Study Of CRISPR/Cas9 Mediated Genome Editing In Drosophila Melanogaster

With the development of the whole genome sequencing,a growing number of insect species ' whole genome sequencing work is finished.In addition to the efforts of so many entomologists,gene annotation work is continuous developing,a large amount of data emerging in front of people.How to study and excavate the gene function hidden behind these information become the key point.An important means of solving this problem is genome editing.CRISPR(Clustered Regularly Interspaced short Palindromic Repeat)system is an immune mechanism to resist of phage DNA in bacteria and archaea.In type II CRISPR system,the Cas9 protein guided by a short period of gRNA to idengtify and cut the specific genome sequence,result in double strand breaks(DSBs).The gRNA recognizes a 20-nt target sequence next to a trinucleotide NGG protospacer adjacent motif(PAM),also the gRNA sequence are the same as 20-nt target sequence.After generating DSBs,orgnasims will repair the double strand breaks through different way and finally achieve targeted genome editing.In this study,we chose model insect fruit fly D.melanogaster(Diptera:Drosophilidae).Targeted editing the white gene which deciding the eye color of drosophila using CRISPR/Cas9 system and multiplex editing CG18208 gene which codoning octopamine receptor using tRNA—gRNA arteracture.1.Mutagenesis of drosophila white gene using CRISPR/Cas9We downloaded CDS sequences of fly white gene from flybase(http://flybase.org/).Analysed it's introns and exons.found and selected the appropriate site through flycrispr Optimal Target Find on flycrispr website(http://flycrispr.molbio.wisc.edu/tools).Then we in vitro transcribed Cas9 mRNA and gRNA.Mixed the Cas9 mRNA and gRNA at the concentration of 400ng/?L and 40 ng/?L,and injected into early embryonic germ line cells of wild-type Canton-S flies.We failed to observe white eye type in G1 flies.This may be casused by RNA degradation during the process of operation.Next,we clone the gRNA into pDCC6 vector,express Cas9 and gRNA with this single plasmid.After injected this single plasmid into flies' early embryonic germ lines,we observed white eye mutant from G1 flies.Sequencing of mutant region observed indel at targeted sites.2.Multiplex targeting CG18208 gene using tRNA-gRNA system.We download CDS sequences of fly CG18208 gene from flybase(http://flybase.org/).Analysed it's introns and exons.Then found and selected the appropriate site through flycrispr Optimal Target Find on flycrispr website(http://flycrispr.molbio.wisc.edu/tools).Next we cloned four different gRNAs into pCFD5 plasmid,making the four gRNA flanked by tRNA.The four gRNAs could be efficiently produced from a single synthetic gene with the tRNA-gRNA architecture after precise excision of transcripts in vivo by the endogenous RNases.In addition we prepare a donor plasmid which have Ga14 and min-whitecontaining two 1 kbp homologous arms.We expected that this two elements will inserted into targeted site at CG18208.Next,we mixed the pCFD5-OA3 and donor plasmid at the concentration of 400 ng/?Land 250 ng/?L,and injected them into early embryonic germ line cells of 51323 flies.The GO flies were outcrossed to TM3/TM6 flies of opposite sex.Finally,we detected mutant at targeted site through Sanger sequencing,TIDE analysing and HRMA.