A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in Cotton (Gossypium hirsutum L.)

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Chao Li (Creator)

Turgay Unver (Creator)

Baohong Zhang (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

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.

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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).

Language: English

Date: 2017

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