NanoCas helps to unlock Mini-CRISPR, first march toward miniature gene editing

By Balambal Suryanarayanan

CRISPR-Cas9 is a repurposed gene editing tool that allows scientists to cut, replace, and insert pieces of DNA in precise regions along the strand. **Courtesy of NIH Image Gallery via Wikimedia Commons**

Genetic disorders, characterized by mutations that alter the genetic material over generations, can be mendelian or chromosomal. The former is identified by an alteration in a single gene and encompasses diseases like hemophilia, thalassemia, cystic fibrosis, Duchenne muscular dystrophy, and sickle-cell anemia. The alteration is traced by performing a pedigree analysis across generations, for by the word of Mendel, dominant and recessive traits capture inheritance at length. Chromosomal disorders, on the other hand, are identified by an absence, excess, or change in the structure of chromosomes and include Down syndrome, Turner’s syndrome, Klinefelter’s syndrome, and Williams syndrome.

One of the solutions to genetic disorders is to reverse mutations by deleting, inserting, replacing, or modifying DNA. On the surface, gene editing can seem like a piece of cake. After all, it’s a code, a sequence of molecules — and all one needs to do is alter that sequence. What could possibly go wrong?

It turns out, a small mistake in gene editing can cause major problems. At its core, genetic editing deals with rewriting the genetic instructions. You could unintentionally target the wrong DNA sequences, introducing a new genetic mutation that could turn fatal. Even if you get the sequence right, you then have to correctly deliver the editing machinery. A correct delivery of the editing machinery might still face challenges from the immune response, and the cycle that follows suit would be a long battle between the genetic makeup and the immune system, two crucial stakeholders that decide the health of a person.

Gene editing strongly relies on precision, accuracy, efficient delivery of the editing machinery, and controlled editing of the output. Keeping in mind these restraints, Professor Jennifer Doudna of UC Berkeley and Professor Emmanuelle Charpentier of Max Planck Unit for the Science of Pathogens revealed that the CRISPR-Cas9 bacterial immune system could be repurposed as a gene editing tool, subsequently winning the Nobel Prize in 2020 for their groundbreaking research. An acronym for Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas9 is a component of the bacterial immune system that scientists adapted to use like a set of molecular scissors to precisely cut DNA, enabling it to be a repurposed gene editing tool.

First discovered in bacterial immune systems by Fransisco Mojica, CRISPR was found to cut the DNA of bacteriophages (viruses that invade bacteria) and disable them. CRISPR consists of two key components: A CRISP-associated (Cas) nuclease that binds and cuts DNA and a guide RNA sequence (gRNA) that guides the Cas nuclease to its target. By modifying Cas9, researchers have been able to control gene editing with focused ultrasound (FUS), turning on certain genes and study them for disease research. This process allows researchers to target and modify different errors that occur in the massive sequence of the human genome, thus marking the first step towards treating genetic disease.

Despite its reputation as an efficient and customizable alternative to other gene editing tools and U.S. regulator approval to perform gene editing in Sickle Cell disease in 2023, the in-vivo delivery of CRISPR using the adeno-associated virus (AAV) — a virus commonly used as a ferry for CRISPR — has been described by researchers as complicated. The main difficulty is the bulky nature of the components of CRISPR, particularly the 1300 amino acid Cas9 enzyme employed to cut the DNA. This amount of amino acids translates to proteins with a weight of 160 kilodaltons (~ 320 kilograms) which is too much to stuff into a 25-nanometer AAV. Researchers tried delivering the CRISPR and the guide RNA through two AAVs, but developments showed no success.

Recently, Mammoth Biosciences — the company co-founded by UC Berkeley’s Nobel prize-winning scientist, Professor Doudna — rigorously screened the genetic sequence data of 176 CRISPR varieties found in microbes, picking NanoCas as the miniature successor to CRISPR. The chief scientific officer and co-founder of the company, Lucas Harrison, and his colleagues have optimized NanoCas by modifying its proteins. Their research is presented in a paper in bioRXiv, titled “Single-AAV CRISPR editing of skeletal muscle in non-human primates with NanoCas, an ultracompact nuclease.” The paper presents NanoCas as having “potent editing capabilities equalling that of first-generation CRISPR systems.”

NanoCas is a much smaller version of CRISPR, with only 425 amino acids — about one-third the size. Its small size makes it easier to squeeze into an AAV. In tests on mice, the team at Mammoth Biosciences found that NanoCas successfully edited the PCSK9 gene (a protein that regulates cholesterol levels) in the liver with about 60 percent efficiency, performing as well as SaCas9, a much bigger CRISPR-based tool. To add to this ability of NanoCas, researchers also found that like SaCas9, NanoCas reduced the PCSK9 protein levels to undetectable limits, which could have important implications for treating and preventing cardiovascular diseases.

Further, in a mouse that exhibited Duchenne muscular dystrophy — a muscle-wasting disease that arises from a mutation in the dystrophin gene — NanoCas was able to insert genetic material in about 10–40 percent of tissue, specifically in the quadricep, calf, and heart muscles when inserted through a single vector AAV.

Finally, for the first time ever, researchers performed single AAV muscle editing in three healthy macaque monkeys. They injected an AAV, which contained the NanoCas, into the monkeys’ bloodstream. NanoCas achieved an efficiency of about 30 percent when focusing on dystrophin mutations in the skeletal muscles of the macaque species with a 15 percent editing efficiency in the heart — a better accuracy than its parent CRISPR with minimum off-target editing.

Trevor Martin, the CEO of Mammoth Biosciences, attributed NanoCas’ achievements to its compact size and compatibility with a wide range of gene editing techniques. He also added that from facing potential problems in editing tissues outside the liver to making a major step towards miniature editing has been remarkable. Terence Flotte, the Dean of University of Massachusetts T.H. Chan School of Medicine and gene therapist, hailed these levels as clinically meaningful and despite expressing concerns about off-target editing, remained hopeful due to its ease of delivery via AAV and compatibility.

Harrington seconds these challenges and has promised that he and his team at Mammoth Biosciences would work on these challenges by allowing it to target other more inaccessible regions relevant to genetic diseases through the AAV. Mammoth’s belief in the therapeutic capabilities of CRISPR and their unwavering dedication and devotion driven by the goal to make treatments accessible for patients with genetic disorders to improve their lives is what has steered them towards presenting this wonderful innovation to the world. Their work may one day make the dream of accurate and precise gene editing through NanoCas a reality.