New approach more than doubles stem cell editing efficiency

A Penn State-led team of interdisciplinary researchers has developed techniques to improve the efficiency of CRISPR-Cas9, the genome editing technique that earned the Nobel Prize in 2020. While CRISPR-Cas9 is faster, less expensive and more accurate than other gene-editing methods, according to project leader Xiaojun “Lance” Lian, associate professor of biomedical engineering and biology at Penn State, the technology has limitations — especially in applications to improve human health.

The researchers developed a more efficient and accessible process to apply CRISPR-Cas9 systems in human pluripotent stem cells (hPSCs), derived from federally approved stem cell lines, which Lian said could greatly advance diagnostics and treatments for genetic disorders. The approach was published Sept. 7 in Cell Reports Methods.

CRISPR-Cas9, which stands for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9, gives scientists the ability to target precise locations of genetic code to change DNA, providing opportunities to create new diagnostic tools and potentially correct mutations to treat genetic causes of disease.

“The human genome is enormous, and CRISPR-Cas9 makes it possible for scientists to find and target a mutated gene for the purpose of studying it,” Lian said.

CRISPR uses a disc of genetic material, known as plasmid DNA, to deliver guided ribonucleic acid (RNA) that positions the Cas9 enzyme at the precise location of the target gene. When the DNA is located, Cas9 binds to it and cuts it out, allowing other DNA to repair the cut. Researchers can then see how the removal changes the gene’s expression. But there are delivery and editing efficiency problems with current DNA-based CRISPR methods, according to Lian.

“Delivery of DNA CRISPR effectors is low,” he said. “Only 20% to 30% of the targeted cells will receive gene-editing DNA when using CRISPR. Delivery of RNA into cells can be more efficient; however, when regular RNA is introduced, cells can see it as a virus. They destroy the RNA before it can make proteins — say, in a matter of a few hours — and, in doing so, destroy the gene editing attempt.”

To improve the outcome, the researchers changed the way the genome editing tools are delivered to the stem cells, using modified RNA (modRNA). The modRNA differs from plasmid DNA in that it replaces one of the base substrates found in RNA with a chemically modified version, and it is stabilized by stronger structural support.

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