Researchers report that a new approach more than doubles the efficiency of stem cell editing

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

Researchers developed a more efficient and accessible way to apply CRISPR-Cas9 systems in human pluripotent stem cells (hPSCs) derived from government-approved stem cell lines, which Lian says could significantly advance the diagnosis and treatment of genetic disorders. The approach was published on September 7th 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 sites in the genetic code to alter DNA and offers opportunities to develop new diagnostic tools and potentially mutations correct to treat genetic causes of disease.

“The human genome is enormous, and CRISPR-Cas9 allows scientists to find and target a mutated gene to study it,” Lian said.

CRISPR uses a slice of genetic material known as plasmid DNA to deliver targeted 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. The researchers can then see how the distance changes the expression of the gene. However, according to Lian, there are delivery and processing efficiency issues with current DNA-based CRISPR methods.

“The delivery of DNA CRISPR effectors is low,” he said. “Only 20% to 30% of target cells receive gene-editing DNA when CRISPR is used. Delivery of RNA into cells can be more efficient; however, when normal RNA is introduced, cells can see it as a virus. They destroy the RNA before it can make proteins — say in a matter of hours — and thus defeat the gene-editing attempt.”

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

“The modRNA proved to be significantly more efficient than plasmid DNA,” Lian said. “Around 90% of the cells received the modRNA from a simple transfection, allowing it to stay in place and do its job.”

The researchers also found that the length of time the modRNA was present was ideal: long enough to modify the cells but not long enough to cause off-target activity. However, according to Lian, modRNA introduced another problem.

When modRNA Cas9 is successfully delivered to the target gene, it creates a double-stranded break in the genome that some cells attempt to repair. Those that repair themselves can pass the repair or “mutation” on to their offspring. This is the process researchers want to better understand, so these are the cells they want to harvest and study. The problem, Lian said, is that most cells with this break identify it as a major problem with the genome and self-destruct rather than attempt to repair themselves.

To reduce Cas9’s toxic side effects and help engineered cells survive, Lian’s team introduced a small protein known to help cell growth. According to Lian, this added protein inhibited cell death and improved Cas9 editing efficiency by up to 84%.

The researchers also found that the modRNA could improve other gene editing techniques, such as base editing. Base editing can turn off genes or correct mutations in the genome by using a protein to change a single nucleotide instead of cutting both strands like CRISPR does.

“We transfected stem cells with either a plasmid-based or a modRNA-based base-editing protein,” Lian said. “Our modRNA-based method was more than four times more efficient at 68% than the plasmid-based technique at about 16% in successfully editing the genome.”

According to Lian, as more gene-editing labs improve the efficiency and effectiveness of gene-editing, researchers will be able to gain a better understanding of genes and their functions more quickly.

“The human body has more than 20,000 genes, but we only study the functions of about 10% of them,” Lian said. “Studying the purpose of each remaining gene, one at a time, could take a lifetime. Using engineered stem cells from our high-efficiency gene-editing techniques can significantly speed up this process.”