Chao Feng, Handong Su , Bai Han , Rui Wang , Yalin Liu , Xianrui Guo , Chang Liu , Jing Zhang , Jing Yuan , James A. Birchler , Fangpu Han

Plant Biotechnology March 23 2018

ABSTRACT

Previous studies revealed that the promoters for driving both Cas9 and sgRNAs are quite important for efficient genome editing by CRISPR/Cas9 in plants. Here we report our results of targeted genome editing using the maize dmc1 gene promoter combined with the U3 promoter for Cas9 and sgRNA, respectively. Three loci in the maize genome were selected for targeting. The T0 plants regenerated were highly‐efficiently edited at the target sites with homozygous or bi‐allelic mutants accounting for about 66%. The mutations in T0 plants could be stably transmitted to the T1 generation and new mutations could be generated in gametes or zygotes. Whole genome re‐sequencing indicated that no off‐target mutations could be detected in the predicted loci with sequence similarity to the targeted site. Our results show that the dmc1 promoter‐controlled (DPC) CRISPR/Cas9 system is highly efficient in maize and provide further evidence that the optimization of the promoters used for the CRISPR/Cas9 system is important for enhancing the efficiency of targeted genome editing in plants. The evolutionary conservation of the dmc1 gene suggests its potential for use in other plant species.

See: https://onlinelibrary.wiley.com/doi/abs/10.1111/pbi.12920

Figure. Schematic illustration of DPC CRISPR/Cas9 expression T-DNA. Maize dmc1 gene and U3 promoters were used for Cas9 and sgRNA, respectively.

(a) Schematic illustration of the target site in the zb7 gene. Black box indicates exons, while the lines between them represent introns. Underlined sequence was selected for targeting; ucleotides marked in blue represent PAM (protospacer adjacent motif). (b) RFLP assay of the genomic DNA of 4 transgene-positive calli (#1-#4) in the first batch. For each callus three independent samples were collected and used. Lane 1-3, calli #1; lane 4-6, calli #2; lane 7-9, calli #3; lane 10-12, calli #4; lane13, control (wild type DNA amplicons digested with PvuII). M, DNA marker. Primer pair zb7-F/zb7-R was used for PCR amplification, Pvu II was used for digestion. (c) Mutation analysis of two transgene-positive calli by cloning followed by Sanger sequencing. #3, #4 were the two calli sample with homozygous or bi-allelic mutations by RFLP assay. (d) Sanger sequencing results of mutations in -regenerated seedlings. #1-3, #1-6, #3-8, #4-1 were selected seedlings used for analysis. Left, sequencing chromatograph and right, the edited sequences at the target site. The yellow box indicates the PvuII site.