DNA Nanoclews Could Thread the Needle of CRISPR

    CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. CRISPR is part of a normally occurring bacterial process, though it has only recently been studied, as bacteria may incorporate foreign DNA in other circumstances and even scavenge damaged DNA from their environment.  Advances in genome editing seem to be happening almost every other day. However, many groups are focused on improving the efficacy of Cas9 target recognition and cleavage—an important criterion for sure—while neglecting the development of efficient delivery methods.

    Now a team of researchers from North Carolina State University (NC State) and the University of North Carolina at Chapel Hill (UNC-CH) have created and utilized a nanoscale vehicle composed of DNA to deliver the CRISPR -Cas9 gene editing complex into cells both in vitro and in vivo.

    “Traditionally, researchers deliver DNA into a targeted cell to make the CRISPR RNA and Cas9 inside the cell itself—but that limits control over its dosage,” explained co-senior author Chase Beisel, Ph.D., assistant professor in the department of chemical and biomolecular engineering at NC State. “By directly delivering the Cas9 protein itself, instead of turning the cell into a Cas9 factory, we can ensure that the cell receives the active editing system and can reduce problems with unintended editing.”

    The findings from this study were published recently in Angewandte Chemie through an article entitled “Self-Assembled DNA Nanoclews for the Efficient Delivery of CRISPR-Cas9 for Genome Editing.”

    The CRISPR-Cas complex is part of a bacterial immune system that protects various microbial species from invaders such as viruses. It does this by creating small strands of RNA called CRISPR RNAs, which match DNA sequences specific to a given invader. When those CRISPR RNAs find a match, they unleash Cas9 proteins that cut the DNA.

    Over the past several years, much attention has been focused on the CRISPR-Cas system for its potential therapeutic use in gene therapy, with the CRISPR RNA identifying the targeted portion of the relevant DNA, and the Cas protein cleaving it. However, for Cas9 to be effective it must first be able to enter the target cells efficiently.

    “Our delivery mechanism resembles a ball of yarn or clew, so we call it a nanoclew,” said Zhen Gu, Ph.D., co-senior author on the study and an assistant professor in the joint biomedical engineering program at NC State and UNC-CH. “Because the nanoclew is made of a DNA-based material, it is highly biocompatible. It also self-assembles, which makes it easy to customize.”

    The nanoclews are made of a single, tightly-wound strand of DNA. The DNA is engineered to partially complement the relevant CRISPR RNA it will carry, allowing the CRISPR-Cas9 complex to loosely attach itself to the nanoclew. “Multiple CRISPR-Cas complexes can be attached to a single nanoclew,” noted lead author Wujin Sun, a Ph.D. student in Dr. Gu’s laboratory.

    When the nanoclew comes into contact with a cell, the cell absorbs the nanoclew completely through typical endocytic mechanisms. The nanoclews are coated with a positively charged polymer, in order to break down the endosomal membrane and set the nanoclew free inside the cell. The CRISPR-Cas9 complexes will then free themselves from the nanoclew structure to make their way to the nucleus. Once the CRISPR-Cas9 complex reaches the nucleus than the gene editing can begin.

    In order to test their delivery method, the investigators created fluorescently labeled cancer cells in culture and within mice. The CRISPR nanoclew was then designed to target the gene generating fluorescent protein in the cells—if the glowing stopped than the nanoclews worked. “And they did work. More than one-third of cancer cells stopped expressing the fluorescent protein,” Dr. Beisel stated.

    The researchers were excited about their findings and are looking ahead at ways to improve their design for exceptional delivery of the genome editing complex.

    “This study is a proof of concept, and additional work needs to be done—but it’s very promising,” Dr. Gu concluded.