Australian researchers have successfully introduced an improved version of Cas12a gene-editing enzyme in mice. Their work establishes a next-generation gene-editing tool that enhances genetic manipulation for cancer and medical research in a preclinical model.
The study, “Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening,” was published in Nature Communications.
“This is the first time Cas12a has been used in preclinical models, which will greatly advance our genome engineering capabilities,” said co-author Eddie La Marca, PhD, a postdoctoral researcher at the Olivia Newton-John Cancer Research Institute (ONJCRI) in Australia.
CRISPR technology, widely used in biomedical research and increasingly tested for clinical applications, relies on Cas enzymes. While Cas9 has been the predominant enzyme in CRISPR applications over the past decade, Cas12a offers advantages.
“In contrast to Cas9, Cas12a can delete multiple genes at the same time with extremely high efficiency,” La Marca said.
To leverage this ability of Cas12a, the researchers from ONJCRI and Genentech engineered a mouse model that expresses an enhanced form of Cas12a with a fluorescent reporter. As they wrote in the study, the researchers were able to “demonstrate efficient single and multiplexed gene editing in vitro” as well as in vivo gene editing in both healthy and cancer-prone stem cells in wild-type mice.
Additionally, the team developed “compact, genome-wide Cas12a knockout libraries,” which can be used with their new mouse model as knockout screens. They write that the libraries are useful for whole-genome screening and are “capable of being used in a variety of biological contexts—in vitro in both primary and transformed cell lines, and in vivo—and for both positive and negative selection-based screens.” These findings contribute to a broader understanding of CRISPR-Cas12a’s potential applications in researching and treating cancer.
“We didn’t know whether enhanced Cas12a would work in a preclinical model, it was only tested previously in tissue cultures. Generating and testing these animal models takes more than a year, so it was a long wait to know if it would be effective and compatible for our preclinical work,” Marco Herold, PhD, CEO of ONJCRI and head of the La Trobe University School of Cancer Medicine, told GEN.
“What excited us the most during this research was confirming that Cas12a is an optimized genome engineering tool, allowing us to knock out multiple genes at once,” Herold shared with GEN.
The study’s implications extend beyond oncology, as Cas12a’s enhanced multiplexing capabilities provide researchers with a more sophisticated toolset to investigate genetic pathways involved in various diseases. The ability to perform multiple gene knockouts and activations represents a significant leap forward in genome engineering.
“We have also crossed our Cas12a animal model with a model that expresses an altered version of Cas9, allowing us to both delete and activate different genes simultaneously,” explained co-lead authors Wei Jin and Yexuan Deng, PhD, both from ONJCRI. “This will allow researchers to use this tool to model and interrogate complex genetic disorders.”
Herold emphasized the potential impact of this work on the broader research community. “We are certain that this work will encourage other research teams to use this Cas12a preclinical model, which, in combination with the screening libraries, represents a powerful new suite of gene-editing tools to improve our understanding of the mechanisms behind many different cancers,” Herold said.
Herold also told GEN that he sees a role for Cas12a as a model for other complex diseases. “Not only can we turn genes on or off in the cancer cells, but we can also turn genes on or off in immune cells, such as T cells, to better understand why these T cells are no longer able to attack cancer cells.”
CRISPR is fast becoming the research and treatment wave of the future. With the approval of Casgevy by the FDA in 2023, and the successful treatment of many patients with sickle cell disease, including Victoria Gray and LaRae Morning, it’s clear that there is a prime place in the clinic for CRISPR.
Herold agrees, and his team is continuing their investigations into how CRISPR-based therapies, including Cas12a, can move into patient settings. “This Cas12a preclinical model will also be instrumental to advancing our understanding of how CRISPR tools could be translated to clinical usage.”
