Question

Describe strategies to delete the Cystic Fibrosis gene and to replace a mutant Cystic Fibrosis gene with a fully functional gene in human cells using the CRISPR/Cas9 system.

Answer 1

Answer: The CRISPR / Cas9 is an effective tool for gene or genome editing, and this technique has been widely used by targeting specific guide RNA (gRNAs) for editing genes or genomic regions. To understand the biological function of a gene this technique is very much useful. Scientists have tried to apply this technique to multiple fields including gene deletion, gene insertion, large fragment deletion, and chromosome translocation. CRISPR / Cas9 is like a molecular scissor, it used to be designed to cut the DNA at a specific position with the help of specially designed gRNA. If you make a cut in the desired genome and if that DNA break is beyond the limit of cell repair then that target gene will disappear in the offspring. In this study, to delete the cystic fibrosis gene from the host cell; first, we need to design some constructs gene-specific guide RNA (sgRNA) and CRISPR/Cas9 vector. The vector backbone will be having a site for the cas9 gene and also the site for a reporter gene like green fluorescence protein (GFP). After the cloning and confirmation, the full construct needs to be transformed is a cell line or the target cell. The process is called transfection. A flow cytometry analysis measuring the percentage of GFP-expressing cells was used to determine transfection efficiencies 2 or 3 days after transfection.

For the generation of cystic fibrosis mutant lines, three subsequent rounds of transfection were performed, in which single genes were targeted by co‐transfection of two CRISPR‐sgRNA vectors. The GFP positive cells were separated 2-3 days after post-infection by using fluorescence-activated cell sorting (FACS). These cells were further allowed to grow in an antibiotic supplemented selection media. To further validate the data you need to do other experiments.