Functional Analysis Of Dicer Family Genes In Locusta Migratoria Mediated By CRISPR/Cas9

Ribonuclease Dicer is mainly responsible for cleaving the precursor of RNA into functional RNA,so that it can perform various physiological functions in the RNAi pathway.Dicer belongs to the RNase ? family and is highly conserved in evolution.Most of them contain an N-terminal domain DEAD,a helicase domain Helicase,and a PAZ domain,two ribonuclease domains RNase ? and a ds RNA binding domain ds RBD,are widely found in plants,mammals,insects and other organisms.The domain of Dicer in different species is similar,but the quantity is different.In mammals,only one Dicer,plays a dual role in the formation of mi RNA and si RNA;most insects have two Dicer,in which Dicer1 cuts pre-mi RNA to produce mi RNA,and Dicer2 participates in cutting ds RNA to form si RNA.However,whether the Dicer family genes of Locusta migratoria have more physiological functions,including LmDicer1,LmDicer2a and LmDicer2b,is the most important problem to be solved in the study of Dicer splicing function.CRISPR/Cas9 technology is an efficient tool to study gene function.In this study,we will use CRISPR/Cas9 technology to construct knockout strains of L.migratoria Dicer family,and detect the biological functions of different Dicer,so as to provide models and tools for the follow-up study of Dicer splicing function.Previous studies of our group found that Dicer,the core enzyme of RNAi pathway,had an important effect on the efficiency of RNAi.In order to further study the physiological function of Dicer,the establishment and phenotypic observation of Dicer family knockout strain of L.migratoria were mediated by CRISPR/Cas9 gene editing technique.The main contents are as follows:1.Design and verification of sgRNA targeting LmDicer family genes based on CRISPR/Cas9 system:The genomic structure and protein domain characteristics of LmDicer1,LmDicer2a and LmDicer2b were analyzed.They all included N-terminal domain DEAD,helicase domain Helicase,PAZ domain and ribonuclease domain RNase ?.LmDicer1 and LmDicer2b also contain ds RNA-binding domain ds RBD,which have 23,20 and 21 exons,respectively.Based on the CRISPR/Cas9 system,one exon was selected for sgRNA design at the front end and back end of the genome,and the effectiveness of sgRNA was analyzed by the method of in vitro Cas9/sgRNA complex cutting targeting DNA.LmDicer1 Exon3 sgRNA1 and LmDicer1 Exon21 sgRNA4;LmDicer2a Exon2 sgRNA1,LmDicer2a Exon3 sgRNA2 and LmDicer2a Exon20 sgRNA3;LmDicer2b Exon5 sgRNA3 and LmDicer2b Exon21sgRNA4 were screened for LmDicer1,LmDicer2a and LmDicer2b respectively,which can be used for the construction of subsequent mutants of L.migratoria.2.Screening of LmDicer family mutants and preservation of eggs:The acquisition of mutant strain and even homozygous strains is a key step in the construction of transgenic insects,and the preservation of eggs provides technical guarantee for insect strain maintenance and experimental synchronization.By injecting the mixture of LmDicer1 Exon3 sgRNA1,LmDicer2a Exon2 sgRNA1,LmDicer2b Exon5 sgRNA3 and Cas9 into the fertilized eggs of L.migratoria,the chimeric mutants of G₀generation were obtained:LmDicer1 has eight,LmDicer2a has six and LmDicer2b has seventeen.Among them,LmDicer1 gene plays an important role in the growth and development of L.migratoria,so homozygous strain can not be obtained after LmDicer1 knockout.The mutants of LmDicer2a and LmDicer2b obtained positive individuals by crossing with wild type,and the positive individuals self-crossed to obtain homozygous strain.Finally,a homozygous strain of LmDicer2a was obtained with a deletion of 10 bp at the target site.Two homozygous strains of LmDicer2b were obtained,and 5bp or 13 bp were missing at the target site.The filter paper cryopreservation technique established in the experiment can ensure that the eggs can be preserved in a low temperature environment for more than 8 months,which provides a guarantee for the maintenance of L.migratoria strains and experimental synchronization.3.Physiological function analysis of LmDicer1:In the process of growth and development of L.migratoria,the viability of LmDicer1 chimeric mutants was lower and the lethality rate was higher than that of wild type at all ages.The hatching rate of G₀generation eggs was low,and it was difficult to molt in the pre-nymph stage;the body surface color of the mutant changed from the 1st instar nymph to the 5th instar nymph stage,and the phenotypes such as leg bending and weakness,broken wings and molting death occurred during molting;the death number of the L.migratoria increased from the 5th instar nymph to the adult stage,and the peak of molting death occurred.Some of the surviving adults also had changes in body surface color,weak legs and curly wings,and the survival days were much lower than those of wild type L.migratoria adults,and the LmDicer1 mutant could not be effectively mutated to offspring.Therefore,LmDicer1 plays a key role in the growth and development of L.migratoria.4.The effect of LmDicer2a/LmDicer2b knockout on the reproductive function of L.migratoria:During the growth and development of L.migratoria,the G₀generation chimeric mutant,G₁generation heterozygote and G₂generation homozygote of LmDicer2a and LmDicer2b were normal at all ages.However,the offspring produced by self-crossing of LmDicer2a homozygote were all female,and the eggs produced by self-crossing of LmDicer2b homozygote could not develop.Further study found that homozygous males of LmDicer2a and LmDicer2b could not pass on their own mutations to their offspring,and their testes were slightly smaller than those of wild type L.migratoria,and their sperm bundles showed different degrees of bending and winding and agglutination,and the sperm was beaded,that is,LmDicer2a and LmDicer2b knockout led to male sterility.