The safety and efficiency of CRISPR genome editing can be improved by identifying the best genome editing option for a specific application.
CRISPR is a powerful approach to genome editing where several different enzymes, known as Cas nucleases, are used to edit the genome. However, each of these enzymes vary in characteristics and choosing the right enzyme is crucial. Researchers from the University of Texas at Austin have tried to make selecting the enzymes for specific applications easier. They established a platform known as NucleaSeq, which tested different Cas nucleases against 10,000 synthetic DNA sequences that they developed, containing DNA variations relative to the guide RNA.
'We designed a new method that tests the specificity of these different CRISPR enzymes – how precise they are – robustly against any changes to the DNA sequence that could misdirect them, and in a cleaner way than has ever been done before,' said Dr Stephen Jones, one of the lead authors.
The research study, published in Nature Biotechnology, provided an insight into the off-target effects of these enzymes – which is an unintended cutting of DNA sites. They examined five engineered Cas nucleases, which are Cas9 variants from the first Cas9 enzymes isolated from Streptococcus pyogenes. They were shown to have increased DNA cutting specificity but not DNA binding specificity. Some of these variants performed better than others, which the researchers stated could be due to the slower cutting step of these enzymes.
However, the low DNA binding specificity of these enzymes can be a limiting factor to certain applications that require high levels of accuracy. This includes genome editing tools that manipulate the expression of genes by deactivating or activating the gene of interest.
Alternatively, Acidaminococcus species Cas12a has been demonstrated in vitro to cut DNA with the same specificity as the original Cas9 nuclease, which is known to have more off-target effects. Cas12a is expected to cut fewer off-targets and have an increased specificity, particular in human cells. The authors suggested that this was due to the DNA cutting rate of Cas12a being slow. This gives the cellular enzymes the opportunity to stop CRISPR enzymes targeting the wrong section of DNA, before they have a chance to start to cut it.
Therefore, depending on the application, this tool enables the scientist to choose the best suited Cas nucleases that they need for genome editing. Dr Jones commented that 'this technique gives us a new way to reduce risk' and further stated that 'It allows gene edits to be more predictable'.