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GEN News Highlights : Dec 13, 2013
Scaled-Up CRISPR-Cas9 Allows Genome-Wide Functional Screening
At present, RNA interference (RNAi) is the most widely used method for studying gene functions. It is limited, however, because it relies on destroying messenger RNA, not DNA. Via RNAi, complete elimination of targeted genes is impossible. To get around this limitation, scientists are rapidly developing an alternative technology, CRISPR, or clustered regularly interspaced short palindromic repeats. CRISPR, already recognized for its ability to completely deplete a given protein in a cell, has been effectively scaled up. As indicated in two studies published December 12 in Science, CRISPR—along with the Cas9 nuclease and single guide RNAs (sgRNAs)—has been used to interrogate gene function on a genome-wide scale.
Both studies employed knockout screening. They showed that it is possible to use CRISPR-Cas9 technology to study all the genes in the genome by deleting a different gene in each of a huge population of cells, then observing which cells proliferated under different conditions.
One study, led by Feng Zhang, the W.M. Keck Career Development Professor in Biomedical Engineering at MIT, a core member of the Broad Institute, and an investigator of the McGovern Institute, is entitled “Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells.” The second study, led by David Sabatini, a professor at the Whitehead Institute and MIT, and Eric Lander, director of the Broad Institute and a professor at MIT, is entitled “Genetic Screens in Human Cells Using the CRISPR/Cas9 System.”
The Zhang team created a library of about 65,000 guide RNA strands that targeted nearly every known gene. They delivered genes for these guides, along with genes for the CRISPR machinery, to human cells. Each cell took up one of the guides, and the gene targeted by that guide was deleted. If the gene lost was necessary for survival, the cell died.
This approach enabled the researchers to identify genes essential to the survival of two populations of cells: cancer cells and pluripotent stem cells. The researchers also identified genes necessary for melanoma cells to survive treatment with the chemotherapy drug vemurafenib.
In their paper, the scientists wrote, “We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening inhuman cells.”
"This is the first work that really introduces so many mutations in a controlled fashion, which really opens a lot of possibilities in functional genomics," said Ophir Shalem, a Broad Institute postdoc and one of the lead authors of the Zhang paper.
The Zhang paper’s authors concluded that “the efficiency of complete knockout, the consistency of distinct sgRNAs, and the validation rate for top screen hits demonstrate the potential of Cas9:sgRNA-based technology.”
The team led by Sabatini and Lander targeted a smaller set of about 7,000 genes, but they designed more RNA guide sequences for each gene. The researchers expected that each sequence would block its target gene equally well, but they found that cells with different guides for the same gene had varying survival rates.
Specific results reported by this team included the following: “A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, while another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes.”
Overall, the team led by Sabatini and Lander concluded that their work showed that “sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs.”
Together, the two papers, said Lander, “demonstrate the extraordinary power and versatility of the CRISPR-Cas9 system as a tool for genome-wide discovery of the mechanisms underlying mammalian biology.”
“And we are just at the beginning,” continued Lander, “we're still uncovering the capabilities of this system and its many applications.”
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