When the nanoclew comes into contact with a cell, the cell absorbs the nanoclew completely — swallowing it and wrapping it in a protective sheath called an endosome. But the nanoclews are coated with a positively charged polymer that breaks down the endosome, setting the nanoclew free inside the cell. The CRISPR-Cas9 complexes can then free themselves from the nanoclew to make their way to the nucleus. Credit: North Carolina State University
CRISPR-Cas system, found in bacteria and archaea, protects bacteria from invaders such as viruses. CRISPR RNA has been used in gene-editing to ID a targeted portion of relevant DNA, and Cas protein cleaves it. But for Cas9 to do its work, it must first find its way into the cell. This work focused on demonstrating the potential of a new vehicle for directly introducing the CRISPR-Cas9 complex – the entire gene-editing tool – into a cell.
“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,” says A/Prof Chase Beisel. “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.”
“Our delivery mechanism resembles a ball of yarn, or clew, so we call it a nanoclew,” says Assistant Prof Zhen Gu. “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 – a CRISPR RNA bound to a Cas9 protein – to loosely attach itself to the nanoclew. “Multiple CRISPR-Cas complexes can be attached to a single nanoclew,” says Wujin Sun.
When the nanoclew comes into contact with a cell, the cell absorbs and wraps the nanoclew completely into an endosome. But the nanoclews are coated with a positively-charged polymer that breaks down the endosome, setting the nanoclew free inside the cell. The CRISPR-Cas9 complexes can then free themselves from the nanoclew to make their way to the nucleus. And once a CRISPR-Cas9 complex reaches the nucleus, gene editing begins.
To test the nanoclew CRISPR-Cas delivery system, the researchers treated cancer cell cultures and tumors in mice to express a fluorescent protein. The CRISPR RNAs on the nanoclews were designed to target the DNA in the cancer cell that was responsible for making the fluorescent proteins. If the glowing stopped, the nanoclews worked. “And they did work. More than a third of cancer cells stopped expressing the fluorescent protein,” Beisel says. https://news.ncsu.edu/2015/08/gu-beisel-clews-2015/
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