Source: Wikimedia Commons/ Ernesto del Aguila III, NHGRI
In the first post on CRISPR-Cas9, I’ve explained how bacteria and archaea create a “database” of infections and use it as a form of prokaryotic immunization. This time, I’m going to concentrate on how biotechnology turns this natural phenomenon into a powerful tool.
CRISPR-Cas9 is probably the most popular of CRISPR systems because of its simplicity. The natural effector complex consists of only three components – crRNA, tracrRNA and Cas9 endonuclease, but the synthetic form is even simpler, engineered as a two-component system by fusing the crRNA and tracrRNA into a single guide RNA (sgRNA or gRNA).
Engineering genomic regions of choice just by supplying two ingredients is a recipe any lab can handle. Directions according to Sander & Joung (2014):
“Twenty nucleotides at the 5′ end of the gRNA (corresponding to the protospacer portion of the crRNA) direct Cas9 to a specific target DNA site using standard RNA-DNA complementarity basepairing rules. These target sites must lie immediately 5′ of a PAM sequence that matches the canonical form 5′-NGG (although recognition at sites with alternate PAM sequences (e.g., 5′-NAG) has also been reported, albeit at less efficient rates). Thus, with this system, Cas9 nuclease activity can be directed to any DNA sequence of the form N20-NGG simply by altering the first 20 nt of the gRNA to correspond to the target DNA sequence.”
Naturally occurring (a) and engineered (b)
CRISPR-Cas systems (Figure 3, Sander & Joung 2014)
CRISPR-based gene drive
Despite all the previously described magical properties of CRISPR-Cas9, its main strength comes with the connection to a gene drive. Gene drive is a genetic element that basically defies the Mendelian laws and via biased inheritance increases in frequency each generation (Champer et al. 2016).
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