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Genome-wide CRISPR-Cas9 Screen Uncovers Genes Involved in Melanoma Treatment Resistance

COLD SPRING HARBOR, NY (GenomeWeb) — Using a CRISPR-Cas9-mediated genome-wide screen, researchers have been able to identify genes linked to skin cancer treatment resistance, according to work presented at the Biology of Genomes conference by Neville Sanjana, a postdoctoral researcher at the Broad Institute.

The CRISPR (clustered regularly interspaced short palindrome repeats)–associated nuclease Cas9 induces double-stranded DNA breaks in the genome, breaks that are targeted to specific spots through a synthetic single-guide RNA (sgRNA). These breaks introduce a frameshift mutation that leads to a loss-of-function allele.

Other genomic engineering approaches rely on protein engineering, Sanjana noted, while the CRISPR-Cas9 system only requires a short bit of RNA.

"Here, the programmability has taken a bit of a quantum leap," Sanjana said.

For their work, Sanjana and his colleagues used lentiviruses to deliver Cas9, sgRNA, and a selection marker to their target cells, where they are then continuously expressed.

They also developed a genome-scale CRISPR knockout library, dubbed GeCKO, using in silico designs of exon targets and tried to minimize any off-target effects. In all, the library contains nearly 65,000 guide sequences targeting some 18,000 genes.

In testing the GeCKO library, the researchers searched for genes essential for cell viability in two cell lines, the A375 cancer cell line and the HUES62 stem cell line. Deep sequencing of the lines two weeks after transduction found a significant reduction in the diversity of sgRNAs in the surviving cells, and many of the depleted sgRNAs targeted genes like ribosomal structural constituents and other essential genes, as Sanjana and his colleagues recently reported in Science.

Sanjana and his colleagues then applied this approach to a melanoma model to search for genes linked to treatment resistance. In more than half of melanoma cases, he noted, the protein kinase BRAF, which regulates the MAPK signaling cascade, is mutated, and in about 70 percent of those cases that mutation is a V600E mutation.

That mutation is targeted by the RAF inhibitor vemurafenib, but while more than half of patients do initially respond to such treatment, resistance to the therapy often develops, Sanjana added.

To find genes associated with escape mutations, Sanjana and his colleagues applied the GeCKO library to the A375 cell line, which is homozygous for the BRAF V600E mutation. They then treated the lines with either DMSO or the drug and searched for variants allowing the cells to withstand treatment after 14 days.

Among their top candidates were both genes previously associated with treatment resistance — NF1 and MED12 — as well as novel ones — NF2, CUL3, TADA2B, and TADA1. Sanjana noted a high level of consistency between guide RNAs that targeted the same gene and a high rate of hit confirmation.

This screen, he said, has led to new leads for cancer researchers.

Sanjana also noted that he and his colleagues have been refining their lentiviral CRISPR tool and genome-wide screening libraries, both of which he estimated will be available on Addgene later this month.

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