High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system

Abstract

Summary Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene‐specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site‐specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2‐edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7–100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode‐based high‐throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1‐edited T0 plants and it matched well with Sanger sequencing results. No off‐target editing was detected at the potential off‐target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing.

Whole genome sequencing reveals rare off‐target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9‐edited cotton plants

“…Recently, we established the CRISPR/Cas9 ( Streptococcus pyogenes Cas9) system in cotton with high efficiency. Interestingly, no off‐target mutations were detected by targeted deep sequencing in the top 26 potential off‐target sites of four sgRNAs, suggesting that the CRISPR/Cas9 system is an efficient and reliable tool for allotetraploid cotton genome editing (Wang et al ., ). To further investigate the specificity of CRISPR/Cas9 system on the genome‐wide scale in cotton genome editing, three target genes – encoding the AP2/B3‐like transcription factor ( AP2 ), MYB44 transcription factor ( MYB44 ) and nucleotide‐binding (NB)‐ARC domain‐containing disease resistance protein ( ARC ) were edited using CRISPR/Cas9 in this study (Li et al ., ).…”

“…Multiple types of mutations were detected at the specific on‐target sites (Figure S1a and Table S3). This editing efficiency is comparable to the efficiency (66%–100%) when editing exogenous transgene Discosoma red fluorescent protein ( dsRed2 ) and an endogenous cotton gene GhCLA1 in our recent report (Wang et al ., ). We found no bias on the editing efficiency between At‐ and Dt‐subgenome loci based on WGS data, but different mutation patterns (Figure S1b).…”

“…For diploid plant species, such as Arabidopsis and rice, the mutation profile is relatively simple, with four potential editing types: homozygous mutations, biallelic mutations, heterozygous mutations and no mutation (Feng et al ., ). However, polyploid species such as cotton potentially have more complex mutation profiles (Wang et al ., ). Off‐target mutations in CRISPR/Cas9‐edited cells can further add complexity to the mutation analysis, and this is emerging as the major concern for this promising technology.…”

Precise editing of CLAVATA genes in Brassica napus L. regulates multilocular silique development

“…Recently, various genome‐editing methods, particularly methods involving sequence‐specific nucleases (SSNs) for creating targeted double‐strand breaks (DSBs), have emerged as major breakthroughs in site‐specific genome editing (Cong et al ., ; Li et al ., ; Wood et al ., ). In particular, CRISPR/CRISPR‐associated 9 (CRISPR/Cas9) is considered the most simple and effective SSN developed thus far and has been used for genome editing in major crops, including rice (Jiang et al ., ; Miao et al ., ; Shan et al ., ), sorghum (Jiang et al ., ), tobacco (Gao et al ., ), wheat (Shan et al ., ; Wang et al ., ), maize (Liang et al ., ), barley (Lawrenson et al ., ), cotton (Wang et al ., ), tomato (Brooks et al ., ), soya bean (Li et al ., ) and camelina (Jiang et al ., ). To date, few studies have reported successful site‐directed mutagenesis using the Cas9/single‐guide RNA (sgRNA) system in rapeseed (Braatz et al ., ).…”

Recent insights into cotton functional genomics: progress and future perspectives

“…In cotton, the application of CRISPR/Cas9 is still at its infancy. Most recently, multiple sites genome editing through CRISPR/Cas9 system in allotetraploid cotton by targeting arginase ( GhARG ), discosoma red fluorescent protein2 ( DsRed2 ) and chloroplast development ( GhCLA1 ) genes proves that it is highly reliable and effective for cotton genome editing (Wang et al ., ,). It is expected that the potential and applications of CRISPR/Cas9 in cotton genome editing are certain to be further developed over time.…”