There aren’t many studies exciting enough to make you jump out your lab coat and rock’n’roll round the lab like it’s the 50s, but a recently reported new application of CRISPR might just start your toes tapping and your heads nodding. This new article does not originate from “Deep down in Louisiana close to New Orleans”, but rather from the laboratory of Stanley Qi at Stanford University (CA, USA), where researchers hoped to discover how changes to nuclear architecture can affect gene regulation and cellular function.
The intrepid team developed a strategy named CRISPR-genome organization (CRISPR-GO) that repositions genomic loci relative to critical compartments within the cell nucleus, such as the nuclear periphery (NP) and Cajal bodies (CBs). Their approach coupled the nuclease-deactivated CRISPR-Cas9 (dCas9) system to target the nuclear-compartment-specific effector domains and utilizes ligand-mediated dimerization for a chemically inducible and reversible approach to the study of genome organization in real-time in living cells.
Here are some of the hits from this raucous new article; go CRISPR-Go go!
- The authors generated two chemically-inducible heterodimerization approaches: an abscisic acid (ABA) inducible ABI/PYL1 system and a Trimethoprim-Haloligand (TMP-Htag) inducible DHFR/HaloTag system
- As an example, in a human bone osteosarcoma cell line expressing dCas9 fused to ABI and PYL1 fused to an NP-specific protein, the introduction of a guide (g)RNA targeting a cyan fluorescent protein (CFP) reporter construct repositions the reporter to the NP, but only in the presence of the ABA inducer (dCas9—ABI—< ABA >—PYL1—NP protein)
- Employing region-specific gRNAs, the authors discovered that the heterodimerization systems could reposition highly-repetitive, less-repetitive, and non-repetitive sequences
- The removal of the specific chemical inducer (g., ABA) reverses any repositioning, allowing for the dynamic analysis of genome organization
- A combination of CRISPR-GO with live-cell CRISPR-dCas9 imaging permits the interrogation of the real-time dynamics of genomic loci repositioning to the NP and CB
- NP relocalization of the CFP reporter (promoted by fusing PYL1 with the nuclear envelope protein Emerin) suggests that NP repositioning occurs by mitosis-dependent and mitosis-independent mechanisms over several hours
- While previous research on genome organization established mitosis-dependent tethering of loci to the NP before, this study demonstrates mitosis-independent tethering for the first time
- While proximal reporter expression diminishes upon relocalization to the NP, distal endogenous gene display no alterations to expression levels
- CB relocalization of the CFP reporter (promoted by fusing PYL1 with Coilin, a marker of CBs) causes significant repression of both proximal reporter gene expression and distal endogenous gene expression
- CB repositioning occurs much faster than NP relocation, taking only a few minutes, perhaps due to the diffuse nature of CBs in the nucleus
- NP relocalization of the CFP reporter (promoted by fusing PYL1 with the nuclear envelope protein Emerin) suggests that NP repositioning occurs by mitosis-dependent and mitosis-independent mechanisms over several hours
“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,” Lead author Qi said. “It suggests that the Cajal body has some unexpected role in controlling transcription.”
The authors end this wild genome organization study by pointing out the future for CRISPR-GO – the adaptation of the system to analyze the interaction of compartments such as nucleoli, nuclear pore, nuclear speckles, and heterochromatin with a more extensive variety of genomic elements, including protein-coding genes, non-coding RNA genes, and regulatory elements.
Go CRISPR-Go go! Read all the liner notes on this rocking new genome organization extravaganza over at Cell, October 2018.