Taming the Transposon Hordes

investigate how transposons affect nearby genes, a vector containing 12 slightly different gRNAs targeted to a particular retroviral element is introduced into cells together with a Cas9-activator or -repressor fusion protein. The Cas9 protein associates with the gRNAs, and the subtle sequence differences of the gRNAs allow almost all copies of the targeted retroviral element to be switched on or off at once.

Researchers repurpose the CRISPR machinery to turn whole classes of transposable elements on or off.

ransposable elements, including remnants of viral DNA retained in the genome for millions of years, make up a substantial fraction of mammalian genomes, and their effects on gene expression are thought to have driven speciation.

However, while there is anecdotal and circumstantial evidence suggesting these elements affect gene regulation—such as transcription factor binding sites in their sequences—techniques for systematically analyzing these elements’ activities are limited, says Joanna Wysocka of Stanford University School of Medicine. (See “Can Viruses in the Genome Cause Disease? The Threat of Ancient Viruses Tucked in the Human Genome.”) “So we don’t know overall how important they may be.”

Using a single short RNA, such as a CRISPR guide RNA, to manipulate transcriptional activity at multiple copies of an element should work in theory, says Wysocka. But in practice, “because over evolutionary time [the elements] accumulate mutations, they are different enough that . . . a single guide RNA [is insufficient].”

Fortunately, Wysocka’s team had previously developed a non-transposon-related imaging approach that involved packing code for multiple guide RNAs into one plasmid. They have now used such a plasmid, called a chimeric array of gRNA oligos (CARGO), containing a variety of subtly different gRNAs, to target multiple copies of the human endogenous retrovirus HERV-K. Expressing gRNAs from the CARGO plasmid together with special Cas9 fusion proteins that cannot cleave DNA but bind it to activate or repress transcription (encoded in a separate plasmid), the team was able to switch on or off almost 90 percent of the 700 full and partial copies of HERV-K in the genomes of cultured human embryonic carcinoma cells at once.

The team identified 275 human genes that were both upregulated by the Cas9-activator and suppressed by the Cas9-repressor, suggesting that these genes, which tended to be close to HERV-K sequences, were directly affected by the retroviral elements. Wysocka and colleagues then confirmed the elements’ roles in six of the genes’ expression by deleting HERV-Ks at the loci and recording their effect on gene activity.

The technique could be extended to a wide variety of transposable elements, says Todd Macfarlan of the National Institute of Child Health and Human Development, who was not involved in the research. Indeed, adds Cornell University’s Cedric Feschotte, also not involved in the study, “it’s really a game changer [for] the potential to interrogate this repetitive fraction of the genome.”

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