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

Wenzhi Jiang,Jen Sheen,Jenifer Bush

doi:10.3389/fgeed.2021.719190

Wenzhi Jiang, Jen Sheen + Show 1 more

Open Access

https://doi.org/10.3389/fgeed.2021.719190

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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.

Highlights

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)
Two reporter genes were developed to facilitate sensitive detection of nonhomologous end joining (NHEJ) or homology-directed repair (HDR) leading to the recovery of GFP fluorescence in transfected protoplasts within 24 h. We demonstrated that this versatile protoplast platform could be applied to four plant species and support precise and efficient mutagenesis by HDR using relatively short single-stranded oligodeoxynucleotide (ssODN) donors without costly modifications
A. thaliana, N. benthamiana, B. rapa and C. sativa, were used in this study to illustrate the selection of optimal leaves at a specific age grown in a defined environmental condition (Figure 1A)

Summary

Introduction

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). The versatile plant protoplast platform with high transfection efficiency (Yoo et al, 2007; Li et al, 2013, 2014, 2015; Marx, 2016) is highly suitable in supporting the systematic efforts required for developing and testing new CRISPR-Cas designs and elucidating relevant molecular mechanisms in broad plant species (Woo et al, 2015; Jin et al, 2021; Zhang et al, 2021)

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