Researchers discovered a version of the Cas protein that fits into adeno-associated viruses (AAVs). This virus can be engineered to deliver targeted therapy to cells and is a common approach for gene therapies. To be able to adopt CAS12a for gene delivery, there is an urgent need for small Cas12a and crRNA combined into one AAV.

The researchers conducted several experiments and found that the EbCas12a protein, a variant found naturally in the bacterium Erysipelotrichia, exhibited DNA cleavage activity outside the body (in vitro) and possessed gene-editing activities. A point mutation (altered DNA sequence) was added by the researchers that created a variant of the protein named (en)EbCas12a. AAV-enEbCas12a was used to target PCSK9 in mice. This gene was previously selected as a therapeutic target that reduces cholesterol levels in the blood.

About the Results

Within 30 days after the injection, the levels of the serum cholesterol in the six mice that received AAV-enEbCas12a had decreased to a significant degree. In contrast, the six other mice that received phosphate-buffered saline stayed the same. Bacteria uses the naturally occurring CRISPR system to go on the defensive against viruses. It is said to be a genetic tool for editing with the CRISPR Cas proteins that are used as scissors to cut the genetic code.

The resulting template DNA will repair the defective gene or will be used to insert a new gene. Professor Wang noted that enEbCas12a appears to be a promising tool for genome-editing and a platform for AAV gene therapy.

Definitions:

• Cas12a cannot be encapsulated with crRNA because of its size
• Small Cas12a is crRNA combined into one AAV
• D141R created enEbCas12a, showing efficiency in gene editing
• (en)EbCas12a is a point mutation (RNA or DNA) added by the researchers creating a variant of the protein.
• Point mutation is a change in one base pair in the DNA sequence.