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

Magdalena Klimek-Chodacka,Rafal Baranski,Miron Gieniec

doi:10.3390/ijms221910740

Magdalena Klimek-Chodacka, Rafal Baranski + Show 1 more

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https://doi.org/10.3390/ijms221910740

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Abstract

The aim of this work was to show an efficient, recombinant DNA-free, multiplex gene-editing method using gRNA:Cas9 ribonucleoprotein (RNP) complexes delivered directly to plant protoplasts. For this purpose, three RNPs were formed in the tube, their activity was confirmed by DNA cleavage in vitro, and then they were delivered to carrot protoplasts incubated with polyethylene glycol (PEG). After 48 h of incubation, single nucleotide deletions and insertions and small deletions at target DNA sites were identified by using fluorescent-PCR capillary electrophoresis and sequencing. When two or three RNPs were delivered simultaneously, long deletions of 33–152 nt between the gRNA target sites were generated. Such mutations occurred with an efficiency of up to 12%, while the overall editing effectiveness was very high, reaching 71%. This highly efficient multiplex gene-editing method, without the need for recombinant DNA technology, can be adapted to other plants for which protoplast culture methods have been established.

Highlights

The development of a genome editing method, as a consequence of the discovery and research on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)and CRISPR-associated (Cas) proteins, was awarded the Nobel Prize in Chemistry in2020
Cleaved DNA products were detected for all three RNP complexes by fluorescent-PCR capillary electrophoresis (Supplementary Figure S1)
The cleavage efficiency depended on the gRNA present in the RNP complex and was the highest for RNP2, for which half of the DNA molecules were cleaved during 15 min of incubation

Summary

Introduction

The development of a genome editing method, as a consequence of the discovery and research on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)and CRISPR-associated (Cas) proteins, was awarded the Nobel Prize in Chemistry in2020 (https://www.nobelprize.org, accessed on 27 August 2021). CRISPR/Cas-based tools, despite their recent development, have been preferentially and widely utilized for genome editing in eukaryotes, including horticultural crops [2], due to their simplicity and efficiency in generating small indel mutations, precise single-base editing, and sequence correction [3].Improved systems utilizing inactivated Cas proteins fused to effectors are being developed to facilitate regulation of gene expression, targeted epigenetic modification, and in vivo labeling [4,5]. CRISPR/Cas-mediated genome editing revolutionizes basic research and opens up new possibilities for the development of plants with improved and novel traits of economic and nutritional importance [6], as exemplified by the gene-edited soybean with high nutritional value that has recently been introduced into agriculture, and whose high oleic oil is available on the US market [7]. The DNA break results from the cleavage activity of a ribonucleoprotein (RNP) complex consisting of a short oligoribonucleotide, a guide

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