CRISPR Digest #4

Lent Term at Cambridge has officially ended and with it the last day of work in the lab and my last ever undergraduate lectures and seminars. Now begins the period of analysing results and trying to formulate a coherent dissertation, but that should be the subject of a different entry.

The last so-called “scientific methodology” workshop we had this term was entitled CRISPR/Cas9 – you can imagine my excitement about this, especially because it turned out that I learnt at least three new facts in that session (quite a lot more than can be said about some of the cancer lectures, which were more or a less a repetition from last year).

  1. The functions of the so-called protospacer adjacent motif (PAM), a short DNA sequence present in the target DNA, just next to the cleavage site, but not the guide RNA; for the widely used Streptococcus pyogenes type II CRISPR/Cas9 system it is NGG:
    1. The PAM possibly provides a means of simplifying the search for the correct target sequence.
    2. Binding of Cas9 may be initiated at the PAM sequence from where the transition to active DNA cleavage may take place.
    3. Most importantly – and this is something I hadn’t at all thought of before – the PAM can provide a mechanism for the discrimination between self and non-self, a fundamental feature of all immune systems. CRISPR systems evolved as adaptive immune systems in bacteria and archaea, targeting invading DNA from bacteriophages (viruses that infect bacteria) and plasmids. The host genome incorporates stretches of the invading DNA and then expresses that as guide RNAs, which in turn direct the Cas nucleases against the invading DNA. But of course this means that the guide RNAs also interact with the DNA that was incorporated in the host genome in the first place, which would lead to cleavage of host DNA – quite the opposite of the desired outcome. Therefore, the host sequence does not contain the necessary PAM sequences so that the Cas nucleases cannot attack their own host! [See, for example, Mali et al. (2013) or Marraffini & Sontheimer (2010).]

  2. Novel methods of regulating the activity of Cas9 variants so that gene expression can be fine-tuned in target cells of interest:
    1. The first of two papers discussed (Nihongaki et al. (2015)) combined the CRISPR system with optogenetics, a tool originally developed by neuroscientists to selectively stimulate or suppress the activity of specific neurons in the brain: in this system Cas9 is catalytically inactive (dCas9; cannot cleave DNA), but is fused to a protein called CIB1. Additionally, a transcriptional activator/repressor is fused to a protein domain of CRY2. Upon irradiation with blue light CIB1 and CRY2 interact, thus bringing the transcriptional regulator in proximity with dCas9, which has been targeted to a region of interest by a guide RNA. This allows controlled expression of genes of interest from their endogenous loci (image copied from their paper):

      Screen Shot 2015-03-17 at 11.58.56

    2. In the second paper by Zetsche et al. (2015) they engineer a split Cas9, in which each half of the protein is fused to a domain from the protein mTOR (normally involved in regulating cell growth). These domains dimerise upon addition of the drug rapamycin, thus activating Cas9 because the two halves are brought together again. This approach allows temporal control of Cas9 activity and can also be used with dCas9 to regulate gene expression. However, the concern I have with the method is that rapamycin is a drug that inhibits mTOR and, to my knowledge, the paper does not address the question of whether the drug treatment in itself is detrimental to the cells.

  3. Lastly, we were introduced to a new, CRISPR-based technique that I had not encountered before: enChIP – engineered DNA-binding molecule ChIP (Fujita & Fujii (2013)). ChIP stands for chromatin immunoprecipitation and in its basic form relies on specific antibodies with which proteins of interest can be “pulled out” together with other proteins and DNA they are interacting with. For example, this technique can be used to find out where in the genome protein X binds – does it bind the promoter regions of certain genes? Does it bind to a specific DNA sequence repeatedly? Etc.
    In enChIP, dCas9 is fused to a short peptide known as a FLAG tag, against which very good antibodies exist. This fusion is then targeted to a DNA sequence of interest (or very close to a sequence of interest) where the protein complexes formed are crosslinked before being “pulled out” with the anti-FLAG antibody. The complexes are subsequently subjected to mass spectrometry, which can resolve the constituent parts of the complex. In a way, therefore, enChIP can provide complementary information to standard ChIP: in the former one is looking for proteins that bind to specific DNA sequences, whereas in the latter one is looking for DNA sequences bound by specific proteins.

Hopefully this digest will keep you all satiated with information on the latest CRISPR news for a while. Now: time to dissertate (yes, the OED assures me that this is indeed a word, if somewhat “unusual”).


Fujita T, Fujii H (2013) Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochemical and Biophysical Research Communications 439: 132-136

Mali P, Esvelt KM, Church GM (2013) Cas9 as a versatile tool for engineering biology. Nature Methods 10: 957-963

Marraffini LA, Sontheimer EJ (2010) Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463: 568-571

Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M (2015) CRISPR-Cas9-based Photoactivatable Transcription System. Chemistry & Biology 22: 169-174

Zetsche B, Volz SE, Zhang F (2015) A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat Biotech 33: 139-142

In silico versus in vitro #2

There is no adequate excuse for not keeping up with regular blog entries. Arguably, working in the lab from 9 a.m. to 8.30 p.m. – in a mad rush to collect some data before the lab meeting next week – in addition to preparing for seminars and the imminent badminton match against Oxford help to explain the recent scarcity of cell/science-related news. Especially when a statistically significant number of experiments in the lab end up looking like this Western blot on the right (image copied from here):

notpub-three kinds of westernsHowever, on the more positive side of things I have learnt a couple more cell/molecular biology techniques since I started working in the lab several weeks ago. Beforehand I only knew about the theory of these methods and how to interpret results, but not how to actually carry out the experiments:

  • Immunohistochemistry (IHC): this technique allows specific staining of (mouse) tissue sections. One can, for example, compare specific markers of proliferation or cell death in healthy skin versus tumours/melanoma lesions.
  • Quantitative reverse transcription polymerase chain reaction (qRT-PCR): this allows analysis of gene expression (specifically transcription) in tumour samples or cultured cells under different conditions. For example, I have been comparing the expression of certain transcription factors in cells that are sensitive to a melanoma drug versus cells that have acquired resistance to that drug.
  • Cell cycle analysis by fluorescence-activated cell sorting (FACS): lastly, this method is used to quantify the proportion of cells within a population that are actively dividing, non-dividing or dead. To do this, the amount of DNA in each cell is stained using a dye such as propidium iodide, or newly synthesised DNA can be labelled using bromodeoxyuridine.

Now on to the simple task of analysing the data…