Monthly Archives: June 2013

CRISPR based genome editing – the future of molecular biology.

A vector for genome editing using CRISPR contains the aforementioned elementes, we put in an insert targeting our gene of interest using the enzyme BsblI, which cuts DNA at precise sites marked by the red arrows in the magnified area. We then put in two single stranded DNA oligos and insert them into the region, and when we express the vector in mammalian cells it produces Cas9 or its variants, needed for editing, as well as the RNA that guides Cas9 to our gene of interest.

It isn’t often that I make such seemingly outlandish claims in the title of a blog-post, but this particular technology, CRISPR-based genome editing, I believe deserves the hype.

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats – what that really means is a long sequence of DNA made of similar repeating sequences grouped together. They serve as part of an immune system that evolves to recognise viruses in bacteria and inactivate viral DNA.

Basically, the system cuts up viral DNA into fragments and puts those fragments into the middle of a CRISPR sequence, which we call a CRISPR array, and bacteria then begin to pump out large amounts of RNA encoding this sequence. Other enzymes in the CRISPR pathway go on to find viral DNA based on the CRISPR with the viral DNA insert and then trigger their degradation – which is neat.

What is neater is that we’re now able to use this in a modified form to edit genes in mammalian cells (including human cells). Instead of putting in a viral insert into the CRISPR array, we can put in a 30-base-pairs long stretch of DNA that targets our genes of interest and express RNA from this using some pretty simple genetic engineering (See figure for a description please).

When expressed in cells this can then result in the Cas9 inducing double strand breaks or inducing a nick in one strand, based on what variant of Cas9 we use, in the gene of interest. The really cool thing about this is we can then either wreck the gene using non-homologous end joining or spike in a mutant sequence using homologous recombination, which are two methods by which double strand breaks are repaired – in non homologous end joining the region with the double strand break is cut out and and the sequences on either side are brought together, resulting in the likely loss of the sequence containing the break. If we have a template with a mutation that varies near the site of the break and sequences of DNA that match the sequences on either side of the break well enough that gets incorporated in place of the broken regions instead (See figure 2)

Mammalian double-strand break (DSB) repair. DNA DSBs are predominantly repaired by either non-homologous end-joining (NHEJ) or homologous recombination (HR) [156]. NHEJ rejoins broken DNA ends, and often requires trimming of DNA before ligation can occur. This can lead to loss of genetic information. In NHEJ, the broken DNA ends are bound by the KU70/KU80 heterodimer, which orchestrates the activity of other repair factors and recruits the phosphatidylinositol 3-kinase DNA-PKcs/PRKDC. DNA-PKcs phosphorylates and activates additional repair proteins, including itself and the ARTEMIS/DCLRE1C nuclease. ARTEMIS and/or the heterotrimeric MRE11-RAD50-NBN complex are thought to process the DNA ends prior to ligation. The DNA ends are joined by the activity of polymerases and a ligase complex consisting of XRCC4, XLF/NHEJ1 and LIG4. In contrast to NHEJ, HR is an error-free repair pathway that utilizes a sister chromatid, present only in the S- or G2-cell cycle phase, as template to repair DSBs. HR is initiated by DNA end-resection, involving the MRE11-RAD50-NBN complex and several accessory factors including nucleases. The MRE11-RAD50-NBN complex also recruits the phosphatidylinositol 3-kinase ATM, which phosphorylates histone H2AX and many other proteins involved in repair and checkpoint signaling. Single-stranded DNA generated by DNA end-resection is bound by RPA, which is subsequently replaced by RAD51. RAD51 promotes the invasion of the single-stranded DNA to a homologous double-stranded DNA template, leading to synapsis, novel DNA synthesis, strand dissolution, and repair. Many more proteins are involved in both NHEJ and HR, which are not depicted here for clarity, as they are not referred to in the main text. For details, see recent reviews by Lieber [81] and San Filippo et al. [80].

 Lans et al. Epigenetics & Chromatin 2012 5:4   doi:10.1186/1756-8935-5-4
This technique has earned some rave reviews recently and one of the really cool things is you can express multiple tracrRNA (RNA containing the crispr array to guide Cas9) from a single vector, and it’s even been used to generate mice carrying multiple mutations in one step – which I think is remarkably cool ( )

Generating mutant versions of genes is one of the things I will be doing in the next few months of my PhD, and I must say these are very exciting times indeed to be a molecular biologist – there is something very exquisite about being able to not just turn off genes temporarily but to delete them or to edit their sequence permanently – it is the sort of stuff that enables us to ask what specific mutations in a gene mean for the development of cancer and I see it contributing to some very good research in the years to come.

Ankur “Exploreable” Chakravarthy.

Update – There’s a new paper out in Nature Biotechnology showing that conventional CRISPR-based systems (the version that induces double strand breaks) can result in promiscuous off-target mutagenesis because the DNA-gRNA coupling can tolerate mismatches.

It will be of interest to see if nickase (single strand break inducing Cas9) variants of CRISPR result in higher fidelity because nicks should be repaired unless there is enough mutant template for that to be knocked in by recombination instead.


A window into acquired resistance to targeted therapies – through the eyes of a MEK inhibitor.


Cancer cells, like all other cells in multicellular organisms, are often dependent on inputs from the environment outside the cell for signals that drive growth and survival, among other things. Since abnormal growth and a failure to die like normal cells do are hallmarks of cancer, it makes sense to try and block signalling pathways that contribute to these features using drugs specific to the proteins in these pathways. This is the fundamental premise behind targeted therapies.

The work I’m going to focus on in this article happens to do with inhibition of MEK, which connects external signals to a set of transcription factors that promote the expression of genes related to cell growth and survival.

The Map-kinase signalling pathway. External growth factor receptor kinases are coupled to transcription of genes promoting cell survival and proliferation by means of the Ras-Raf-MEK-ERK signalling cascade. Kinases are proteins that add an inorganic phosphate group to other proteins or in some cases, lipids. 

This pathway is of interest because b-Raf is found to have a very particular mutation, V600E, in a majority of malignant melanomas – something so characteristic of the disease that there is a drug that specifically targets the mutant version of this protein, but responses are often short-lived because cells learn to get round the blockade of the protein. Interestingly, this pathway is also involved in colorectal cancer, often involving the same mutations or a mutation in the protein that comes before b-Raf, called k-ras, which is a very potent oncogene.

Simon Cook and his group at the Babraham Institute tried to figure out how cancer cells that depended on this pathway would come to acquire resistance to a MEK inhibitor (which is downstream of B-raf and k-ras). To do this, they took two colorectal cancer cell lines with a b-Raf mutation and two with a k-ras mutation and cultured them in the presence of ever increasing concentrations of a MEK inhibitor : AZD6244. They fundamentally found that resistant cells seemed to acquire amplifications in the number of copies of b-Raf or k-Ras, thus serving to maintain the same signal intensity downstream of MEK in the presence of the drug as they would have in the absence thereof.

Resistance to a MEK inhibitor in a B-raf mutant cell line is explained by amplification of B-raf at the DNA level, which is reflected at the protein level and increased activation of ERK1 (P-ERK1/2). The graph at the bottom right shows that knocking down b-Raf levels using RNA interference reverses resistance to the MEK inhibitor.

They also found that in Ras mutant cell lines, amplification of k-Ras was to blame for the phenotype. The problem with that is Ras is a pain in the rear to develop drugs against, but with b-Raf there are inhibitors available and it should be possible to resensitize resistant cells to the MEK inhibitor by hitting it at both points in the pathway.

The trouble with this MEK inhibitor is that it leads to cell cycle arrest being the major response as opposed to cell death, so it would be sensible to see if, for already dampened levels of ERK activation through MEK inhibition, it should be possible to increase the proportion of cancer cells that actually fuck off and die instead of just waiting for the drug to wear off.

Apoptosis is a process mediated by a combination of pro-apoptotic proteins and anti-apoptotic proteins and when a threshold is reached in terms of dominance of pro-apoptotic proteins it sets of a cascade of signalling events that leads to the destruction of the cells. One of the reasons cell cycle arrest is favoured over cell death is the hyperactivity of anti-apoptotic proteins such as Bcl2. There is a protein called BH3 which can bind to and disable Bcl2, rendering cells much more susceptible to apoptosis if the MEK pathway is hit, so they looked at combining a drug that mimics the structure of BH3 with the MEK inhibitor and promptly found that apoptosis was greatly enhanced and the emergence of resistance delayed .

Finally, of course, it is worth considering the fact that in malignant melanoma, drug holidays, where treatment is not administered for a while, has been shown to reverse resistance well in line with what we’d expect – the overexpression of oncogenes is associated with oncogene induced senescence and what might maintain the activity of the pathway in the presence of the drug might activate the pathway too much when the drug is taken away – like how a car might crash if you suddenly took the brakes off while the pedal was still pressed to the same extent as when driving with the brakes on. This means that drug resistance is favoured only because of a selective pressure imposed by the drug and might actually be a detriment in competition with drug sensitive cells when the drug is absent. Take the drug away when the cells that are most dependent form the bulk of the tumour, and they really do crash dramatically

That’s all from me until next time.


PS – additional papers…



Intro to Schizo

A while ago, I saw an Indian movie in which the female protagonist was afflicted with visual and auditory hallucinations, bizarre paranoia and delusions. Of course, there was a song and dance sequence to accompany it. This movie spurred me to go online and look for more information regarding this ‘madness’. I learnt about Schizophrenia and later about Autism spectrum disorder. There is a TED talk by Jill Bolt Taylor who talked about the most fascinating things in a very interesting manner. You can find that here.

Ever since, I have made an effort to know more about this interesting disorder and today, I wish to introduce this topic to you. Before we do, I believe it is important to understand the structure of the brain. You can find some information here which has a fun, interactive way of learning about the basics of brain and its function.

It is important to know that Disassociative identity disorder (Usually referred to as split personality or multiple personality disorder) is different from Schizophrenia. People afflicted with this disorder, as described above, suffer from hallucinations, delusions, behavioural problems etc. which makes it difficult for them to be accepted in the society.

Most medications prescribed for patients with this disorder belongs to the class of molecules which have a capacity to suppress the activity of dopamine (sometimes serotonin as well) receptors. Clinical studies relating schizophrenia to brain dopamine metabolism have ranged from controversial to negative, with HVA levels in the CSF the same for schizophrenics and controls. There is a dopamine hypothesis theory on the internet, have a look.

Dopamine receptors (D1-D5) are G-protein coupled receptors meaning they work through secondary messenger system (like Adrenalin). Serotonin receptor aka 5-HT receptors are ion gated channels. I remember writing a post about ion channels. Check it out!

Let us see where dopamine is present in high quantity and what it does to the system. The ventral tegmental area (VTA, situated in mid brain) contains the largest group of dopamine neurons in the human brain. The main function of dopamine is to set a threshold for executing behaviors. Meaning, if a certain high level of dopamine activity occurs, a lower impetus is enough to evoke a given behavior. As a consequence, high levels of dopamine lead to high levels of motor activity and “impulsive” behavior; conversely, low levels of dopamine lead to torpor and slowed reactions. Another function of dopamine is to ‘teach’. If an action is followed by an acitivty, it alters the brain in such a way that same action becomes easier to execute if performed at a later time. There are several theories surrounding how this occurs. Most of them involve basal ganglia modifications.

In my next post, I shall talk about the controversy surrounding usage of anti – psychotic drugs for treatment of Schizophrenia.

Till then,