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CRISPR Deletion-Based Screen Identifies Functional Long Non-Coding RNAs

NEW YORK (GenomeWeb) – A new study published today in Nature Biotechnology turns high-throughput CRISPR/Cas9-based knockout screening towards long non-coding RNAs to find functional elements.

A Chinese-American collaboration, led by first author Shiyou Zhu and senior author Wei Li  of Peking University and first author Wensheng Wei and senior author Xiaole Shirley Liu of the Dana-Farber Cancer Institute, designed a paired guide RNA (gRNA) screening method that identified 51 lncRNAs positively or negatively regulating cell growth in Huh7.5 OC cells.

It's a deletion-based strategy, Neville Sanjana of the New York Genome Center and New York University told GenomeWeb, where two gRNAs simultaneously direct cleavage to cut out a chunk of DNA.  "I think this is a nice preview of how we can scale up to cover larger intervals of DNA to tackle the non-coding genome.

"Using this method, we screened approximately 700 human lncRNAs and identified lncRNAs that have oncogenic or tumor suppressor activities in cancer cells," the authors wrote. "The vast majority of all mammalian genomes comprise non-coding regions, many of which have important regulatory roles. Functional analyses of non-coding regions have been challenging, and an effective screening strategy for non-coding regions based on genomic deletion was, until now, lacking."

The researchers followed up by looking more closely at five lncRNAs essential to cell proliferation and four lncRNAs detrimental to cell growth. The validation experiments confirmed all nine hits, the authors said, but added that the biological significance is not readily apparent. They added that more than 30 percent of the identified lncRNAs were located in introns. "Disrupting introns may perturb splicing or other regulatory elements and have deleterious effects on cell proliferation," they wrote.

"LncRNAs are exciting non-coding elements and several groups are applying CRISPR screens to interrogate them," Sanjana, an expert on CRISPR screening who is also involved with a separate screening study of non-coding elements, said.

It's the latest CRISPR screening method to look for functional elements of the genome. High-throughput CRIPSR-based knockout screens first found their way into the literature in late 2013, when two separate groups led by Broad Institute researchers Eric Lander and Feng Zhang, respectively, published papers in the same issue of Science. Since, then, several groups have used knockout screens to parse the genome, looking for functional elements amongst enhancers, non-coding RNAs, and transcription factors.

In the Nature Biotechnology publication, the researchers noted several limitations to their study. Deleting lncRNAs may affect nearby functional elements like enhancers and microRNAs, they said, and the screen could not reveal a mechanism of action for the lncRNAs identified, requiring follow-up study.

The researchers performed analysis of screening data with the model-based analysis of genome-wide CRISPR/Cas9 knockout (MAGeCK) algorithm developed by Liu and Zhang.