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

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.

Wang P ，Zhang J ，Sun L ，Ma Y ，Xu J ，Liang S ，Deng J ，Tan J ，Zhang Q ，Tu L ，Daniell H ，Jin S ，Zhang X ... -  《-》

High-efficient and precise base editing of C•G to T•A in the allotetraploid cotton (Gossypium hirsutum) genome using a modified CRISPR/Cas9 system.

The base-editing technique using CRISPR/nCas9 (Cas9 nickase) or dCas9 (deactivated Cas9) fused with cytidine deaminase is a powerful tool to create point mutations. In this study, a novel G. hirsutum-Base Editor 3 (GhBE3) base-editing system has been developed to create single-base mutations in the allotetraploid genome of cotton (Gossypium hirsutum). A cytidine deaminase sequence (APOBEC) fused with nCas9 and uracil glycosylase inhibitor (UGI) was inserted into our CRISPR/Cas9 plasmid (pRGEB32-GhU6.7). Three target sites were chosen for two target genes, GhCLA and GhPEBP, to test the efficiency and accuracy of GhBE3. The editing efficiency ranged from 26.67 to 57.78% at the three target sites. Targeted deep sequencing revealed that the C→T substitution efficiency within an 'editing window', approximately six-nucleotide windows of -17 to -12 bp from the PAM sequence, was up to 18.63% of the total sequences. The 27 most likely off-target sites predicted by CRISPR-P and Cas-OFFinder tools were analysed by targeted deep sequencing, and it was found that rare C→T substitutions (average < 0.1%) were detected in the editing windows of these sites. Furthermore, whole-genome sequencing analyses on two GhCLA-edited and one wild-type plants with about 100× depth showed that no bona fide off-target mutations were detectable from 1500 predicted potential off-target sites across the genome. In addition, the edited bases were inherited to T1 progeny. These results demonstrate that GhBE3 has high specificity and accuracy for the generation of targeted point mutations in allotetraploid cotton.

Qin L ，Li J ，Wang Q ，Xu Z ，Sun L ，Alariqi M ，Manghwar H ，Wang G ，Li B ，Ding X ，Rui H ，Huang H ，Lu T ，Lindsey K ，Daniell H ，Zhang X ，Jin S ... -  《-》

Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant.

Li B ，Fu C ，Zhou J ，Hui F ，Wang Q ，Wang F ，Wang G ，Xu Z ，Che L ，Yuan D ，Wang Y ，Zhang X ，Jin S ... -  《Cells》

Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system.

Chen X ，Lu X ，Shu N ，Wang S ，Wang J ，Wang D ，Guo L ，Ye W ... -  《Scientific Reports》

Efficient genome editing of Brassica campestris based on the CRISPR/Cas9 system.

Xiong X ，Liu W ，Jiang J ，Xu L ，Huang L ，Cao J ... -  《-》