CRISPR
Summary
CRISPR/Cas9 is a gene editing technique that allows scientists to “cut and paste” genes resulting in a gene “knockout” or insertion of new DNA or specific sequence changes, depending on how the technology is used. CRISPR/Cas9 was discovered within bacteria, which use it to fight viruses.
Also known as:
CRISPR/Cas9, clustered regularly interspaced short palindromic repeats
Samples needed
Cells to be modified, guide RNA to lead Cas9 to DNA sequence of interest
Controls
Having controls while using CRISPR can be difficult due to it being basically invisible while inside the cell. A transfection control could use green fluorescent protein mRNA, which would allow researchers to see if the Cas9 protein was successful in arriving at the correct location. A negative control would result in no change of the phenotype of the sample.
The most important control should show scientists that the DNA has been modified in the correct place. This can involve DNA sequencing, or if the gene is being disrupted, use of a Western blot to show loss of target protein expression.
Method
CRISPR/Cas9 can be used to delete or modify existing DNA within a cell or insert new DNA at a specific location. Scientists utilize a “guide RNA” (gRNA) to guide the Cas9 enzyme to the position where editing of the gene should take place. Cas9 unzips the DNA and allows the RNA to bind to it, then Cas9 will cut both strands of the DNA. In response to this DNA break, the cell will attempt to repair the sequence. This can result in either correct repair, incorrect repair leading to a disabled gene, or insertion of a new DNA sequence from a repair plasmid also provided by the researchers.
Anne E. Ebersold
Sources:
https://medlineplus.gov/genetics/understanding/genomicresearch/genomeediting/
https://www.sciencenewsforstudents.org/article/explainer-how-crispr-works
Last updated on April 25, 2022