A different CRISPR enzyme may make genome editing sharper

The key enzyme used in CRISPR genome editing has competition.

The enzyme Cas12a, also known as Cpf1, is more precise for use in genome editing than the currently widely-adopted Cas9, suggests research published in Molecular Cell. Concerns over the accuracy of CRISPR/Cas9 were raised by research last year (see BioNews 903).

'The overall goal is to find the best enzyme that nature gave us and then make it better still, rather than taking the first one that was discovered through historical accident,' said study co-author Dr Ilya Finkelstein, assistant professor at the University of Texas at Austin.

For genome editing, scientists have repurposed the CRISPR-guided Cas proteins from the immune systems of bacteria, where they target and cut the DNA of invading viruses. To do this, the enzymes are highly specific to the DNA sequences they bind, and it is this capability that researchers have harnessed to precisely target and modify the genomes of other organisms. Although most work has centred around Cas9, other enzymes exist.

In questioning whether CRISPR/Cas9 is the best choice for genome editing, Dr Finkelstein and the team set out to test if another system – CRISPR/Cas12a – might be a better candidate in terms of specificity and reducing off-target effects. To do this, they closely studied its basic biochemistry.

The specificity of CRISPR/Cas targeting is achieved via a molecule in the enzyme complex: guide RNA. Guide RNA 'decides' which region of genomic DNA the enzyme should bind to and subsequently cut. Cas9's guide RNA 'reads' a sequence of 20 DNA bases before binding.

In theory, this should be sufficient to generate a unique combination which only binds to one location in a genome. In practice, this is not always the case. Cas9 can tolerate a certain proportion of 'mismatches' in the target sequence. What this means is it can bind and cut at regions other than the one desired, inducing off-target effects – a key concern surrounding medical applicability of CRISPR/Cas9.

This is where the research team found Cas12a differs: it also binds to a 20 base sequence, but in a different manner. While Cas9 only reads the first seven or eight bases of the 20 letter guide sequence before binding, Cas12a reads them all: and will not bind if a single one is wrong. This choosiness ensures that it is much less likely to bind anything except the specified sequence, and off-target effects occur to a much smaller extent.

However, Cas12a may not be a perfect successor to Cas9. A study published in Science in 2015 showed that while it is highly specific in cutting double-stranded DNA, the normal state of DNA in cells, it is an indiscriminate cutter when DNA is single-stranded, which is the form of DNA present when replication is occurring.

Dr Finkelstein told Axios that Cas12a tends to be tightly associated with one DNA strand after the genome is cut, and that they do not know how this would affect genome editing.