Hyeran Kim, Jisun Choi & Kang-Hee Won

BMC Plant Biology volume 20, Article number: 449 (2020) 

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.

See: https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-020-02665-0

Figure 2: In vitro cleavage assay for CRISPR/Cas9 or CRISPR/Cpf1 RNP-mediated CaMLO2 gene in two peppers. a Target locus of CaMLO2 gene, four designed guide RNAs (sgRNA1 and sgRNA2 for Cas9 and crRNA1 and crRNA2 for LbCpf1) and a specific primer pair, F and R. b Target sequences of the four guide RNAs. c In vitro cleavage assay with preassembled Cas9-only as a control, Cas9-sgRNA1 and Cas9-sgRNA2 for CaMLO2 gene of CM334 or Dempsey. d In vitro cleavage assay with preassembled LbCpf1-only as a control, LbCpf1-crRNA1 and LbCpf1-crRNA2 for CaMLO2 gene of CM334 and Dempsey.