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A streamlined guide RNA screening system for genome editing in Sorghum bicolor

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Title
A streamlined guide RNA screening system for genome editing in Sorghum bicolor
Author(s)
Jeong Sun Lee; Su-Ji Bae; Kim, Jin-Soo; Kim, Changsoo; Beum-Chang Kang
Publication Date
2023-08
Journal
Plant Methods, v.19, no.1
Publisher
BioMed Central Ltd
Abstract
Background: Genome editing tools derived from clustered regularly interspaced short palindromic repeats (CRISPR) systems have been developed for generating targeted mutations in plants. Although these tools hold promise for rapid crop improvement, target-specific guide RNAs exhibit variable activity. To improve genome editing, a rapid and precise method for evaluating their efficiency is necessary. Results: Here we report an efficient system for screening single guide RNAs (sgRNAs) for genome editing in sorghum using a transient protoplast transfection assay. Protoplasts were isolated from leaves from sorghum plants cultivated under three different conditions. Cultivation for three days of continuous darkness following seven days with a 16-h light and 8-h dark photoperiod resulted in the highest yield of viable protoplasts and the highest protoplast transfection efficiency. We tested both plasmid-mediated and ribonucleoprotein-based delivery to protoplasts, via polyethylene glycol-mediated transfection, of CRISPR components targeting the sorghum genome. The frequencies of small insertions and deletions induced by a set of sgRNAs targeting four endogenous sorghum genes were analyzed via targeted deep sequencing. Our screening system induced indels in sorghum protoplasts at frequencies of up to 77.8% (plasmid) and 18.5% (RNP). The entire screening system was completed within 16 days. Conclusions: The screening system optimized in this study for predicting sgRNA activity for genome editing in sorghum is efficient and straightforward. This system will reduce the time and effort needed for sorghum genome editing.
URI
https://pr.ibs.re.kr/handle/8788114/14277
DOI
10.1186/s13007-023-01058-2
ISSN
1746-4811
Appears in Collections:
Center for Genome Engineering(유전체 교정 연구단) > 1. Journal Papers (저널논문)
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