CRISPR or clustered, regularly interspaced, short palindromic repeat sequences are commonly found in bacteria and function as part of their innate immune system to counter foreign nucleic acids such as viruses and plasmids. CRISPR DNA sequences are translated into CRISPR RNAs (crRNAs) which complex with Cas or (CRISPR-associated) proteins to bring about cleavage of invading DNA. These systems were first described in 1987 but their precise function as an immune defense was only well-established by 2008.
At the same time, gene editing was being explored with the use of zinc finger proteins and transcription activator-like effector nucleases (TALEN). These proteins can basically be engineered to bind any DNA sequence, inducing double stranded breaks that trigger the cell’s physiological DNA repair mechanism which is prone to introducing errors. This leads to introduction of DNA modifications that often generate a dysfunctional/altered protein, or no protein at all. Other methods of knocking down genes include RNA interference where short RNA sequences of 21-25 bases are introduced into the cell and bind to a complementary endogenous mRNA sequence, leading to their cleavage or inhibition of translation. RNA interference methods however differ from genetic/DNA editing by being transient and incomplete, where low levels of protein function may still exist. They have also been plagued by off-target effects where imperfect matches between siRNA (or silencing RNA) can also lead to gene knockdown.
The use of CRISPR has caught the scientific world’s attention due to its ease in introducing genetic alterations. They have now been modified down to a simple two component system that can be introduced via a single vector: the Cas9 nuclease and the guide RNA (gRNA) consisting of a crRNA and tracRNA (or transactivating RNA; necessary for modulating Cas9 function) fusion. By modifying the guide RNA sequence, one can target any genetic sequence for cleavage by Cas9.
How CRISPR works (From Sander and Young, 2014 “CRISPR-Cas systems for editing, regulating and targeting genomes”, Nature Biotechnology, 32,347–355)
Indeed, several genetic screens have been performed with CRISPR using large gRNA libraries (64,000-87,000) producing knock-out cell lines within 7-14 days. Complete gene knockout was observed creating clear phenotypes as seen from a complete loss of fluorescence from GFP-expressing cells. What about off-target effects? Principally, CRISPR and RNAi work on the same premise of targeting short (20 base long) sequences that might occur in several places within the genome. However, siRNAs utilize endogenous proteins Argonaute and Dicer to achieve their effects, whereas the details on how gRNA-Cas9 work together to achieve genetic alteration has not been sufficiently well-characterized. The jury therefore is still not out yet on their potential off-target effects. Certain interesting observations however have been described. For example, open chromatin structures may potentially lead to more Cas9 binding and cleavage, while cleavage efficiency of a gRNA was found highly determined by Cas9 affinity. The CRISPR system also affords the added advantage of not just being used for gene knockouts but also gene activations as catalytic dead versions of Cas9 may be used to provide other perturbations to DNA.
But in case you get carried away and start acting like a CRISPR-obsessed maniac, take note of the limitations. For pharma companies in particular, what drug can provide 100% inhibition of a target protein? In that respect, siRNA screening as compared to CRISPR screening may provide a more accurate representation of the actions of small molecule drugs. Also, the fact that cell lines have to be engineered and lentiviral systems established may impose greater challenges on time and resources compared to siRNA screening where one simply throws on siRNAs onto cells and see effects within 2-3 days.
In the next blogpost, we shall talk more on how best to counter the off-target effects of siRNA screens. A subject quite dear to my heart.