About a year and a half ago I wrote here about new uses of CRISPR/Cas9 as an imaging tool. In particular, I was excited about the possibility to use enzyme-dead Cas9 (dCas9) as an RNA binding protein for live imaging of mRNA. Unfortunately, in my hands this did not work (the dCas9 has exited the nucleus with non-targeting guide RNA at the same rate as with the specific guide RNA).
Last week, a new CRISPR tool was published in Nature, from Feng Zhang’s lab.
The hottest buzz-word in biology today is CRISPR: an adaptive immune system in bacteria and archea. At its basis is a nuclease, named Cas9, which is targeted to DNA by a short single-guide RNA (sgRNA). This turned out to be a very useful system for genome engineering in any organism due to its specificity (provided by the sgRNA) and its simplicity (all you need is to express the Cas9 and sgRNA in the cell). However, this system can also be used for other purposes. One such use is modulation of gene expression, for example by targeting a nuclease dead Cas9 (dCas9) fused to a transcription activator or repressor to promoter regions. Another such use is for imaging.
Here, I’ll described how Cas9 can be used to visualize specific DNA loci or specific RNA transcripts in fixed and live cells.
Posted in CRISPR/Cas9, FISH, Gene expression, Genetics, Journal club, Whole tissue imaging
Tagged CASFISH, CRISPR, DNA FISH, HaloTag, Mammalian cell, quantitative microscopy, RCas9, Singer lab, stress granules
(part 1, part 2)
I ended part 2 Monday night. It was an exciting day with many excellent talks, but the best talk (mine, of course!) was due the next day.
Tuesday started with the seminar on engineering cells and tissues. There was the mandatory CRISPR talk as the great new thing in bio-engineering these days. Jennifer Doudna talked about the discovery, then went on to discuss new experiments (using Halo-tag to track Cas9 in live cell nuclei to study movement & binding kinetics) and improved technologies (transfect cells with pre-assembled Cas9-gRNA for quick editing & less off-target cleavage).