In silico versus in vitro

For the past five days at least I have felt bad for not updating my blog with some fact about CRISPR or the latest controversy concerning “cancer stem cells”. The reason for this involuntary hiatus is, of course, lab work. All the lab work. All the time. How should I put this; there are distinct benefits to working in silico: leaving the lab/office whenever is convenient, being able to continue work at home, and actually getting home on time for dinner.

Here I am, genuinely happy to be doing work in cell culture (isn’t the dark blue of that lab coat excellent?):

image3

And then running samples in pre-cast 20-well gels ready to do a Western blot after having treated melanoma cells with a plethora of small molecule inhibitors:

image1

But then disaster had to strike. It was all going too well. Due to the bubbles rising from the electrodes in the above picture I didn’t have a clear view of the second gel running behind it. What a disappointment:

image2The sad smiley face accurately represents my emotions concerning this gel. Well, at least I know what I’m doing tomorrow.

So I’ll end with a mini CRISPR update: Tsai et al. (2014) developed a new method last year to reduce non-specific cleavage of DNA during genome-editing. They require expression of RNA-guided FokI nucleases, which also cleave DNA, but are only active when dimeric. Each single FokI molecule is guided to its target by a guide RNA, but only when two guide RNAs each bring a FokI to the desired locus do the enzymes become active. This drastically reduces off-target effects because both the sequence and spacing has to be correct. The original CRISPR/Cas9 system has considerable off-target effects, as shown by Lin et al. (2014), for example.

Reference:

Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, Wile BM, Vertino PM, Stewart FJ, Bao G (2014) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Research 42: 7473-7485

Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotech 32: 569-576

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3 thoughts on “In silico versus in vitro

    • Oh no – don’t be sorry! The bubbles were meant to be there (they’re a sign that voltage is actually being applied to the protein gel, pulling the sodium dodecyl sulfate (SDS)-coated proteins towards the positive pole)!

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      • Ah I see, fair enough.
        Incidentally, SDS-coated protein is an application of one of my studies into the effect of surfactants on the micro-scale fluid dynamics. So I’m glad this sort of thing is actually useful!

        Liked by 1 person

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