Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
5513594 | Methods | 2017 | 7 Pages |
â¢CRISPR-Cas genome editing has been employed successfully in many areas of biological research.â¢Among the remaining challenges in optimizing this platform is how best to deliver the Cas9 protein and guide RNA.â¢This paper describes procedures for producing and delivering pre-formed Cas9 ribonucleoprotein to cells.â¢The approach is highly effective and avoids pitfalls encountered with other delivery methods.
The CRISPR-Cas genome editing system is very powerful. The format of the CRISPR reagents and the means of delivery are often important factors in targeting efficiency. Delivery of recombinant Cas9 protein and guide RNA (gRNA) as a preformed ribonucleoprotein (RNP) complex has recently emerged as a powerful and general approach to genome editing. Here we outline methods to produce and deliver Cas9 RNPs. A donor DNA carrying desired sequence changes can also be included to program precise sequence introduction or replacement. RNP delivery limits exposure to genome editing reagents, reduces off-target events, drives high rates of homology-dependent repair, and can be applied to embryos to rapidly generate animal models. RNP delivery thus minimizes some of the pitfalls of alternative editing modalities and is rapidly being adopted by the genome editing community.