In Vivo Gene Editing By CRISPR/Cas9 In Drosophila

In vivo gene editing was considered as the most difficult and essential problem in biology.Through editing target gene,people can learn more detail about its function and the relevant signal pathways.In this study,we edited target genes by knocking DNA fragment encoding photoconvertible fluorescent protein into various loci through CRISPR/Cas9 in Drosophila.The bacterial CRISPR/Cas system has proven to be an efficient tool for genetic manipulation in various organisms.CRISPR is referred to clustered regularly interspaced short palindromic repeats which is a family of DNA sequences containing snippets of DNA from viruses that have attacked the bacterium.These snippets are used by the bacterium to detect and destroy DNA from similar viruses during subsequent attacks.Together with CRISPR-associated proteins,such as Cas9,these sequences play a key role in the bacterial defense system.By engineering CRISPR/Cas9,the type ? CRISPR/Cas,in vivo gene editing has become more efficient.Morphogens,ligands of TGF-? family signaling pathway,contribute to growth regulation,patterning and stem cell fate determination.Morphogen could decide cellular differentiation by the gradient of their concentration which was formed during their diffusion.Therefore,it is critical to study the pattern of their distribution and diffusion.Fusion protein,by adding morphogenetic protein with some tags,can present the distribution and movement of morphogen directly and precisely,but the success rate of building endogenous fusion morphogen protein is very low.In our studies,we intended to insert photoconvertible fluorescent protein dendra in front of the C terminal of Dpp,the N terminal of Wg and the stop code of mEFTu1 in order to label them.We observed that,unlike the integration of dendra in the Dpp N terminal exon region,Dpp marked with dendra in C terminal resulted in severe defects in Drosophila imaginal disc but surprisingly had less effect on adult compound eyes and wings.For Wg which was marked by dendra protein in N terminal,we failed to get the KI lines.And we incorporated a visible selection marker when we knock dendra in the gene mEFTul.We did get positive KI lines,but with a frame-shift.Our failure is consistent with the recent reports showing that CRISPR/Cas9 system might induce undesired indel mutations and off-target insertion which restricts its application.On the other hand,we also found that loci display differential suggestibility to CRISPR/Cas9-mediated editing.