Which are the best CRISPR-CAS9 strategies?

The CRISPR-CAS9 system may well have opened Pandora’s box, but it is also definitely the cornucopia of genome editing.

We can do what we want in the genome: settle a mutation, correct a mutation, insert a fluorescent tag to a protein, add an exogenous gene, delete an endogenous function, suppress a cis-regulatory region, add a reporter…. I might just not have enough imagination!

The main challenge is to define a good strategy, taking in account the specifics of the project and being aware of the corresponding limitations.

Figure 1: CRISPR-CAS9 system is 1 guide RNA with the CAS9 endonuclease to generate a DSB
CRISPR-CAS9 system is 1 guide RNA with the CAS9 endonuclease to generate a DSB. Source: Nature Protocols (DOI : 10.1038/nprot.2013.143)

The principle of the CRISPR-CAS9 has already been exposed numerous times. The protein CAS9 (from the bacteria Streptococcus pyogenes) is an endonuclease that generates a Double Stranded Break (DSB). Its activity is targeted by one single small RNA, called guide RNA (gRNA or sgRNA). The DSB is repaired by the cell (via the NHEJ pathway) leading to knock-out (KO) to the site targeted by the gRNA molecule.

In this overview, I won’t go into details of the structure, the mechanism or history, in order to stay focused on the biotechnology and the concerns of the end-users from a practical point of view.

Feel free to contact me for further information and with any questions!

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