Efficient Gene Editing with Compact Cas12a Protein

Researchers have created an innovative version of a critical CRISPR gene-editing protein that has effective editing activity and is small enough to be bundled into a non-pathogenic virus capable of delivering it to targeted cells. Hongjian Wang and colleagues from Wuhan University in China disclosed their findings on May 23^rd, 2024 in the open-access journal PLOS Biology.

In recent years, there has been an explosion of research seeking to harness CRISPR gene-editing systems, which are naturally found in many bacteria as a defense against viruses, to use them as possible cures for human disease. These systems rely on so-called CRISPR-associated (Cas) proteins, the two most often employed varieties being Cas9 and Cas12a, each with their own set of characteristics and advantages.

One potential approach is to package CRISPR proteins within a non-pathogenic virus, which might then deliver the proteins to target cells and edit particular DNA sequences to treat disease. However, the frequently employed adeno-associated virus is small, and while certain Cas9 proteins can fit inside, Cas12a proteins are usually too big.

Recently, a very small version of Cas12a known as EbCas12a has been discovered by Wang and colleagues in a naturally occurring bacterium species belonging to the Erysipelotrichia class. They increased its gene-editing efficacy by purposefully substituting one of the protein’s amino acid building blocks with another.

This modified protein, called enEbCas12a, exhibits gene-editing effectiveness similar to two other Cas12a proteins known for extremely accurate gene editing when applied to mammalian cells in a lab dish.

The researchers then proved that enEbCas12a is small enough to be employed in adeno-associated virus-based gene therapy. They tweaked enEbCas12a to target a specific cholesterol-associated gene, packed it within the virus, and gave it to mice with elevated cholesterol.

One month later, scientists discovered a substantial drop in blood cholesterol levels in the treated mice compared to animals that did not get the virus.

More study is needed to establish whether enEbCas12a can one day be utilized to treat human diseases. Nonetheless, the findings imply that adeno-associated virus might be used to deliver Cas12a proteins in gene therapy.

The authors added, “The novel compact enEbCas12a, along with its crRNA, can be packaged into an all-in-one AAV system for convenient gene editing in vitro and in vivo with high-fidelity, which can be very beneficial for future clinical applications and more tool developments including all-in-one AAV- based multi-gene editing, base editing, primer editing, etc.”