Establishment And Optimization Of The Gene Editing System Of CRISPR/Cas9 In Sea Island Cotton

CRISPR/Cas9 has become a leading tool for targeted editing and precise repair of eukaryotes.Genome editing using CRISPR/Cas9 has been shown to be possible in all biological systems,from bacteria to complex eukaryotes like humans.This provides a key technical tool for studying functional genomics and deciphering biological genetic codes.However,great variation in the efficiency of on-targret and off-target mutagenesis between different organisms and at different target gene loci has been observed,even sometime no editing on target site.This has a negative effect on the wide application of CRISPR/Cas9 gene editing technology.Xinjiang is the only commercial production base of island cotton in China.The purpose of this study is to establish a CRISPR/Cas9 gene editing technology system in island cotton and optimize the system in order to improve the editing efficiency of genome and reduce the off-targget effect.The gene editing system will provide technical support for functional genomics research,cotton molecular design breeding and create new cotton germplasm.The main results are as follows:(1)Using two GbU6 promoters cloned from Cotton Island Xinhai 16,we constructed several CRISPR/Cas9 gene editing vectors with DNA fragments of Xinhai 16 endogenous gene target site.The core DNA fragments of CRISPR/Cas9 gene editing vector were enriched by PCR.Protoplasts from embryogenic callus of Xinhai 16 were prepared and were transformed using the core DNA fragments of CRISPR/Cas9 by PEG transient transformation.The genomic DNA of protoplast was extracted and digested first,then PCR,or direct PCR,and finally the PCR products were cloned and sequenced.The results showed that the mutation of endogenous target gene was detected successfully.The type of sequence mutation was mainly base substitution and a few base deletion.The results showed that the CRISPR/Cas9 gene editing system based on native U6 promoter could realize the function of target gene editing in Sea-island cotton,indicating that the system could be used for creation cotton mutants and molecular design breeding of cotton.(2)On the basis of the above-mentioned CRISPR/Cas9 genome editing technology system,gene editing vectors with two types of codon optimization of Cas9,two kinds of number of PAM loci(1 and 2),two kinds of number of target loci(1 and 2)were constructed to compare and analyze the effect of different Cas9 gene sequences,the number of PAM loci and the number of target loci on efficiency of on-target and off-target mutagenesis.The results showed that there was no significant difference on editing efficiency and off-target effect between two different optimized Cas9 gene sequences;Editing efficiency of the target sequence with double PAM loci(No Shift)was higher than that of the target sequence with single PAM locus(Shift);The editing efficiency of all target sequences in the gene editing vector with double target sequence was higher than that of the gene editing vector with single target sequence.The off-target efficiency of double target sequence is lower than that of single target sequence.Our results provides an important theoretical basis for the design of sgRNA in CRISPR/Cas9 gene editing system.