Genome Editing Mediated By CRISPR/Cas9 In Caenorhabditis Elegans

As the third generation of artificial endonuclease,CRISPR/Cas9 system has some advantages:it is easy to use and has a wide range of targets,it also has target specificity and a high efficiency.In the past few years,CRISPR/Cas9 system has become a powerful tool for genome editing and it has been widely used in various organisms.As a model organism,Caenorhabditis elegans(C.elegans)is small,transparent for ease of manipulation and observation,and has a short lifespan and simple structure.As a result,C.elegans is often used to study the mechanisms of lifespan regulation and stress response.It was known that in C.elegans,AMPK,14-3-3 and HSP70 proteins are likely to participate stress response.Given these proteins are highly conserved in evolution,studying their function will help to understand the regulation mechanism of stress response in other species.AAK-1 and AAK-2 proteins encoded by aak-1 and aak-2 are a subunits of AMPK;FTT-2 encoded by ftt-2 belongs to one of two 14-3-3 proteins in C.elegans;f44e5.5/f44e5.4 are two adjacent genes which encode two identical HSP70 proteins in C.elegans.In this study,we attempted to obtain aak-1,aak-2,ftt-2,f44e5.5/f44e5.4 mutants by using CRPIPR/Cas9 mediated genome editing for studying their function in stress response.First of all,we utilized the sgRNA efficiency testing system constructed by our laboratory to assay the effectiveness of sgRNAs used for editing following nonessential genes:aak-1,aak-2,ftt-2,f44e5.5/f44e5.4 in C.elegans.We designed two sgRNAs for each gene and tested their effectiveness.One of two sgRNAs locates at the 5' end of gene and another one locates at the 3' end.Next,we coinjected two sgRNAs,the plasmid with Cas9 coding sequence along with the marker gene into the gonad of C.elegans.If the exogenous Cas9 guided by dual sgRNAs could cut the genomic DNA in germ cells,it would be possible to obtain F1 mutants with deletion,which can be screened by PCR.And we tracked the deletion in F2 worms to confirm that we got stable mutants.In order to increase screen efficiency,we used two strategies:Co-CRISPR strategy and the strategy of combining Co-CRISPR and the sgRNA efficiency testing system.Our results showed that the combination of Co-CRISPR and the sgRNA efficiency testing system could enrich gene editing events compared to Co-CRISPR.Finally,we analyzed thermotolerance of aak-2(xmu2),f44e5.5(xmu3),dpy-13(xmu7),f44e5.5 f44e5.4(xmu4)mutants.Data showed that the heat resistance of aak-2(xmu2)has decreased significantly inconsistent with previous studies,while the heat resistance of f44e5.5/f44e5.4 single or double mutants did not have obvious change.In conclusion,this study provided useful guidance to genome editing mediated by CRISPR/Cas9 in C.elegans.