Drosophila Gene Knock-in By CRISPR/Cas9

The studies of biological processes are depended on genetic manipulation.Genes’role can be inferred through analysing the phenotypes which are casued by the interference of related genes activity.Gene knock-in can help us to detect or manipulate the expression of target genes more accurately,and help to understand biological processes.Though Drosophila is well known as model organism,because of a wide range of available genetic strategies and tools.It’s difficult to achieve gene knock-in at the interested site without introducing unnecessary exogenous fragment.Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)systems,which can site-specifically recognize and cut exogenous DNA with the help of guide RNAs(gRNAs),carry out the adaptive immuse responses.The system has been boardly applying in genomic engineering since TypeⅡ CRISPR/Cas9 system resconstructured.Three different positions,3’UTR,intron and exon,are successfully marked with fluorescent tags through this method.And visible markers are successfully introduced for screening.We inserted fluorescent protein mCherry in the 3’UTR of PINK1 to generate PINK1-mCherry allele in Drosophila,and the allele functions as normal PINK1.The distribution of red fluorescence can help understand the localization of endogenous PINK1 in cells.At the same time,Dendra was inserted in the Dpp exon region.Exogenous Dendra insertion did not affect Dpp functions.However,the identififcation of these genes are required PCR screening,which is time-consuming and larborious.Therefore,we introduces visible marker gene,3p3-DsRed,and Dendra flanked by SA and SD elements into the Hh intron.Red fluorescence can be detected in the 3p3-DsRed Drosophila’s eyes,and it’s convenient to screen positive insertions.After successfully identification,Hh was labeled by Dendra,without affecting the normal function of Hh.Hh and Dpp are morphogen,both of which were fused with Dendra.It gives us a great convenience to study how morphogen gradients are shaped.In addition,we also knockin another visible marker yellow,which affect the Drosophila body color,and followed with a attP site.This attP site enables the introduction of any given DNA sequence into the gene of interest.We inject the plasmids into the ubiquitously-expressing Cas9 embryos,and this approach is effecient.At the same time,we found the presence of off-target phenomenon in the Cas9 system.