Being only announced five years ago, CRISPR-Cas9 still feels like a revolutionary new technique – but the next generation of CRISPR-Cas9 technology has already been created.
A team of scientists from the Broad Institute, the USA, headed by David Liu, have recently published research in Nature detailing their next step in CRISPR-Cas9 technologies; their attempt to engineer out limitations.
CRISPR-Cas9 is a technique for editing a genome. It’s based on a system used by bacteria to protect themselves from viruses.
In the technique, two short Ribonucleic acids (RNAs) with a sequence complementary to a target sequence combine with the Cas9 enzyme. When the two RNAs find their target, the Cas9 cuts the DNA in the target location. Once cut, the cell attempts to repair the gene, but often makes mistakes causing a mutation.
Though the excitement surrounding the CRISPR-Cas9 system may make it appear like a magic bullet in a bioscientist’s gun, in reality it is far from perfect.
One major issue is that the system leads to a lot of off-target mutations, thought to be because it has evolved to be promiscuous, in bacteria, for defensive reasons.
Another major issue is the Cas9 enzyme’s requirement for a protospacer adjacent motif (PAM), a short sequence of specific base pairs immediately after a target site. The most commonly used Cas9 is from Streptococcus pyogenes, and its PAM sequence is NGG.
Though it still allows the targeting of many sites, this is limited to around 1/16th of the human genome. As a result, the user is profoundly limited in treating human diseases.
Rational engineering has previously been attempted to change this PAM sequence, and therefore allow a much greater list of potential targets, but thus far the work has not been hugely successful.
This work from the Liu group has thankfully been different.
The team used evolution to develop their xCas9, in which the mutating Cas9 gene is transferred from cell to cell in a way dependent on the activity of interest. Therefore, only the ‘best’ Cas9 is transferred every round over hundreds of rounds.
xCas9 is able to recognise different PAM sequences, including NG, GAA, and GAT. Engineered in a flexible fashion, it can allow the system to target up to four times more sites than previously, making the technology much more versatile.
The obvious concern with engineering a Cas9 which has a shorter PAM sequence is the risk of more off-target mutations. However, incredibly, this does not seem to be the case with xCas9; it shows greater specificity and less off-target mutations in current experiments.
A CRISPR-Cas9 variant which can target four times more of the genome, with less off-target effects, would certainly be useful. However, xCas9 has not yet been tested thoroughly, which it will need to be to confirm whether it’s as good as it seems.
“I’m not 100% sure xCas9 is going to be flat out better than [Streptococcus pyogenes] Cas9,” Liu said to Science’s John Cohen in an interview. “I want everyone to test it because I want to know the answer.”
This author is certainly keeping his fingers crossed.
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