CRISPR, (short for “clustered regularly interspaced short palindromic repeats”), is a groundbreaking gene-editing tool that empowers scientists to finely manipulate DNA, whether by inserting, deleting, or swapping genetic materials. This technology harnesses programmable enzymes, acting as molecular scissors, to precisely cut DNA at targeted locations. Among these enzymes, Cas12a stands out for its specificity and accuracy in its genome editing capabilities compared to the more widely used Cas9 enzyme. However, Cas12a's efficiency in making these edits currently falls way short of Cas9.

In a recent study published in Nature Communications, researchers at the University of Illinois Urbana-Champaign unveiled a novel strategy to bolster Cas12a's activity while preserving its accuracy. Led by Huimin Zhao (BSD theme leader/CABBI/CGD/MMG), Steven L. Miller Chair of chemical and biomolecular engineering, the team included graduate students Guanhua Xun and Zhixin Zhu, postdoctoral researcher Jingxia Lu, and automation engineer Nilmani Singh from the Carl R. Woese Institute for Genomic Biology.

CRISPR relies on programmable editing enzymes called nucleases, sourced from microorganisms like bacteria and viruses, to cut DNA. These enzymes, which include Cas12a and Cas9, are guided to their DNA targets by CRISPR RNA. Cas12a exhibits superior accuracy, displaying fewer off-target effects compared to Cas9, as well as the ability to edit multiple genome locations simultaneously. Yet Cas12a’s high accuracy is a double-edged sword, as it can hinder its genome editing activity.

“The human genome is large, around 3 billion base pairs. But if you just want to hit a specific 20 base pairs, there’s a high chance you won’t hit just that,” explained Singh. “That’s a big hurdle. CRISPR offers lot of potential for drug development, but these won’t make it to clinical trials unless you minimize off-target effects.”

Prior efforts by other researchers to enhance Cas12a's efficiency involved tinkering with the enzyme itself, but the Illinois team took a different approach, focusing on the guide crRNA instead.

The researchers incorporated 2-aminoadenine, also known as base Z, into the structure of the crRNA. This alteration fundamentally transforms the base pairing dynamics through the substitution of base adenine for base Z. The resulting Z – thymine bond is stronger due to the three-hydrogen bonds, increasing the binding efficiency of crRNA to the target DNA. This, in turn, enhances Cas12a's activity while maintaining its specificity, rivaling the efficiency of Cas9 in mammalian cells.

“Our guide RNA engineering is very unique, because we incorporate a natural occurring, non-conical base,” said Zhao. “When we think of DNA we think of the typical ATCG bases, but a few years ago we discovered some organisms have base Z instead of an A in their genome. Ever since then we’ve been building off that discovery, exploring new applications for this.”

The team says this new approach has many implications for therapeutics, cell engineering, and in-vitro diagnostics, as it addresses a significant hurdle in gene editing efficiency. In fact, the team had previously used this strategy to integrate base Z into mRNA used in the COVID-19 vaccine, substantially bolstering antigen-specific immune responses. Xun explained that base Z holds promise for vaccine development due to its ability to decrease immunogenicity, or the bodies incorrect immune response to antibodies, of the vaccine.

“Compared to protein engineering strategies which alter the Cas nuclease, our strategy is very simple,” said Xun, who is first author on the study. “We showed previously that this strategy was effective in creating a Z-based COVID-19 vaccine, and we think it has many more potential uses across both academia and industry.”

“It was surprising to me how such simple engineering could lead to a huge improvement in efficiency,” said Zhu. “Other methods like protein engineering require many rounds of screening, but with our method we can move the process along faster.”

CRISPR technology has countless potential applications, from treating genetic diseases, to creating genetically modified organisms, to advancing biomedical research. Its ease of use, precision, and versatility have made it one of the most powerful tools in modern biotechnology. By extending this strategy to other genome editing tools, the team envisions more efficient targeting of disease-associated genes, fostering new frontiers in precision medicine.

The study was funded by the NIH and the Steve L. Miller Endowed Chair fund. The paper can be found at https://doi.org/10.1038/s41467-024-48012-x