A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in Cotton (Gossypium hirsutum L.)
Loading...
Date
2017-03-03
Access
Authors
Zhang, Baohong
Li, Chao
Unver, Turgay
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
The complex allotetraploid genome is one of major challenges in cotton for repressing gene expression.
Developing site-specific DNA mutation is the long-term dream for cotton breeding scientists. The
clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is emerging as a robust biotechnology for targeted-DNA mutation. In this study, two sgRNAs, GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, were designed in the identical genomic regions of GhMYB25-like A and GhMYB25-like D, which were encoded by cotton A subgenome and the D subgenome, respectively, was assembled to direct Cas9-mediated allotetraploid cotton genome
editing. High proportion (14.2–21.4%) CRISPR/Cas9-induced specific truncation events, either from GhMYB25-like A DNA site or from GhMYB25-like D DNA site, were detected in 50% examined transgenic cotton through PCR amplification assay and sequencing analyses. Sequencing results also demonstrated that 100% and 98.8% mutation frequency were occurred on GhMYB25-like-sgRNA1
and GhMYB25-like-sgRNA2 target site respectively. The off-target effect was evaluated by sequencing
two putative off-target sites, which have 3 and 1 mismatched nucleotides with GhMYB25-like-sgRNA1
and GhMYB25-like-sgRNA2, respectively; all the examined samples were not detected any off-targetcaused mutation events. Thus, these results demonstrated that CRISPR/Cas9 is qualified for generating DNA level mutations on allotetraploid cotton genome with high-efficiency and high-specificity.
Description
Keywords
Citation
Li, C. et al. A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in Cotton (Gossypium hirsutum L.). Sci. Rep. 7, 43902; doi: 10.1038/srep43902 (2017).
DOI
10.1038/srep43902