| In recent years,the gene editing technology of directed modification of genomic DNA sequence has provided a great impetus to advance the basic research in the field of life science.Gene editing mainly includes Zinc-Finger Nuclease(ZFN),Transcription Activator-Like Effector Nuclease(TALEN)and Clustered Regularly Interspaced Short Palindromic Repeat(CRISPR)technology,among which CRISPR/Cas9 technology has advantages of simple operation,high coverage and low miss rate.It has rapidly become the mainstream technology of genome editing and has been widely used.Functional analysis of the whole genome is the main trend of current scientific research.Cas9-based editing system has gradually become a powerful tool for functional analysis of the whole genome.The construction of genome-wide knockout individual library will rapidly promote the research on gene function,germplasm creation and genetic modification,as well as the prevention and treatment of diseases and other related studies.Large-scale gene knockout at the individual level has been achieved in Danio rerio,Drosophila,Oryza sativa,and other species.Several key genes that have important effects on growth and development have been discovered,which has greatly promoted the development of basic research,medicine,agriculture and other fields.As an important type organism of Lepidoptera,the study of silkworm is of great significance to the basic research of entomology and pest control.The whole genome sequencing of Bombyx mori has been completed earlier and a variety of genetic manipulation technology platforms have been established,but the functions of the vast majority genes are still unknown.Therefore,using gene editing technology to construct the whole genome knockout library can screen functional genes with high throughput and accelerate the research and annotation of functional genomes.Our laboratory has established the genome-wide CRISPR screening library in BmE cells.The cell library screening is easy to operate and has a short cycle.Currently,essential/non-essential genes,temperature-responsive genes,and key genes in the infection process of Bombyx mori Nucleopolyhedrovirus have been screened,confirming that the genome-wide CRISPR screening library is simple and efficient in silkworm cell studies.Although the construction of knockout individual library of Bombyx mori takes a long time and involves a lot of work,it is more conducive to the discovery of mutant strains and screening of functional genes.Therefore,the construction of an individual library is also crucial.In this study,we injected a mixed plasmid library and constructed a mutant library,and 520 sgRNA×Cas9 hybrid strains were obtained.The sgRNA and target sites of each individual in the silkworm mutant library were sequenced.In order to provide better theoretical and technical support for the construction of the silkworm individual library,editing experiment research and sgRNA optimization design,we further explored the molecular detection strategy of mutant library and analysed silkworm editing rules systematacially,which will probably promote the process of silkworm genome editing research.The main results and conclusions of this study are as follows:1.The construction of knockout individual library of Bombyx mori and largescale detection and analysis of sgRNAThe construction of the silkworm mutant library adopts the method of "large-scale injection,G1 generation positive screening,multiple backcrossing to obtain G3 generation,crossing with Cas9 to obtain F1 generation hybrid strains",and 520 knockout strains were obtained.In order to reduce the workload,the mixed injection method of sgRNA library(94000 sgRNAs,covering 16198 genes)was adopted,so it was necessary to identify the sgRNA of each subsequent transgenic strains.We tested the sgRNA of1560 individuals from 520 knockout strains respectively by using the method of Sanger Sequencing and Next Generation Sequencing(two males and one female as three replicates in each strain are divided into group 1,2 and 3),and the sequencing results were statistically analyzed.A total of 267 kinds of sgRNAs covering 188 genes were detected and some individuals had multiple sgRNAs in the individual database.Through statistical analysis,only 43% of single sgRNA individuals in G1 generation was found,the proportion of single sgRNA individuals in G3 generation individuals increased to 75%,and the proportion of single sgRNA individuals in their offspring F1 generation increased to 86%.The comparison of the inheritance of multiple sgRNA individuals from G3 generation to sgRNA in F1 generation showed that 74% of the sgRNA was separated in F1 generation.The above results indicate that the multiple sgRNA individuals obtained by injecting mixed CRISPR vector library could be separated by backcross to get single sgRNA individuals,which would greatly reduce the workload of constructing silkworm mutant library.2.Cas9-mediated double-strand breaks repair of target site in knockout individual library of Bombyx moriWe systematically analyzed the editing results of 520 knockout strains,and further studied the main repair types and repair mechanisms in silkworm after editing.The results showed that the overall average mutation efficiency in the silkworm knockout strains was63.8%.We found that the mutation types were dominated by base deletion types,accounting for 70.54% of the total mutation types,which was higher than the insertion rate(4.80%),substitution rate(6.92%),and other mutation types.Next,we further analyzed the insertions and deletions respectively.The results showed that the base deletions tend to occur in the upstream region of the cleaved site,and the deletions are mostly small fragments of 1-20 bp,and the base insertions are mostly single base,accounting for 62.56% of insertion types.The majority of small fragments inserted in 1to 2 bp are repaired using upstream nucleotide as a template.These results indicated that the double-strand breaks caused by gene editing in Bombyx mori has a certain tendency in the repair process.3.Analysis of related factors affecting the mutation efficiency in knockout individual library of Bombyx moriThe large amount of mutation data from the mutant library provides a basis for the analysis of the factors affecting the mutation efficiency of silkworm individuals.We analyzed the overall mutations of the target s ites of the three groups of samples and several factors affecting the editing efficiency.The correlation value of the mutation rate among the three groups is relatively high.The analysis of GC content at the target site showed that the mutation efficiency of the target site was positively correlated with the GC content.The analysis of PAM sequence showed that when PAM was AGG or CGG,it was more favorable for CRISPR/Cas9 system to edit target sequences.It was found that the position of guanine at-1 and-2 near the PAM sequence,and the position of adenine at the 3' end of the PAM sequence were favorable for target site editing.Thymine at the5' end of the target(position-17 to-20)is favorable for target editing,while it may inhibit editing at the 3' end(position-1 to-7).We further analyzed the potential relationship between the distribution of 2-nt and 3-nt motifs in the 12-nt seed region and the mutation efficiency in all the tested target sites.It was found that target sites containing motifs such as "A/CA/C" dinucleotides were more likely to induce mutation,while the trinucleotides containing "TT" are not conducive to induce mutation.In addition,we investigated the relationship between the mutation efficiency and the apparent modification of the target site,and analyzed the DNA-6m A level near the target site(3kb before and after).It was found that the higher the DNA-6m A modification level in the silkworm,the lower the gene editing efficiency.It is speculated that DNA methylation may affect the binding of Cas9 protein to the target site.Combined with the transcriptome data of silkworm embryos,the analysis showed that the mutation efficiency had no significant relationship between the expression level of targeted gene and sex of silkworm.The results above provided a reference for us to design and optimize sgRNA in Bombyx mori and the subsequent large-scale germplasm creation.In summary,a small-scale silkworm knockout individual library was prepared in this study.By systematically analyzing the sgRNA and target site editing results of 520 silkworm knockout strains,we found the regularities of sgRNA integration and heredity,the editing characteristics of CRISPR in silkworm,and discussed the Cas9-mediated double-strand break repair rules and the related factors affecting editing efficiency in Bombyx mori,which provided a reference for design and optimization of sgRNA and subsequent large-scale germplasm creation. |
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