Large DNA sequences more accurately inserted into cells

A CRISPR-based form of genome editing that can replace entire genes may offer a new way to treat some genetic diseases.

Many genetic conditions may be linked to a single gene but can be caused by a variety of mutations in different locations within that gene. For such conditions, developing genome-editing-based therapies could be expensive, because they would have to be targeted to the specific mutation, meaning the development of many separate therapies which would each benefit only a small number of patients.

Researchers at Massachusetts General Hospital suggest that a genome-editing approach which can replace much longer sequences of DNA could be used to replace the whole gene with an unaffected version, meaning a single therapy could be used for many patients. Senior author Dr Ben Kleinstiver said: 'Installation of large genetic sequences at targeted locations could endow cells with new capabilities while obviating safety, efficacy, and manufacturing issues resulting from traditional random integration approaches,'.

The study, published in Nature Biotechnology, focused on a CRISPR-based genome editing approach called HELIX, which can replace DNA sequences up to 10,000 bases in length. It uses the CRISPR RNA guide system to target the correct part of the genome to edit, but instead of using a Cas enzyme to cut the DNA, it uses a CAST (CRISPR-associated transposase) enzyme to integrate the new sequence at the target site.

Existing CAST enzymes were not accurate enough to be considered for use in gene therapies – there were issues with integrating the wrong sequence at the target site, as well as casing off-target effects where the genome is changed in places other than the target site.

The researchers were able to improve both of these issues by modifying the structure of the CAST enzyme and supplementing the system with nicking homing endonucleases – DNA-cutting enzymes which avoid the double-stranded breaks more likely to cause harmful chromosomal abnormalities.

'We created HELIX systems with greater than 96 percent on-target integration specificity – increased from approximately 50 percent for the naturally occurring wild-type CAST system,' said lead author, PhD student Connor Tou. 'We also determined that HELIX maintains its advantageous properties in human cells.'

The researchers hope their improved HELIX technology can be used to develop therapies for diseases with multiple causative mutations, sidestepping the need for bespoke therapies and enabling patients to receive treatment more quickly and affordably.

Other approaches for large-scale DNA insertions are being developed, including 'PASTE', which uses a different type of enzyme derived from viruses instead of CAST (see BioNews 1170).