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Stanford Researchers Use CRISPR to Manipulate Genome Organization in 3D

NEW YORK (GenomeWeb) – A team of Stanford University researchers studying spatial genome organization and its effect on cellular function has developed a CRISPR-based system to reorganize the genome.

In a paper published in Cell today, the researchers described CRISPR-Genome Organization (CRISPR-GO) — a system that can control the positioning of genomic loci relative to specific nuclear compartments, including the nuclear periphery, Cajal bodies, and promyelocytic leukemia (PML) bodies. Further, the system can mediate rapid de novo formation of Cajal bodies at desired chromatin loci and can cause significant repression of endogenous gene expression over distances of 30 to 600 kilobases.

"CRISPR-GO is chemically inducible and reversible, enabling interrogation of real-time dynamics of chromatin interactions with nuclear compartments in living cells. Inducible repositioning of genomic loci to the nuclear periphery allows for dissection of mitosis-dependent and -independent relocalization events, and interrogation of the relationship between gene position and gene expression," the authors wrote.

Cajal bodies have been implicated in the biogenesis of small nuclear RNA, ribonucleoprotein assembly, and telomerase biogenesis, are essential for vertebrate embryogenesis, and are abundant in tumor cells and neurons, the researchers noted. PML nuclear bodies are also associated with tumorigenesis and defense against viral infection. But the relationship between nuclear body or chromatin colocalization and gene expression remains poorly understood — microscopic and chromosome conformation capture-based techniques cannot show causal links between genome organization and function.

For this study, the researchers combined a deactivated Cas9 and nuclear-compartment specific proteins to create CRISPR-GO. To test if the system was able to alter the position of chromosomes, they then introduced a single-guide RNA that targeted a highly repetitive endogenous region within chromosome 3q29 and added an independent CRISPR-Cas9 imaging component.

After two days, the researchers observed that the percentage of chromosome 3 loci that localized to the nuclear periphery increased from 19 percent to 87 percent, and the percentage of cells showing at least one locus in the nuclear periphery increased from 27 percent to 95 percent. This confirmed that CRISPR-GO was efficient in repositioning highly repetitive endogenous genomic loci in human cells.

They further tested repositioning other highly repetitive endogenous genomic loci, as well as less repetitive sequences, to the nuclear periphery and found that CRISPR-GO was efficient in doing so.

The team then asked if the CRISPR-GO system could also reposition non-repetitive genomic loci. Specifically, the researchers targeted the non-repetitive XIST gene, located at ChrXq13.2, and designed 13 sgRNAs spanning the XIST genomic region. They observed that the percentage of periphery-localized XIST loci increased from 39 to 79 percent, and the percentage of cells with periphery loci increased from 59 to 90 percent.

Additional experiments using various numbers of sgRNAs and a single sgRNA further confirmed that CRISPR-GO was able to mediate efficient relocalization of non-repetitive loci to the nuclear periphery.

Importantly, when the researchers used CRISPR-GO to colocalize genomic loci with the nuclear periphery or Cajal bodies, they found that adjacent reporter gene expression was altered as a result.

"We were super-excited to see this; it's the first time that researchers have evidence to show the Cajal body can have a direct gene-regulation effect, in this case repressing gene expression," senior author Stanley Qi said in a statement. "It suggests that the Cajal body has some unexpected role in controlling transcription."

When the researchers used CRISPR-GO to move DNA from the middle of telomeres to the nuclear periphery, the telomeres stopped growing. But when the telomeres were moved closed to the Cajal bodies, they grew, and cell viability increased.

"Notably, colocalization of endogenous loci with Cajal bodies significantly represses distal gene expression from the target site," the authors wrote. "The CRISPR-GO method will facilitate a deeper understanding of the functional role of spatiotemporal genome organization."

They also concluded that the system could potentially be adapted to other nuclear compartments such as nucleoli, nuclear pore complexes, nuclear speckles, and heterochromatin, and that its programmable feature could allow its use in targeting protein-coding genes, non-coding RNA genes, and regulatory elements.