Posted on 22 November 2015
Gene editing has incredible potential and could give us an unprecedented control of the biological world. The newest addition CRISPR offers unprecedented speed and ease, but there have been questions over its accuracy and reliability. New data hints we can relax a little; it’s safer than we thought.
A brief overview of CRISPR
CRISPR has made gene editing big news in very little time. While it may not be the most accurate method, the system is customisable, cheap and fast and has clear advantages over its predecessors. The system is essentially made of two parts: an enzyme called Cas9 which snips the target DNA sequence, and a guiding sequence made up of RNA which binds to a matching DNA sequence. The system also needs a small 3 letter sequence called PAM, which is required next to the site for Cas9 to cut. Together these can accurately target a specific sequence in the genome, allowing you to make tiny changes or insert a new sequence by hijacking the cell’s own repair systems.
A cautious beginning
CRISPR has shown promising results in laboratory settings, but researchers have been cautious about moving onto humans, due to fears the complex may cut other sites and cause unwanted mutations elsewhere. While CRISPR is an accurate system, the human genome is enormous and even one off-target snip could cause problems.
Some good news
Because of these remaining fears and questions, researchers from UC Berkeley including the co-discoverer of CRISPR Jennifer Doudna, set to analyse and test just how accurate the CRISPR-Cas9 system actually is.
Their new report lays out how CRISPR scans billions of base pairs to find its target, and that the Cas9 enzyme has at least 3 check points before it can cut – meaning the system is much more reliable and accurate than was feared. This is potentially great news for the field.
“CRISPR-Cas9 has evolved for accurate DNA targeting, and we now understand the molecular basis for its seek-and-cleave activity, which helps limit off-target DNA editing”
Putting some fears to rest
One of the biggest concerns about CRISPR was off-target binding, and whether this leads to any additional cuts to the sequence – which might lead to harmful mutation. It seemed likely CRISPR would form some interactions with other sites in the genome, but they didn’t know if this was enough for the Cas9 enzyme to snip. The new research suggests it’s not, and that while off-target binding does happen, it doesn’t lead to a cut.
“It’s crazy that the Cas9 complex manages to scan the vast space of eukaryotic genomes. There is a lot of off-target binding by Cas9, but we found that these interactions are very brief—from milliseconds to seconds—before Cas9 moves on. If Cas9 bound for tens of seconds or minutes at each off-target site, it would never, ever be able to find a target and cut in a timely manner”
A triple check
The research showed that only when the correct sequence is found will the binding change the shape of Cas9 enough to allow it to edit a given sequence, like a proof-reading step. The Cas9 must recognise a PAM sequence (which occurs about 300 million times in the human genome), and the exact target sequence the RNA guide binds to, before it will clamp down and act. The team found that there’s an added checkpoint too; the interaction must be long enough. If the sequence isn’t a correct fit, it won’t bind long enough to induce Cas9 activity.
This is positive news, and could herald more clinical trials involving CRISPR in the near future. There are also many other alternative enzymes to Cas9 in development or that already exist, which act in subtly different ways and may increase safety even further.
Read more at Genetic Engineering News