The discovery of CRISPR as the bacterial immune system and its DNA-cutting ability kickstarted the gene editing revolution that began around 2012. CRISPR has two components: a guide RNA and a nuclease. The RNA brings the nuclease to matching sites around the genome, and the nuclease cuts DNA at these sites. This can disrupt genes or replace sequences.

Before the nuclease cuts into the genome, it checks for a short nearby sequence called the protospacer adjacent motif (PAM). In bacteria, this checking step stops CRISPR from cutting self DNA by accident. This PAM-checking step has been carried over into gene editing systems that use CRISPR, but it undermines their effectiveness.

“If you want to target a genetic disease, but you don't find the PAM next to the target sequence, you cannot treat that mutation,” said Ylenia Jabalera Ruz, a postdoctoral researcher at the Center for Cooperative Research in Biosciences.

Researchers have some flexibility because CRISPR enzymes — such as different versions of the Cas9 and Cas12 enzymes — have different PAM sequences, but the search for a PAM-less CRISPR system is ongoing.

Jabalera and her colleagues, writing in Nature Biotechnology, said that they have developed what they call a “PAM-flexible” enzyme — by going back in time (1). They used a technique called ancestral sequence reconstruction (ASR) that rewinds the evolutionary clock to design an enzyme that opens up more regions of the genome to editing.

The sequence of amino acids that make up proteins evolves over time. The research team sequenced dozens of existing variants of a popular gene editing protein called Cas12a found in hydrobacteria. By predicting the likely mutations that would have produced these diverse proteins, they could deduce their ancestral protein sequence.

The resulting protein, dubbed ReChb, resembles what Cas12a would have looked like inside the common ancestor of modern-day hydrobacteria three billion years ago. When the team put ReChb to work, they instantly noticed that it performed differently from current Cas12a.

This is our focus: reaching the unreachable.”
- Raúl Pérez-Jiménez, Center for Cooperative Research in Biosciences

They challenged ReChb to edit gene targets alongside a version of Cas12a engineered by Benjamin Kleinstiver and his team at Massachusetts General Hospital (2). This, said Jabalera, is the “gold standard” editing tool in the field. ReChb performed admirably against its descendent. The two enzymes’ editing efficiency was similar, but ReChb could cut at target sites with PAMs that the newer enzyme could not recognize.

The tool was equally capable of cutting different types of genomic targets: It sliced through regular double-stranded DNA with ease and matched that performance against single-stranded DNA and RNA.  

Kleinstiver, who was not involved in the current study, was impressed by the ancient protein’s performance. “The authors demonstrate the ability of ReChb to target certain sites in human cells that encode PAMs not accessible to other previously described enzyme,” he wrote in an email. While acknowledging that this initial study analyzed a modest number of gene editing sites, he added that “their data does support that ReChb could be useful to eliminate editing blindspots in the genome.”

The team plans to extend the ancestral reconstruction approach to other gene editing systems, such as base editing, which acts like a genetic “find and replace” tool for errors in the genome.

“We can catalyze the next generation of genome editors,” said Jabalera. The team wants to trial the enzyme’s ability to correct mutations in rare diseases like amyotrophic lateral sclerosis. But before using the tool in humans, researchers must safety test it. A less stringent target selection process raises the risk of ReChb making off-target edits, said Kleinstiver. Jabalera agreed that this was a risk, but she said that their analysis had not revealed any evidence of ReChb making these unintended edits.

The new enzyme could ultimately expand the suite of gene editing tools that has already revolutionized genomics. Raúl Pérez-Jiménez, Jabalera’s group leader and the paper’s senior author, said, “This is our focus: reaching the unreachable.”

References

1. Jabalera, Y. et al.  A resurrected ancestor of Cas12a expands target access and substrate recognition for nucleic acid editing and detection. Nat Biotechnol  1-10 (2024).
2. Kleinstiver, B.P. et al.  Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat Biotechnol  37, 276-282 (2019).