We are planning to target a locus we believe to be an enhancer by multiplex CRISPR-based targeting and the dCas9KRAB and dCas9VP64 systems.
Our final vector will include 4 different sgRNAs (expression driven by 4 different promoters) that will target the potential enhancer. They are designed to do so by "tiling" that enhancer. The dCas9KRAB and dCas9VP64 will act as the repressors or activators respectively. We would like to test the sgRNA's accuracy and specificity before committing to the official experiment. One obvious way it to simply use an active Cas9 with each individual sgRNA and testing their efficacy a using genome editing test kit.
I am interested in a potentially quicker and more robust approach.
its important to note that we are only hypothesizing that region is an enhancer (based on previous data we have) and so testing expression of our gene of interest is not a viable option to test sgRNA efficacy.
The CRISPR/Cas9-sgRNA system has recently become a popular tool for genome editing and a very hot topic in the field of medical research. But CRISPRpred, for efficient in silico prediction of sgRNAs on-target activity which is based on the applications of Support Vector Machine (SVM) model. And also CRISPRpred is enough flexible to extract relevant features and use them in a learning algorithm.
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