11 August 2016
The latest gene editing technique outperforms the conventional method to perform large-scale screening of gene functions, according to research conducted at The Netherlands Cancer Institute. The insight shows how the CRISPR–Cas9 system could help to identify genes involved in diseases such as cancer.
Determining the genetics of a cellular process or disease usually involves functional genetic screening, in which genes are suppressed one by one to demonstrate their contribution. These large-scale experiments can be used for a variety of research purposes and provide large amounts of useful information.
“An ideal genetic screen provides a list of genes that are involved in a certain process,” explains Bastiaan Evers, lead author of the new study. “For example, one could study whether disruptions of any gene confers resistance to cancer therapies. That information could be used to better understand, predict and intervene in treatment failure.”
Conventional functional genetic screening is performed with a technique called RNA interference (RNAi), which disrupts the process of translating genes into proteins. However, this technique often disrupts genes other than the intended target, or incompletely suppresses the target gene so its function is masked. Evers and colleagues wanted to determine whether a newer technique, called CRISPR–Cas9 gene editing, is more reliable.
CRISPR–Cas9 is an immune system in bacteria and archaea (single-celled micro-organisms lacking nuclei) that cuts foreign genetic material and can be exploited to target specific genes in human cells. “CRISPR technology allows very rapid and cheap editing of genomes in ways that easily result in complete functional knockouts of genes,” explains Evers. “In addition, CRISPR technologies seem to suffer less than RNAi from off-target activity.”
Evers and colleagues compared the performance of RNAi, CRISPR–Cas9, and a variant technique called CRISPR interference (CRISPRi), in which DNA is not cut but a gene is suppressed. They assessed the ability of all three techniques to identify 46 genes that are known to be essential for cellular life. They found that the CRISPR–Cas9 system was more reliable than both RNAi and CRISPRi at identifying the genes, largely because of fewer off-target effects and greater consistency between reagents targeting the same gene.
References
- Evers, B., Jastrzebski, K., Heijmans, J. P., Grernrum, W., Beijersbergen, R. L. et al. CRISPR knockour screening outperforms shRNA and CRISPRi in identifying essential genes. Nature Biotechnology (2016). | article
