgene-free and improved germplasm that can be more easily commercialised, it still faces numerous technological constraints and challenges.…”

Integrating CRISPR/Cas systems with programmable DNA nanostructures for delivery and beyond

“…Homology dependent gene editing frequencies are typically low, with the exception of a recent report of tobacco transformation that displayed ∼10% successful gene targeting ( Huang et al., 2021b ; Puchta et al., 2022 ). The plant cell wall can be removed with enzymes to produce protoplasts that are amenable to electroporation or polyethylene glycol-mediated approaches and transformation with preassembled Cas-RNP complexes resulted in ∼5% gene targeting frequencies ( Jiang et al, 2021b ). An exciting new development demonstrated in vivo biolistic transformation of wheat with Cas-RNPs, targeting meristem cells and avoiding the requirement for plant regeneration ( Kumagai et al., 2022 ).…”

Cytokinin biosynthesis in cyanobacteria: Insights for crop improvement

“…Combining rapidly evolving advancements in the CRISPR/ Cas tool with knowledge of the IPT or CKX gene function could speed up the development of new, higher-yielding cultivars that could shape future agricultural practices (Jiang et al, 2021).…”