A stable DNA-free screening system for CRISPR/RNPs-mediated gene editing in hot and sweet cultivars of Capsicum annuum
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RESEARCH ARTICLE
Open Access
A stable DNA-free screening system for CRISPR/RNPs-mediated gene editing in hot and sweet cultivars of Capsicum annuum Hyeran Kim* , Jisun Choi† and Kang-Hee Won†
Abstract Background: DNA-free, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) ribonucleoprotein (RNP)-based genome editing is a simple, convincing, and promising tool for precision crop breeding. The efficacy of designed CRISPR-based genome editing tools is a critical prerequisite for successful precision gene editing in crops. Results: This study demonstrates that soil-grown leaf- or callus-derived pepper protoplasts are a useful system for screening of efficient guide RNAs for CRISPR/Cas9 or CRISPR/Cas12a (Cpf1). CRISPR/Cas9 or Cpf1 were delivered as CRISPR/RNP complexes of purified endonucleases mixed with the designed single guide RNA, which can edit the target gene, CaMLO2 in two pepper cultivars with whole genome sequenced, Capsicum annuum ‘CM334’ and C. annuum ‘Dempsey’. The designed guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) are conserved for CaMLO2 in both CM334 and Dempsey and cleave CaMLO2 in vitro. CRISPR/Cas9- or /Cpf1-RNP complexes were transfected into purely isolated protoplasts of the hot pepper CM334 and sweet pepper Dempsey by PEG-mediated delivery. Targeted deep sequencing analysis indicated that the targeted CaMLO2 gene was differentially edited in both cultivars, depending on the applied CRISPR/RNPs. Conclusions: Pepper protoplast-based CRISPR guide-RNA selection is a robust method to check the efficacy of designed CRISPR tools and is a prerequisite for regenerating edited plants, which is a critical time-limiting procedure. The rapid and convincing selection of guide RNA against a target genome reduces the laborious efforts for tissue culture and facilitates effective gene editing for pepper improvement. Keywords: Pepper genome editing, Capsicum annuum CM334, C. annuum Dempsey, CRISPR/Cas9, CRISPR/LbCpf1, Pepper leaf protoplasts, Pepper callus protoplasts
Background Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas), CRISPR/Cas9 has emerged as the first RNA-guided genome-editing tool to introduce a target mutation in any sequenced genome after being reported as a programmable molecular scissor in 2012 [1]. Since obtaining SpCas9-based CRISPR tools * Correspondence: [email protected] † Jisun Choi and Kang-Hee Won contributed equally to this work. Department of Biological Sciences, Kangwon National University, Kangwondaehak-gil 1, Chuncheon 24341, South Korea
from Streptococcus pyogenes, various tools have been developed from different strains, such as Staphylococcus aureus Cas9 [2], Francisella novicida Cas9 [3], Streptococcus thermophilus Cas9 [4], and Campylobacter jejuni Cas9 [5]. These developed CRISPR-based tools have been promptly applied to all kinds of research areas from generating knock-out cell lines and organisms to biotechnology of animals [6, 7], plants [8–11], and humans [12–14]. CRISPR/
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