Modeling of Designing SgRNA for hTERT Gene Silencing in breast cancer Stem cells with CRISPRoff System

Cancer is one of the oldest diseases ever described by medicine.Breast cancer among women in the vast majority of countriesworldwide, representing a quarter of all cancers diagnosed inwomen. The broad field of gene therapy promises a numberof innovative treatments that are likely to become importantin preventing deaths from cancer. Clustered Regularly InterspacedShort Palindromic Repeats (CRISPR) systems and theirCRISPR associated proteins (Cas proteins) have allowed for anunprecedented ability to manipulate the genome. Key amongstthe applications of these systems is their use in gene editing fortargeted gene knockout, knockin, and modification. An importantstrategy to battle excessive activity of CRISPR complexeswould be to develop CRISPR-OFF switches. This supramolecularstrategy can be used for heritably gene-silencing for atleast ۵۰ days post-transfection, with over ۸۰% of transfectedcells. Transient expression of CRISPRoff writes an epigenetic program that human cells maintain for more than ۴۵۰ cell divisions,highlighting that this form of gene silencing is stableand heritable. Here, we intend to Design High specific SgRNAfor CRISPRoff system to target Human Telomerase ReverseTranscriptase (hTERT) Gene in BCSCS, which has a role inEpithelial-to-Mesenchymal transition (EMT) and Metastasis.hTERT also promotes chemoresistance properties, tumorsphereformation and other important functional CSC properties. ThegRNAs were designed with the help of Chop-Chop tool and theresults of many of them were checked and their qualificationwas evaluated. Then the corresponding gRNA with maximumeffect and on-target was selected with the help of data analysis.Next, the plasmid related to CRISPRoff-v۱.۲ was selected totarget the hTERT gene with the help of AddGene's reviewedResults: At the end, gRNA was analyzed by CRISPRoff webservertool regarding the on/off target ability and CRISPRspec,and finally, the highest level of effectiveness was shown

کلیدواژه ها:

Breast Cancer ، CRISPRoff ، SgRNA ، hTERT ، EMT