CRISPR/Cas9-Mediated High-throughput Mutant Library In Cotton For The Efficient Screening Of Candidate Endogenous Plant Host Insect-resistance Genes

Cotton is not only an important fiber crop,but also an important oil crop.However,the cotton planting growth period is long,from sowing to harvest in the growth and development of each period,it may be harmed by a variety of pests.The pest effect has become one of the important factors affecting cotton yield and quality.As one of the four major genetically engineered crops,the application of Bt cotton can at some levels reduce the damage of a few lepidopteran pests,whereas,Bt non-target pests such as sap-sucking insects have emerged as the major insect pests in most cotton production regions.With the increasing damaging of Bt non-target pests,the large-scale use of chemical insecticides has led to the increased resistance of many pests to insecticides,which not only increases the cost of agricultural production,but also damages the ecological environment.On the other hand,with the expansion of planting area and the extension of planting years of Bt cotton,the risk of insect resistance to Bt cotton should not be ignored.Therefore,a new strategy is needed to study insect resistance in cotton.The identification of endogenous genes for insect-resistance breeding is a promising strategy in cotton,which could be complementary to the use of Bt.Therefore,an efficient and reliable strategy for the high-throughput functional analysis of genes is desirable for cotton functional genomics.The CRISPR/Cas9 genome editing system has become a popolar and universal tool.Fortunately,the successful establishment of an efficient and precise CRISPR/Cas9 genome editing system for cotton,making it possible to build a large-scale mutant library for cotton genomics research.Here,we describe a high-throughput screening method for endogenous insect-resistance related genes in cotton by a CRISPR/Cas9 mediated large-scale mutant library.This study provides a new and efficient strategy for functional genomics research and breeding research in cotton.The main results were as follows:1.Design of sgRNA and construction of plasmid vector libraryIn this study,528 DEGs were selected from our previous transcriptome and metabolomics analyses.Through genome-wide comparative analysis in cotton,a total of 968 highly specific sgRNAs were screened to target 502 genes.The construction of vector library by the method of mixed primer pools only requires primer design,PCR amplification and vector cloning once,takes about the same time as for constructing a single vector,and therefore is a fast and reliable method.2.Construction of cotton genome editing mutant library and identification of target GenesThe mutant library contained up to 5000 independent embryonic calluses and the regeneration of more than 2,000 independent transgenic T0 plants following Agrobacterium-mediated transformation and tissue culture.The target genes of T0 plants were detected by high-throughput sequencing based on barcodes.We detected the presence of sgRNAs in 1380 T0 plants and 576 calluses and identified their sequences.A total of 555 different sgRNA sequences targeting 412 genes were identified and the coverage of the mutant library represented 82.07% of the total target genes.3.High editing efficiency and heritability Through high-throughput sequencing,software analysis and manual screening,a total of 369 effective genome editing data in T0 plants were obtained.The results showed that the effective genome editing rate(>1%)of T0 plants was up to 97.29%,75.61% showed a highly efficient mutation rate,with editing occurring in more in than 80%,and short DNA deletions accounts for the largest proportion of edits.The average heritability of cotton genome editing was up to 84.78%.4.Low risk of off-target 9 potential off-target sites were detected by high-throughput sequencing,but no offtarget sites were found in this study,which confirmed the high specificity of CRISPR/Cas9 system in cotton genome editing.5.Efficient phenotypic identification rates In insect bioassays of T1 and T2 generation from 200 lines,more than 10% of genes exhibited altered resistance to insect infestation.This suggests that screening a genome editing mutant library,targeting a specific trait(insect resistance in this case),is a promising strategy to identify candidate genes.6.Candidate genes related to insect resistance The mutant materials of GHMLP423,GHDML1 and GHZAT10 showed consistent phenotypes associated with insect resistance,which played an important role in plant responses to abiotic and biotic stresses.Through preliminary verification,it was found that GHMLP423 may be involved in JA signaling pathway related to insect resistance in cotton.The methodology described in this study uses the CRISPR/Cas9 system to construct a library of pooled vectors,by using nearly a thousand sgRNAs targeting hundreds of genes on plant genome at one time,and rapidly generates a high coverage mutant library for phenotypic screening,and identified a number of insect-resistance genes.The technology provides a solid foundation for cotton functional genomics.This method is applicable to other polyploid plant species with complex genomes,and will be a valuable reference for other crop species study.