CRISPR/Cas System Induced Mir-505Knockout In Mammalian Cells

Objective：CRISPR/Cas system is a novel genome-editing tool,the Cas9nuclease is guided tothe specific genomic loci by a20nt guide sequence and makes DNA double strandbreak.MicroRNAs are a kind of non-coding small RNAs in a length of22nt, and function asregulators in a variety of bioprocesses,such as cells growth, proliferation and apoptosis.In thiswork,we designed two sgRNAs targeted to mir-505locus to induce mir-505genomic locimodified by CRISPR/Cas systems.And investigated the influencing factors of cleavage efficiencyin this system.In order to further investigate the biological function of mir-505,we tried to get ridof mir-505gene completely. CRISPR/Cas system induced homologous recombination was carriedout to replace the whole mir-505gene,and mir-505knockout cells were obtained.Afterwards,wetest some biological charaters in mir-505knockout cells.Methods：Two sgRNAs with different GC%were designed according to the online software,and then inserted into a sgRNA expression vector and a Cas9/sgRNA expression vector,respectively. The resulting Cas9/sgRNA expression vector could express the sgRNA and Cas9nuclease simultaneously, carried out the codelivery of sgRNA and Cas9nuclease.The resultingvectors were transfected into mammalian cells, the cleavage efficiency was assayed in T7E1assayand DNA sequencing.The results showed CRISPR/Cas system worked at mir-505target site anddifferent CRISPR/Cas system has different cleavage efficiency. In order to replace the wholemir-505gene,we transfected a donor vector together with the CRISPR/Cas system vectors,toinduce homologous recombination repair pathway,and used G418resistance screening to obtainthe mir-505knockout cells.Real-Time PCR was applied to detect mir-505gene copy number andMTT method was used to compare the growth rate differences between mir-505knockout andwild type cells.Results：The CRISPR/Cas system we designed was able to target mir-505genomic locus, andcleave double strand DNA at target site.The results revealed that higher concentration of CRISPRvectors, and sgRNAs with its gene cloned in the same vector together with Cas9nuclease genecould increase the cleavage efficiency.On the other hand, the one of the two sgRNA targeting thesame mir-505locus with higher GC content could also increase the ratio of cleaved DNA.By cotransfecting the donor vector with CRISPR/Cas system vector into mammalian cells,we gainedthe mir-505knockout cells after G418resistance screening.Having detected the mir-505genecopy number by Real-Time PCR,we found only mono-allelic,other than bi-allelic knockout cellscould be achieved by homologous recombination induced by CRISPR/Cas system.Compared withwild type cells,mir-505mono-allelic knockout cells grew much slower,and the growth retardationworsened with time lasting.In order to gain the mir-505completely abolished cells,we tried theknockout process again in mono-allelic knockout cells to bulid mir-505bi-allelic knockout cells.Conclusion：CRISPR/Cas system could target mir-505genomic locus in mammalian cells,and induce mutagenesis of the gene. The cleavage efficiency of CRISPR/Cas system was affectedby the dosage of sgRNAs and Cas9nuclease, the way of codelivery of sgRNAs and Cas9nuclease,and the GC%of sgRNAs.The growth rate of mir-505knockout cells was significantlydecrease,which revealed the mir-505played a role in the regulation of cell growth andproliferation.