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

International Journal of Molecular Sciences

Magdalena Klimek-Chodacka¹  Rafal Baranski¹  Miron Gieniec¹ 

[1] Department of Plant Biology and Biotechnology, Faculty of Biotechnology and Horticulture, University of Agriculture in Krakow, AL. 29 Listopada 54, 31-425 Krakow, Poland;

关键词: Cas9 protein;    Daucus carota;    genome editing;    PEG;    targeted mutagenesis;   

DOI  :  10.3390/ijms221910740

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown