Conversations with Stephen Tsang: Putting CRISPR to the test

CRISPR-cas9 has revolutionized the fields of genetics and genome editing. Since it’s discovery, researchers have adopted a full-speed-ahead approach to employing the tool in the lab. And rightly so. It has provided researchers with a precise and easily reproducible way of making alterations to the genes of any organism they want. It’s been used to engineer everything from human pluripotent stem cells to farm crops like sorghum and rice. The day is close at hand when drugs developed using CRISPR-cas9 will hit the market and clinical uses of the technology will be an everyday occurrence.

While the scientific community has generally accepted CRISPR’s gifts with open arms, there are some who’ve suggested moving forward a bit more carefully. They maintain that more needs to be learned about how CRISPR’s insertions affect off-target genes. Of course, that will take time, effort, and some honest judgement.

A recent study by Columbia University’s Stephen Tsang provides an important and timely addition to the discourse forming around CRISPR-cas9 technology, its uses, and its safety. SCINQ caught up with him to discuss his findings.

SCIENTIFIC INQUIRER: Firstly, for those still unfamiliar with CRISPR-Cas9, can you briefly describe it and its uses.

STEPHEN TSANG: CRISPR-Cas9 is a tool scientists can use to correct mutated genes. Inherited diseases are often caused by one or more mutations in a gene, and CRISPR-Cas9 can be designed to target and repair the mutation, potentially leading to amelioration of the disease. This system was originally discovered in bacteria as an immune defense strategy. Scientists then modified it to make it easier and more efficient to apply in research and potentially clinical settings as well. It is now considered the front-line technique in genome engineering.

Dr. Stephen Tsang

SCINQ: Previous efforts to check for mutations employed predictive algorithms to identify possible mutations, what called the results into question?

ST: These predictive algorithms seem to do a good job when CRISPR is performed in cells or tissues in a dish, but whole genome sequencing has not been employed to look for all off-target effects in living animals. While in the past we relied on algorithms to estimate the location of potential unintended editing by Cas9, we were curious about the accuracy of those predictions. Especially as CRISPR draws closer to being translated into actual patients, it is more important than ever to determine the safety of this tool and any unintended genetic alterations it might produce.

SCINQ: Rather than using algorithms, you turned to whole genome sequencing. What advantages did it offer?

ST: Whole genome sequencing is an unbiased and comprehensive way to assess the true off-target effects of CRISPR and confirm or refute the algorithm-based predictions regarding unintended gene editing by Cas9.

SCINQ: How was the experiment designed and what was the objective?

ST: A large number of genetic variants have been identified in this era of next-generation sequencing, but whether they cause disease must be tested, which is a costly and time-consuming endeavor. The objective of our original study on this topic, published in Molecular Therapy in 2016, was to illustrate how CRISPR can speed the process by testing for and validating the pathophysiology of sequence variants.

We used targeted gene editing by CRISPR/Cas9 to test variant pathogenicity and resolve a century-long debate regarding the molecular origin of retinal degeneration in Pde6brd1/Pde6brd1 mice. Despite being the most extensively studied RP model, no one has ever been able to prove definitively whether a murine leukemia virus (Xmv-28) insertion or nonsense mutation (C to A transversion in codon 347, Y347X) causes the retina of this mouse model to deteriorate. Our report finally puts this controversy to rest. We show that CRISPR repair of the nonsense mutation restores retinal survival and function. Since the Xmv-28 insertion remains in our CRISPR-treated mice, Y347X is shown to be the causative variant.

Our most recent paper in Nature Methods utilized whole genome sequencing data from our experimental mice in the original study to explore the off-target effects of CRISPR-Cas9. Our objective was to determine whether CRISPR-Cas9 introduced mutations in unexpected sites in the genome.

SCINQ: What did it reveal about CRISPR?

ST: Our original study demonstrated that CRISPR can perform precise genome surgery, that it can be a useful tool for easily distinguishing between pathological and benign genetic variants, and that it has the potential to prevent retinal degeneration from occurring. Our follow-up paper, however, does raise questions as to whether Cas9 cleavage sites can be faithfully predicted using algorithms alone and suggesting a need for further whole genome sequencing studies following treatment with CRISPR-Cas9.

SCINQ: Was this in line with what you expected?

ST: The most commonly used method of off-target analysis is in silico prediction software. But this software focuses solely on homologous regions to the gRNA. Our studies indicate the in silico software may be good for targeting guides but not for predicting off-target effects, since there was little homology between the guide and experimentally verified off-target mutations.

SCINQ: What are the broader implications of the study’s findings? Does this mean that clinical use of CRISPR to treat disease needs more scrutiny before moving forward?

ST: Nothing in science can be conclusive without repeated testing. Our study suggests a need for further examination of the off-target effects of Cas9 and a more rigorous assessment of the accuracy of currently available algorithms for predicting off-target effects.

SCINQ: On a personal level, what brought you to a life in the sciences and genetics in particular?

ST: I was drawn to science by a desire to change the status quo, to develop treatments for untreatable conditions, and to make a difference in the world. I embraced the quote, “The Truth Shall Set You Free.” In many regards, I believe that science can point us towards truth, and it has even been described as ‘the language of God’ by NIH director, Francis Collins. Genetics is a subset of truth within science: genes don’t lie. The pursuit of truth is what compelled me to study science and genetics.

SCINQ: Can you tie this experiment into your past endeavors? How does this influence future investigations?

ST: Geneticists have dreamed of being able to accurately and easily modify the genome at the base-pair level. To do so efficiently and safely could potentially cure countless inherited diseases, reducing human suffering and improving overall health. While we have used zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) previously in the lab, CRISPR arguably holds the greatest potential for being able to achieve this dream. We still plan to continue to explore this technique. Optimizing it and ensuring its safety and efficacy are primary goals of our future experiments.

SCINQ: What role do you believe the scientist should play in the world? How do you fit into that?

ST: Scientists ideally should be the ambassadors of knowledge to society. Their purpose is to learn about the world, how it works, and how it can be improved, and to share their findings and interpretation in an objective manner. There are so many issues and unanswered questions, and so many different opinions, that it can be difficult to know what to believe or where to start in changing the world for the better. Scientists, ideally, should be the ones providing unbiased, rigorous analysis of these problems and suggesting reliable, innovative options, backed by sound science and an unwavering commitment to deepening our knowledge of ourselves and our world. I aspire every day to live up to this commitment.

SCINQ: Finally, what did you want to be when you were in grade school?

ST: I was hospitalized for pneumonia during kindergarten and then released that there was no greater gift than health.

I can tell you what I didn’t want to be: I didn’t want to be an engineer like my father. I also didn’t like humanities and couldn’t really see myself as a lawyer or writer. By process of elimination, that left me with biomedical science, which turned out to be a perfect fit.

For more about Stephen Tsang and his work. [MORE]

Alternatively, listen to him in his own words.

Learn more about CRISPR-cas9 by listening to this TED talk with one of its inventors, Jennifer Doudna.

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