Princeton and MIT gene-editing pioneers uncover clues for improving CRISPR and other tools

Gene-editing technologies such as CRISPR have shown promise as both research tools and therapies for a range of diseases. But existing tools don’t always alter the genome as intended, and they can cause unwanted and potentially dangerous mutations.  

To better understand the mechanisms behind gene editing, scientists at Princeton University and the Massachusetts Institute of Technology developed a screening method called Repair-seq, which allows researchers to identify genetic elements and associated cellular processes that occur during gene editing.

In two studies published in the journal Cell, the researchers used Repair-seq to generate insights they say could help in the design of more accurate and efficient gene-editing systems. They mapped out the contribution of several pathways in the process by which cells repair double-stranded DNA breaks induced by CRISPR/Cas9 or Cas12a. They also worked with gene-editing pioneer David Liu at the Broad Institute of MIT and Harvard to pinpoint a cellular process that impedes another gene-editing method called “prime editing,” which doesn't cause double-stranded breaks. 

Repair-seq pairs CRISPR-based screens with site-specific deep genetic sequencing to profile mutations at targeted DNA lesions, according to the team. In one Cell study, the team used Repair-seq to disable 476 genes involved in the repair of double-stranded breaks and examine various outcomes from editing with the Cas9 and Cas12a enzymes. The experiments provided a high-resolution atlas that links repair outcomes with specific genetic factors.

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“Systematic exploration of this atlas revealed that repair outcomes with superficially similar sequence architectures can show marked differences in genetic dependencies,” the researchers wrote in the study. For example, the researchers showed that the DNA1 and MCM10 genes modulate how fragments of genomic sequences repair double-stranded breaks.

In a separate Cell study published last week, the team worked with Liu to study prime editing using a Repair-seq screen. Liu’s team first described prime editing in 2019. The platform involves a guide RNA called pegRNA that sends Cas9 to its target, where the enzyme cuts one strand of DNA. It uses an RNA template to install the desired sequence at the site of the nicked DNA, which then serves as the template to remake the other strand, completing the edit.

The team turned off each of 476 DNA repair genes and examined the outcomes of prime editing. They found that the DNA mismatch repair process—which corrects base errors during DNA replication—interferes with prime editing. Based on that discovery, the researchers designed new prime editing systems that expressed a protein called MLH1dn to temporarily mute mismatch repair.

Liu and colleagues combined the novel prime editing tool with engineered pegRNAs, as described in a recent Nature Biotechnology study, and achieved markedly enhanced editing efficiency, they said.

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Liu previously worked with the same team to deploy Repair-Seq toward identifying factors that affect base editing, which tweaks one nucleotide base rather than changing a long piece of DNA. The results were shared in a Nature Biotechnology study published in June. Liu was a co-founder of Beam Therapeutics, which is working on base-editing therapies for a range of disorders including blood diseases and cancers.

The new research is stocked with gene-editing pioneers, in fact. The co-corresponding author of one of the Cell studies is Cecilia Cotta-Ramusino, who helped get another CRISPR startup co-founded by Liu, Editas Medicine, off the ground. She joined “gene writer” startup Tessera Therapeutics in 2019 as head of platform development.

Liu is now directing his attention to moving prime editing forward. He co-founded Prime Medicine, which decloaked in July with $315 million. The biotech is now incorporating the new findings on pegRNA and prime editing into its platform, it said Tuesday.