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Programmable CRISPR-Based Tool 'Pastes' in New Genes While Avoiding Double-Strand Breaks

NEW YORK — Researchers at MIT have developed a programmable tool that combines CRISPR and integrases to cut out unwanted stretches of DNA and paste in a replacement.

While CRISPR-Cas9 gene editing can make such swaps, it is limited by the need for double-stranded DNA breaks, which can induce undesired insertions, deletions, or rearrangements. Instead, the researchers sought to develop an editing approach that does not require any such double breaks.

As they reported last week in Nature Biotechnology, the researchers fused a CRISPR-Cas9 nickase with a serine integrase — which can insert large amounts of DNA — and a reverse transcriptase to target any site in the genome without inducing any double-stranded breaks. The approach, dubbed programmable addition via site-specific targeting elements (PASTE), can insert sequences as large as 36 kilobases. In addition to potentially serving as a research tool, PASTE could also be developed to treat diseases stemming from genetic mutations.

"It's a new genetic way of potentially targeting these really hard-to-treat diseases," co-senior author Omar Abudayyeh from MIT's McGovern Institute for Brain Research said in a statement. "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."

To develop their tool, the researchers harnessed serine integrases, which typically integrate into the genome at certain attachment sites, known as landing pads. The researchers designed CRISPR-based prime-editing guide RNAs (pegRNAs) to include the attachment site sequence for serine integrases, rebranding them as attachment site-containing guide RNAs (atgRNAs). This way, the PASTE tool could be programmed to first insert the attachment sites at the desired sites in the genome through the CRISPR atgRNAs and then enable the serine integrase to deliver its cargo at those spots. That occurs without any double-stranded breaks as one strand of the cargo is added using a fused reverse transcriptase, followed by the complementary strand.

After developing PASTEv1, which the researchers found could efficiently insert an enhanced GFP transgene, they worked to improve the tool's editing abilities and generated a number of different versions of the tool. PASTEv3, for instance, combined the improved integration efficiency of PASTEv2 with a new atgRNA and could integrate DNA templates about 36,000 base pairs in size with about 10 percent to 20 percent integration efficiencies.

The researchers additionally benchmarked PASTEv3 against editing tools that require double-stranded DNA breaks, particularly homology-independent targeted insertion (HITI) technology or homology-directed repair (HDR) approaches. Through this, they found PASTE had better gene insertion efficiencies than HITI and had similar or, for one of the seven tested genes, slightly lower insertion efficiencies as HDR. However, PASTE generated fewer indels than the other approaches and fewer off-target integrations.

"We see very few indels, and because we're not making double-stranded breaks, you don't have to worry about chromosomal rearrangements or large-scale chromosome arm deletions," Abudayyeh added.

In a range of human cell types, the researchers tested the ability of PASTE to insert about a dozen genes, including ones with therapeutic potential. These genes could be pasted into nine different genomic regions, with efficiencies between 5 percent and 60 percent.

"We think that this is a large step toward achieving the dream of programmable insertion of DNA," co-senior author Jonathan Gootenberg, also from MIT, added in a statement. "It's a technique that can be easily tailored both to the site that we want to integrate as well as the cargo."