The Crispr/Cas9 genome editing system has risen to fame so quickly it appears to be suffering from a case of the bends. Researchers at Johns Hopkins University however have just given it a bit more room by to breathe by overcoming an inherent bias that limits sequences the system can target.
By modifying the short guide RNA (sgRNA), which directs Cas9 to specific targets, the team was able to expand on the number of targetable sites in the genome. Here are the hurdles they overcame:
- Limited by the nature of sgRNA expression, the team identified the commonly used U6 promoter as the ‘limiting reagent’, as it requires a G nucleotide to initiate transcription. This posed a big restriction on the possible targets in earlier systems due to a constrained motif.
- The team went out and made their own upgraded system, with the H1 promoter driving expression of the sgRNAs while also being able to initiate transcription with the classical G as well as A.
- The new A targeting site motif occurs 15% more frequently than G’s and is enriched in some interesting genes.
- They topped it off with a demonstration of gene editing of the retinitis pigmentosa gene in human stem cells.
First author, Vinod Ranganathan shares that “Since 15 percent more genes that have target sites start with an A rather than a G, simply using this expanded guide RNA approach more than doubles the number of sites we can access.” Senior author, Donald Zack concludes that “The old technique is like an express train that can only make stops every few miles along DNA. This new technique is like a local train — we can generate mutations more efficiently than we ever could before.”
Double your targeting odds at Nature Communications, August 2014