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Whole gene replacement possible with new gene-editing tool

Researchers have developed a new tool that could potentially ‘cut’ out faulty genes and ‘paste’ in full-length copies of corrected genes. This could be used to treat genetic conditions with a large number of mutations. The study, published in Nature Biotechnology, described the development of a new gene editing tool that enabled the precise and efficient insertion of large DNA sequences into the genome.

Gene editing is risky

Gene editing is a method used to modify the sequence of DNA. The approach theoretically could cure genetic diseases by repairing or replacing the mutated gene. However, the technology is still quite risky.

Current approaches are based on the repair of double-stranded breaks by cellular responses such as homology-directed repair (HDR) or non-homologous end joining (NHEJ). HDR is inefficient in non-dividing cells. Most cells in the adult human body are non-dividing. NHEJ has off-target effects because the DNA can get cut in the wrong place. As a result, sequences are inserted in the incorrect place. Current genome technologies have not been able to effectively overcome these challenges.

Researchers at the Massachusetts Institute of Technology (MIT) developed PASTE (programmable addition via site-specific targeting elements). PASTE inserts large sequences of DNA into a cell’s genome without causing double-stranded breaks. Therefore, it does not rely on error-prone and low efficiency repair mechanisms to alter large parts of the genome for gene therapy.

Dr Omar Abudayyeh, co-senior author and gene therapy researcher at MIT said, “We wanted to work toward what gene therapy was supposed to do at its original inception, which is to replace genes, not just correct individual mutations.”  

Cut and ‘PASTE’

The PASTE approach is based on CRISPR-Cas9 technology. Cas9 was fused to two enzymes – a reverse transcriptase and a serine integrase. Site-specific integration of DNA sequences was achieved without causing large double stranded breaks in the genome.

First, 46 base-pair attB sequences were added to RNA molecules called attachment site-containing guide RNA (atgRNA). These were copied into the genome via reverse transcription and flap repair. The attB sequences created a ‘landing site’ (blue triangle in figure 1) for the integrase enzyme. It served as a specific genomic site that could be recognised by the integrase. The transgene contained a corresponding attP sequence (orange triangle in figure 1). The integrase was then able to insert the attP-coupled transgene into the target area with the attB attachment site.

Figure 1: Schematic of gene insertion with PASTE. A fusion protein of Cas9, reverse transcriptase and integrase was created. attP-attached landing sites (blue triangle) were inserted by Cas9-directed reverse transcriptase. Cas9-directed integrase recognised the landing sites using attP sequences (orange triangle) and inserted the transgene. Source: Published in Nature Biotechnology.

The researchers also used metagenomic mining to find the most efficient integrase/attachment site combination.

The researchers showed that PASTE could achieve high levels of integration efficiency with large sequences. Sequences that ranged from 779 bp to 36,000 bp were inserted in primary human liver cells and T cells with efficiencies of 4-5% and in cell lines with efficiencies of 50-60%. The team also found that the approach was more specific than existing gene editing technologies because it had almost no off-target effects. The size range of sequences that were successfully inserted using PASTE means that over 99.7% of human genes could be transgenes. This suggests that almost every gene is replaceable.

Dr Abudayyeh said, “We see very few insertions/deletions, and because we’re not making double-stranded breaks, you don’t have to worry about chromosomal rearrangements or large-scale chromosome arm deletions.”

Applications in therapy and biotechnology

PASTE technology could be a valuable tool in both therapy and research. It can be used to treat genetic diseases that are driven by a large number of mutations, such as cystic fibrosis and Leber’s congenital amaurosis. The full-length functional gene could be inserted across all loci. As a biotechnology tool, PASTE could have applications in gene-tagging, investigating genes with unknown function and developing disease models.

“We think that this is a large step toward achieving the dream of programmable insertion of DNA,” said Dr Jonathan Gootenberg, co-senior author and Research Scientist at 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.”