CRISPR/Cas 9 – Genome editing technique
Modulating the expression of one or multiple genes to determine its effect on an organism’s phenotype is an indispensable tool in molecular biology. Nowadays researchers claim to have overpassed more ancient techniques such as siRNA resorting to CRISPR/Cas9. Is it a myth or a reality?
CRISPR/ Cas9 technique
CRISPR refers to Clustered Regularly Interspaced Short Palindromic. Its a gene editing technique that uses three components: the CRISPR associated protein 9 (Cas9) and a guide RNA (gRNA) which includes CRISPR repetitive sequences and DNA target called spacer. This gRNA sequence will guide an expressed Cas9. Once, the so called gRNA has bind to the complementary DNA sequence, the Cas9 will cleave the double strand. However, to enhance the specificity and avoid a maximum of off-target breaks, the Cas9 must recognize a unique but redundant DNA reading frame from 2 to 5 nucleotides near the target. This recognition pattern is a protospacer adjacent motif PAM and is repeated each 8 bps in the genome for the SpCas9 PAM for example (cf Ramakrishna et al., 2014).
A wide range of Applications
Researchers actually work on Cas9 variants able to recognize different PAM sequence or modulate gene expression differently. For instance, some mutations degenerate the two Cas9 domains responsible for double strand breaks to promote gene activation or repression. Thus, the main advantage of this technique is its versatility : mutation in Cas9 allow us to process different gene editing from activation, interference, gene editing, to gene target, epigenetic applications or gene purifications. However the main difficulties remain in the delivery methods and the stability of the system which depends a lot of the experimentation (type of cell, tissue, in vivo or in vitro experiments)… Recent studies have shown that CRISPR Cas9 used against drepanocytose could increase the risk of cancer (Le Monde, édition Science et Médecine, 13/06).
See also : CRISPR: Gene editing and beyond from Nature