Systematic Analysis Of CRISPR/Cas9 Mediated Genome Editing Of Bombyx Mori

Since the 1970 s,scientists have been devoted to exploring genome editing technology.Subsequently,with the discovery of Zinc-Finger Nuclease(ZFN)in 1996 and Transcription ActivatorLike Effector Nuclease(TALEN)editing method in 2009,many important discoveries and advances have been made in genome and gene function-related studies.The emergence of Clustered Regularly Interspaced Short Palindromic Repeat(CRISPR)/ CRISPR-associated 9(Cas 9)technology in 2013 opened a new stage of research on genome editing technology.CRISPR/Cas9 and its derivatives are still the most widely used and researched editing method.They have been widely used in the study of gene function of multiple model organisms and the treatment of target diseases due to their high mutation efficiency and ease of operation,bringing revolutionary changes to the development of life science.However,the current understanding of the editing results of Cas9 is still limited.It is generally believed that CRISPR/Cas9 will result in random base insertion or deletion(indel)at the target site after editing.Homologous recombination(HDR)generated by artificial introduction of exogenous DNA is precisely repaired.Other repair patterns,such as non-homologous terminal ligation(NHEJ),are random and unpredictable.But other scientists have suggested that the results are consistent regardless of the repair pathway.The results of this study suggest that deep sequencing reveals regularity in the CRISPR/Cas9 editing spectrum in Bombyx mori.By analysing of a total of 31161 editing events from 9 sgRNA target sites in 16 individuals,we found mutations generated by Cas9 editing had a certain regularity.In combination with cell experiments,it was also found that the deletion sequences produced by Cas9 editing were more often found in the upstream of the target sites.In order to further understand Cas9-mediated mutations and provide theoretical and technical support for the design of CRISPR/Cas9 editing experiments,the specific results obtained through further research are as follows:1.Editing efficiency and mutation type of CRISPR/Cas9 are different in Bombyx moriAfter microinjection of Cas9 and the designed target site sgRNA complex,16 individuals with phenotype were selected for statistical analysis.Individual editing efficiency ranges from 2.16% to 13.15%.The mutation types are mainly small fragment deletion,with small substitution and insertion.The average percentages of these three mutation types are 65.55%,6.77% and 13.26%,respectively.By analyzing the indel of 9 targeting sites,it is found that the deletion is mainly based on single base deletion.In addition to single base deletion,when there are micro homologous sequences in the targeting sites,the repair tends to microhomology-mediated end joining,which is consistent with the CRISPR/Cas9 editing results of zebrafish and human.In addition,the insertion sequence of each target site was analyzed,and it was found that the insertion sequence of 1,6 and 8 bases accounted for 51.36% of the total insertion sequence.Further analysis showed that the insertion of a single T,accounted for 47.50% of the insertion of a single base sequence.Insertion is formed by a variety of mechanisms,including direct acquisition of free genomic fragments,replication of cleaved ends,replication of adjacent sequences,and random addition of free nucleotides.Through the analysis and comparison of different individuals and sgRNAs,it was found that the editing efficiency and mutation type of CRISPR/Cas9 varies with sgRNA sequence,direction and individual in Bombyx mori.2.CRISPR/Cas9 editing range is non-random and predictableBy analyzing the mutation types of mutation sequences at 9 targeting sites of 16 individuals,it is found that mutation types with high proportion of mutation sequences occur in multiple individuals at the same time,such as the targeting sites of sgRNA9:CATACTACAGATACTTTCTT.The top three mutation types are deletion of 4,21 and 3 bases.These three mutation types exist in 16 individuals,and the highest proportion of mutation sequences among different individuals are 75.47%,69.06% and 59.33%,respectively.The top 200 mutation types of 9 targeting sites were selected for mutation sequence proportion and statistics on the number of individuals appearing at the same time.The percentage of mutation sequences of the top mutation types was high and existed in multiple individuals which as same as those obtained on sgRNA9.In addition,the mutation types of the top 20 were calculated.These mutation sequences accounted for 83.07% of the total mutation sequences,of which mutation sequences that existed simultaneously in 13 and more than 13 individuals accounted for 53.99% of the total mutation sequences.We found that the mutation types of single target site are mainly affected by the sequence of target sites,so the repair outcomes of target sites can be predicted when designing sgRNAs.3.CRISPR/Cas9 mediates Bombyx mori deletion sequence tending to be deleted upstream of the target siteIn order to obtain high-efficiency CRISPR/Cas9 editing results,sgRNA of 4 different targeting genes and Cas9 are further designed to be co-transfected into silkworm cell lines BmE,BmNs and BmN-sidI.the cell editing efficiency ranges from 18.83% to 57.30%,the deletion sequence accounts for 46.77% to 88.43% of the total mutation sequence,and the deletion sequence upstream of the targeting site accounts for 69.44% to 97.22% of the deletion sequence,with an average of 80.38%.Compared with 42.65%,48.39% and 31.48% data of human,mouse and zebrafish downloaded and analyzed on NCBI,it has an obvious special tendency,which the upstream of the target was missing.In addition,the coincidence degree of different targeting sites of a single cell line and different cell lines of a single targeting site is analyzed,and the coincidence degree of the former is lower than that of the latter,which proves that CRISPR/Cas9 repair at the cell level is not random,and repair is mainly affected by the sequence of targeting sites.This reflects the similarities and differences of editing results mediated by CRISPR/Cas9 between silkworm and other species.The overall repair is consistent and the deletion of target site is special.It provides design ideas for silkworm genome editing experiments,helps predict the editing results,and has some reference value for the research of silkworm DNA repair pathway.