Newswise – HOUSTON – (October 24, 2022) – A Rice University lab is leading efforts to reveal potential threats to the efficacy and safety of therapies based on CRISPR-Cas9, the award-winning gene-editing technique Nobel Prize, even when it appears to be working as intended.
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 while off-target DNA modifications have long been a concern, the invisible modifications that accompany Targeted changes must also be recognized — and quantified.
Bao noted that a 2018 Nature Biotechnology paper indicated the presence of large deletions. “That’s when we started looking at what we could do to quantify them, using CRISPR-Cas9 systems designed for the treatment of sickle cell disease,” he said.
Bao has been a strong proponent of CRISPR-Cas9 as a tool for treating sickle cell disease, a quest that has brought him and his colleagues one step closer to a cure. Now, researchers are concerned that large deletions or other undetected changes due to gene editing could persist in stem cells as they divide and differentiate, with long-term health implications.
“We don’t fully understand why a few thousand DNA bases at the Cas9 break site can disappear and DNA double-strand breaks can still be joined efficiently,” Bao said. “That’s the first question, and we have a few guesses. The second is, what are the biological consequences? Large deletions (DL) can reach neighboring genes and disrupt the expression of both the target gene and neighboring genes. It is unclear whether LDs could drive the expression of truncated proteins.
“You could also have proteins that fold badly or proteins with an extra domain due to large insertions,” he said. “All sorts of things can happen, and cells can die or have abnormal functions.”
His lab has developed a procedure that uses single molecule real-time sequencing (SMRT) with two unique molecular identifiers (UMI) to find and quantify unintended LDs as well as the large insertions and local chromosome rearrangements that accompany small insertions/deletions (INDEL) at a Cas9 target cut site.
“To quantify large genetic changes, we need to perform long-range PCR, but this could induce artifacts during DNA amplification,” Bao said. “So we used 18-base UMIs as a sort of barcode.”
“We add them to DNA molecules that we want to amplify to identify specific DNA molecules as a way to reduce or eliminate artifacts from long-range PCR,” he said. “We also developed a bioinformatics pipeline to analyze SMRT sequencing data and quantify LDs and large insertions.”
Bao’s lab tool, called LongAmp-seq (for long amplicon sequencing), accurately quantifies both small INDELs and large LDs. Unlike SMRT-seq, which requires the use of a long-playback sequencer often only available at a central facility, LongAmp-seq can be performed using a short-playback sequencer.
To test the strategy, the lab team led by former Rice student Julie Park, now an assistant research professor of bioengineering, used Streptococcus pyogenes Cas9 to edit beta-globin (HBB), gamma-globin (HBG) and B-cell lymphoma/leukemia 11A (BCL11A) in hematopoietic stem and progenitor cells (HSPC) of 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 at high frequency in HSPCs: up to 35.4% in HBB, 14.3% in HBG and 15.2% in BCL11A genes, as well as on the PD-1 gene (15.2%) in T-cells.
Since two of the specific CRISPR guide RNAs tested by the Bao lab are being used in clinical trials to treat sickle cell disease, he said it was important to determine the biological consequences of large genetic 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 carries the code for ribosomes to make proteins. “Then we’ll move on to the protein level,” Bao said. “We want to know if these large deletions and insertions persist after transplantation of the gene-modified HSPCs into mice and patients”
Co-authors of Rice’s study are graduate students Mingming Cao and Yilei Fu, alumni Yidan Pan and Timothy Davis, research specialist Lavanya Saxena, microscopy/bioinstrumentation specialist Harshavardhan Deshmukh and Todd Treangen, assistant professor of computer science, and Vivien of Emory Sheehan University, associate professor of pediatrics.
Bao is the head of the Foyt Family Bioengineering Department and Professor, 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 summary at https://www.science.org/doi/10.1126/sciadv.abo7676.
This press release can be viewed online at https://news.rice.edu/news/2022/even-good-gene-edits-can-go-bad.
Follow Rice News and Media Relations via Twitter @RiceUNews.
Associated 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 Lab: http://bao.rice.edu
Department of Rice Bioengineering: https://bioengineering.rice.edu
George R. Brown School of Engineering: https://engineering.rice.edu
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