Functional Analysis Of Exopalaemon Carinicauda Molt Inhibiting Hormone Through Targeted Gene Knockout Using CRISPR/Cas9 Genome Editing Tool

Molting is a crucial process for crustaceans which has a close relationship with growth.Early researches showed that molt-inhibiting hormone(MIH)was a negative-regulatory factor that suppresses its molting process in the complicated crustacean molting pathway.But because of the limited crustacean functional gene research platform,no studies on crustacean MIH gene in vivo have been reported so far.We have successfully applied the CRISPR/Cas9 technology on the genome editing of the ridgetail white prawn Exopalaemon carinicauda in our previous studies.In this study,transcriptome of E.carinicauda were analyzed and the molt-inhibiting hormone gene(Ec MIH)information was confirmed by PCR.The expression profile of Ec MIH was detected by q PCR,and it was successfully knocked out through the genome editing tool CRISPR/Cas9.The obtained mutations had a three days shorter time before developing into juveniles compared with blank and control groups,which verified the Ec MIH function preliminarily.In addition,the secretory expression vector p CT7-CHISP6H-Cas9 was constructed and the recombinant Cas9 protein was prepared.Moreover,the biological activity of recombinant Cas9 in genome editing was studied in vitro at the same time.1.Based on the transcriptome of E.carinicauda,the full-length c DNA sequence of Ec MIH was obtained.The ORF of it encoded 119 amino acids with a predicted molecular weight(MW)about 13747.82 Da.The theoretical isoelectric point(PI)of Ec MIH was 8.61.It consists of three exons and two introns.The expression profiles of Ec MIH were analyzed by real-time PCR in different tissues,different stages and different molting periods.Ec MIH g RNA was designed in the second exon and was transcript synthesized in vitro,then it was injected into E.carinicauda one-cell embryos combined with m RNA of Cas9 nuclease.The detection primers were designed and the genomic region flanking the Ec MIH g RNA target was amplified.Both the sequencing results of PCR products and monoclonal assay analysis showed that Ec MIH successfully knocked out by using CRISPR/Cas9 system.The frameshift mutant E.carinicauda individuals were obtained,and the mutation ratio reached 42.9%.In addition,the larvae development time of these mutants was reduced from 11 days to 8 days,while no other significant differences were observed.2.The mature peptide sequence of Cas9 was amplified with designed p CT7-CHISP6H-Cas9F/R as primers and Cas9P-1EA as template.Then according to the In-fusion?R HD Clontech kit,the PCR products and the linearized p CT7-CHISP6 H plasmids digested by Pma C I were put together to construct the recombinant secretory expression plasmid p CT7-CHISP6H-Cas9.And the secretory recombinant Cas9 was obtained after the p CT7-CHISP6H-Cas9 plasmid was transformed into BL21(DE3).The r Cas9 protein was purified by affinity chromatography and desalting methods.Moreover,the recombinant enzyme could cleave the target plasmid under the guidance of g RNA in vitro.