New CRISPR approach inserts large DNA sequences into genome

A 'drag-and-drop' genome editing approach has succeeded in inserting long DNA sequences into the human genome, offering a potential treatment for genetic disorders such as cystic fibrosis.

A Massachusetts Institute of Technology (MIT)-based research team has developed the PASTE (Programmable Addition via Site-specific Targeting Elements) technique, which combines the traditional CRISPR/Cas9 approach with viral proteins called serine integrases. This strategy enabled the incorporation of large DNA sequences up to 36,000 DNA base pairs at nine different loci in human cells with a five to 60 percent success rate.

'We think that this is a large step toward achieving the dream of programmable insertion of DNA,' said co-senior author, Dr Jonathan Gootenberg from MIT's McGovern Institute for Brain Research. 'It's a technique that can be easily tailored both to the site that we want to integrate as well as the cargo.'

Integrases were previously considered for gene therapy; however, the system requires highly specific target sequences (~46 base pairs) for DNA insertion. PASTE overcomes this by adapting the CRISPR/Cas9 approach to insert these sequences, enabling the integrase proteins to operate almost anywhere in the genome.

Moreover, PASTE avoids creating double-stranded breaks, reducing the risk of chromosomal rearrangements – where pieces of chromosomes are accidentally deleted, duplicated, or moved around when the cell tries to repair the broken DNA.

Speaking about the future of the method, Dr Gootenberg commented 'One of the fantastic things about engineering these molecular technologies is that people can build on them, develop and apply them in ways that maybe we didn't think of or hadn't considered'.

Currently, over 4000 genetic diseases are caused by loss-of-function mutations and PASTE's inventors hope it could eventually be used to develop single-dose gene therapies which can introduce a working copy of a gene. They will need to improve its delivery and efficiency in vivo (just 2.5 percent of target cells were successfully edited in mouse livers) to make this a reality.

The study was published in Nature Biotechnology.