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 ( http://www.sciencedirect.com/science/article/pii/S0092867413004674 )

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.

Cheers,
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. http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.2623.html?WT.mc_id=TWT_NatureBiotech

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.

Advertisements

One response to “CRISPR based genome editing – the future of molecular biology.

  1. Pingback: CRISPR based genome editing - the future of mol...

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s