Even good gene edits can go bad

Newswise – HOUSTON – (October 24, 2022) – A Rice University lab is leading efforts to uncover potential threats to the efficacy and safety of therapies based on CRISPR-Cas9, the Nobel Prize-winning gene editing technique, even if it appears work as planned.

Bioengineer Gang Bao of Rice’s George R. Brown School of Engineering and his team point out in a paper published in Science Advances that although off-target edits to DNA have long been a concern, unseen changes that associated with on-target edits are also required to be recognized – and quantified.

Bao noted that a 2018 Nature Biotechnology article indicated the presence of large deletions. “That’s when we started investigating what we can do to quantify them, thanks to CRISPR-Cas9 systems developed to treat sickle cell disease,” he said.

Bao has been a strong proponent of CRISPR-Cas9 as a tool to treat sickle cell disease, a quest that has brought him and his colleagues ever closer to a cure. Now researchers fear that large deletions or other undetected gene-editing changes could persist in stem cells as they divide and differentiate, and thus have long-term health implications.

“We don’t have a good understanding of why a few thousand DNA bases can be lost at the Cas9 cleavage site and the DNA double-strand breaks can still be efficiently rejoined,” Bao said. “That is the first question and we have some hypotheses. The second is what are the biological consequences? Large deletions (LDs) can reach neighboring genes and disrupt expression of both the target gene and nearby genes. It is unclear whether LDs could result in the expression of truncated proteins.

“You could also have proteins that fold incorrectly, or proteins with an extra domain because of large insertions,” he said. “All sorts of things could happen, and the cells could die or have abnormal functions.”

His lab developed a method that uses real-time single molecule sequencing (SMRT) with dual unique molecular identifiers (UMI) to find unintended LDs along with large insertions and local chromosomal rearrangements associated with small insertions/deletions (INDELs) and to quantify a Cas9 on-target interface.

“To quantify large gene changes, we need to do long-range PCR, but that might introduce artifacts during DNA amplification,” Bao said. “So we used UMIs of 18 bases as a kind of barcode.”

“We add them to the DNA molecules that we want to amplify to identify specific DNA molecules to reduce or eliminate artifacts due to long-range PCR,” he said. “We also developed a bioinformatics pipeline to analyze SMRT sequencing data and quantify the LDs and large insertions.”

Bao lab’s tool called LongAmp-seq (for sequencing long amplicons) accurately quantifies both small INDELs and large LDs. Unlike SMRT-seq, which requires the use of a long-read sequencer that is often only available at a central facility, LongAmp-seq can be performed using a short-read sequencer.

To test the strategy, the lab team, led by Rice graduate student Julie Park, now an assistant professor of bioengineering, used Streptococcus pyogenes Cas9 to test beta-globin (HBB), gamma-globin (HBG), and B-cell lymphoma/leukemia 11A (BCL11A) enhancer in hematopoietic stem and progenitor cells (HSPC) from patients with sickle cell disease and the PD-1 gene in primary T cells.

They found that large deletions of up to several thousand bases occurred with high frequency in HSPCs: up to 35.4% in HBB, 14.3% in HBG and 15.2% in BCL11A genes and on the PD -1 gene (15.2%) in T cells.

Since two of the specific CRISPR guide RNAs tested by the Bao lab are used in clinical trials to treat sickle cell disease, it is important to determine the biological consequences of large gene changes due to Cas9-induced double-strand breaks.

Bao said the Rice team is currently looking downstream to analyze the consequences of long deletions on messenger RNA, the mediator that codes for ribosomes to make proteins. “Then we move on to the protein level,” Bao said. “We want to know whether these large deletions and insertions persist after the gene-edited HSPCs have been transplanted into mice and patients.”

Rice’s study co-authors are graduate students Mingming Cao and Yilei Fu, alumni Yidan Pan and Timothy Davis, research specialist Lavanya Saxena, microscopist/bioinstrumentation specialist Harshavardhan Deshmukh and Todd Treangen, an assistant professor of computer science, and Vivien from Emory University Sheehan, associate professor of pediatrics.

Bao is a department head and Foyt Family Professor of Bioengineering, Professor of Chemistry, Materials Science and Nanoengineering, and Mechanical Engineering, and a CPRIT Fellow in Cancer Research.

The National Institutes of Health (R01HL152314, OT2HL154977) supported the research.

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Read the abstract at https://www.science.org/doi/10.1126/sciadv.abo7676.

This press release is available online at https://news.rice.edu/news/2022/even-good-gene-edits-can-go-bad.

Follow Rice News and Media Relations on Twitter @RiceUNews.

Related Materials:

Rice Lab offers new strategies, tools for genome editing: https://news2.rice.edu/2016/02/08/rice-lab-offers-new-strategies-tools-for-genome-editing-2/

New genetic weapons challenge sickle cell disease: https://news2.rice.edu/2019/06/03/new-genetic-weapons-challenge-sickle-cell-disease-2/

Bao Laboratory: http://bao.rice.edu

Rice Department of Bioengineering: https://bioengineering.rice.edu

George R. Brown School of Engineering: https://engineering.rice.edu

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