The important part of using CRISPR/Cas9 method is the selection of adequate sgRNA that binds target region on DNA. Optimal performance of CRISPR/Cas9 technology can be accomplished only with use of highly efficient and specific sgRNA. Numerous web tools that propose possible sgRNA sequences are available. However, the sgRNA efficieny should be evaluated experimentally. Several assays for sgRNA validation based on different approaches are available. This thesis decribes validation of four sgRNA targeting TLR10 gene with different in-vitro validation methods. After validation the sgRNAs were succesfully used for activation of TLR10 transcription using CRISPRa. A549 cells were transfected with transcription activators-fused dCas9 and different sgRNAs. Transcription regulation with CRISPR technology offers solid basis for further research on TLR10, which is the least known receptor of the TLR family with no known ligand.
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