Optimization Of Mutant Locust Construction Based On CRISPR/Cas9 Technology And Functional Analysis Of LmdsRNase2 Deletion Strain

The key to the acquisition of transgenic insects are efficient gene editing tools,effective delivery technology and stable hybrid genetic system.The CRISPR/Cas9 system contains Cas9 protein and sg RNA,which has the advantages of efficient cutting,simple structure and convenient operation,etc.,and has been applied in the construction of transgenic strains of Drosophila melanogaster,Bombyx mori and Locusta migratoria.However,the improvement of CRISPR/Cas9 efficiency is still the primary problem to be solved in the construction of transgenic locust.This article aims to construct a migratory locust strain sensitive to feeding double-stranded RNA（dsRNA）,select the Lmds RNase2 gene that is highly expressed in the midgut and degrade dsRNA as the target,through the screening of high efficiency in vivo and in vitro sg RNA loci,optimization of micro injection system and establish an effective hybrid strategy,to establish and optimize a CRISPR/Cas9 technology to build efficient transgenic locust system;finally obtained a stable Lmds RNase2^-/-transgenic line,and the silencing efficiency of different tissues to feeding different genes in locust was analyzed.Through the dsRNA feeding experiment,clarifies the mechanism of midgut Lmds RNase2 degradation of feeding pathway dsRNA,and also provides a new locust model for gene function study by feeding dsRNA.The main research results are as follows:1.Construction of CRISPR/Cas9 gene editing system in vitro and in vitro and screening of effective sg RNAFirst,three kinds of sg RNAs（sg RNA-545,sg RNA-685 and sg RNA-726）were designed based on the locust Lmds RNase2 gene,and the cleavage vector p Ac-sg RNA-Cas9expressing sg RNA and Cas9 and the reporter vector p IE4-Rep.e GFP containing the target sequence was constructed.Used the dual-fluorescence reporter vector system,we screened highly efficient sg RNA-545 and sg RNA-726 at the cellular level,and the mixture of sg RNA and Cas9 protein（RNP）at the two sites was incubated with the target gene fragment in vitro,which confirmed its high cleavage efficiency in vitro.Secondly,the cleavage efficiency in vivo was determined by injecting RNP into the fertilized eggs.RNP-sg RNA-545 and RNP-sg RNA-726 were injected into the fertilized eggs of migratory locusts.On the 5th day of the egg stage,the target site sequence was obtained by PCR,and digested with the T7nucleic acid（T7E1）.Detection and DNA sequencing showed that both RNP-sg RNA-545and RNP-sg RNA-726 can generate typical insertion and deletion mutations（indels）at the target site.2.In vivo microinjection egg screening and gene editing system optimizationThe key to obtaining mutant locust is to improve the hatching rate of eggs after microinjection.The newly born eggs of locust are wrapped in the oocyst.If the eggs are taken immediately after laying,the oocyst will be destroyed and the less tanned eggs will appear yellow.If the egg was wrapped in the oocyst for about 20 minutes,then the eggshell would be tanned and turned brown.Microinjection of less tanned yellow eggs and tanned brown eggs showed that the hatching rate of brown eggs was higher and the efficiency of mutant acquisition was increased,and the mutants produced could be effectively inherited to the next generation,indicating that microinjection of tanned brown eggs could improve the acquisition efficiency of mutant locust.The early social division occurred after 4h after oviposition,much longer than the time of egg tanning,which was the reason why the brown eggs were able to produce highly heritability mutations.The results of stress test and electron microscopic observation showed that the high hatching rate of tanned eggs was due to the hardening of eggshell,the increased density of eggshell and the enhanced anti-infection ability of eggshell.Therefore,during the construction of gene-edited migratory locusts,brown eggs tanned within 20 minutes after spawn should be selected for microinjection,and microinjection operation should be completed within 4 h,which can effectively improve the acquisition efficiency of transgenic locust.3.Establishment of mutant hybrid system of locust and acquisition of Lmds RNase2^-/-strainRNP-sg RNA-726 was selected for microinjection of tanned eggs of locust.After detection of the egg stage,it continued to be reared to adults.After amplification and sequencing by clipping antennae,12 mutant individuals were screened in G₀ generation,and each individual was paired with wild type to obtain G₁ generation oocysts.After that,sequencing was performed in the oocyst,and the oocysts with mutations were incubated.At the fifth instar,mutant individuals were sampled for detection,and the mutant individuals from the same oocyst were hybridized to obtain the oocysts of G₂ generation.The oocysts with mutations were incubated.Homozygous mutant individuals were sampled and tested at the fifth image.Homozygous mutant individuals with the same mutation type were self-crossed,and finally a homozygous mutant strain was obtained.Therefore,the effective methods for obtaining mutant lines were as follows:G₁ generation was obtained by hybridization of G₀ generation mutants with wild type;Homozygous G₂ generation was obtained by self-crossing of positive individuals in G₁ generation.Selfing of positive individuals between G₂ to expand the population.By using this hybrid system,a 5 bp deletion Lmds RNase2^-/-locust strain was obtained.4.Function verification of Lmds RNase2^-/-locustMidgut juice of Lmds RNase2^-/-was incubated in vitro with dsRNA.The results showed that dsRNA could stable exist in the midgut fluid without being degraded,indicating that Lmds RNase2 was a key enzyme for the degradation of dsRNA in the midgut.By feeding ds Lm Cht10,a key epidermal chitin gene Lm Cht10,it was found that ds Lm Cht10 effectively silenced the expression of target gene Lm Cht10 in epidermis,foregut,midgut and hindgut,and could effectively kill locusts during molting,further indicating that Lmds RNase2^-/-ocusts improved the sensitivity to feeding dsRNA.In addition,feeding ds Lm CDA2 also effectively silenced the expression of target gene Lm CDA2,indicating that the sensitivity of Lmds RNase2^-/-feeding dsRNA of locusts was universal.In conclusion,the successfully constructed Lmds RNase2^-/-locusts can effectively improve the RNAi efficiency by feeding dsRNA,and can be used as a new model for the study the gene function of locust.