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Human Practices

Beginning of a Journey

Since the advent of CRISPR-Cas, there has been much interest surrounding human DNA editing. One benefit of DNA editing is that this results in a permanent correction of the pathogenic gene to a wild-type copy. Depending on the cell type that is edited, it may result in a heritable change (on germ cells) or a non-heritable but functional correction in the person (somatic cells). However, major issues regarding human DNA editing remain. This includes safety concerns from possible permanent off-target effects as well as ethical and regulatory considerations.

In contrast, changes in the RNA are transient but functional. One particular RNA editing platform is the dCas13-ADAR2_DD fusion protein for gRNA targeted A-to-I RNA base change. In the literature, the dCas13b-ADAR2_DD(E488Q) was found to have high editing efficiencies. However, it also had many off-target activities.

Although both DNA and RNA editing systems present therapeutic benefits, their shortfalls could hinder public acceptance and clinical adoption. To better understand the knowledge and concerns about nucleic acid editing, and to develop a platform that suits the requirements of the public (who would be adopters of our future technology), we first conducted a survey before embarking on our project.

Initial Pilot Study

As the beginning of our project coincides with the Life Hacks 2.0! event collaboration with Team NUS_Singapore 2019, we decided to take this opportunity to educate the students on CRISPR-Cas and get a gauge of their attitude towards this technology through a pilot survey. In preparation for the event, we created two posters for a poster presentation on Cas9 (DNA binding) as well as the newly-discovered Cas13 (RNA binding). We also prepared a PowerPoint presentation on DNA and RNA editing, as well as their applications. You may read more about the event at our Outreach page. After giving a general introduction to the topic, we conducted a pilot survey of 40 randomly selected participants.

Our Takeaway

On DNA editing, most participants (75%) were keen on its uptake, but only for therapeutic purposes. None of the participants supported the use of DNA editing for cosmetic enhancements. On RNA editing, almost all of the participants (95%) were keen to see its implementation in the near future, citing its transient hence reversible nature as an attractive feature. However, some of the participants commented that our survey could be difficult to understand, especially to the general public with no scientific background. Encouraged by the preliminary results and general feedback on the survey, we decided to consult an expert on survey design (preferably with a background in scientific communications) for its distribution to a wider audience.

Xiao Yu sharing about Cas9 to the students at Life Hacks! 2.0

On Gene Editing: Survey of the General Public in Singapore

Our meeting with Asst Prof Christopher Cummings!

Survey Design

We decided to examine the perception, attitudes and concerns about nucleic acid editing of the general public at the start of our project design in order to develop an RNA editing tool that is good, responsible and accepted for use by the public. To investigate this, we crafted a survey in consultation with Asst Prof Christopher Cummings from the Wee Kim Wee School of Communication and Information in NTU. His understanding of public scientific communication, as well as his research interest in the public engagement of synthetic biology, has been very helpful in providing us with a guideline for survey design. With feedback from our pilot survey, we also included a short introduction on DNA and RNA editing to provide some context to participants with no scientific background.

We utilised a 7-point Likert scale to collect information on public perception. Surveys were conducted electronically so that it is accessible to a wider audience. The survey was conducted anonymously to minimise social desirability and biased responses. We had also placed demographic questions at the end of the survey as suggested by Asst Prof Cummings to prevent these questions from influencing their responses. A sample of our survey may be accessed here.

Survey Results

We collected a total of 395 responses. The basic demographic data are shown in Figures 1 (gender distribution), 2 (age group) and 3 (education levels). There were more female (58.5%) than male (41.5%) respondents. Most of the respondents were aged 21-30 years old, and have a university degree.

Figure 1. Pie Chart of Gender Distribution.

Figure 2. Bar Graph of Age Groups.

Figure 3. Bar Graph of Education Levels.

On the applications of gene editing (Figure 4), a majority of participants chose healthcare (52.4%) over other options. This indicates that health is of an important concern and having an alternative treatment option is desirable. Many have also indicated their preference of using gene editing technologies for research and development, such as understanding disease progression and pathology. This also correlates with the desire to improve healthcare.

Figure 4. Prioritised Applications of Gene Editing.

Next, we gave the participants a series of questions to assess their general perception and attitudes toward gene editing (Figure 5). Most participants were confident in making an informed choice to undergo gene therapy should the need arise. They are also less willing to accept germline editing which results in heritable changes. This may be due to the uncertainty of how gene editing would affect the development of a child and the recent controversy surrounding gene edited babies. Many were also agreeable in using gene editing for medical purposes.

Figure 5. Perception and Attitude of Gene Editing.

Our next question assessed the underlying factors affecting their choice in adopting gene editing for therapeutic uses (Figure 6). Of note, safety was of a major concern, followed by the availability of other treatments, cost and their personal beliefs.

Figure 6. Factors Affecting Uptake of Gene Editing.

Our final set of questions assessed the preference for DNA or RNA editing (Figures 7-9). We also asked about their perceived risk of either method. As seen, RNA editing was associated with less risk, possibly due to its transient nature and reversibility of side effects by stopping treatment.

Figure 7. Preference for DNA or RNA Editing.

Figure 8. Perceived Risk of DNA Editing.

Figure 9. Perceived Risk of RNA Editing.

Our Takeaway

From the survey, we were encouraged to pursue RNA editing due to its overwhelming preference for adoption among the general public of Singapore. Although it is transient and poses less of a risk than DNA editing, we felt that the current platform of RNA editing may be further improved to reduce off-target effects. We also noted that safety is an important consideration that strongly influences the acceptance of gene editing, which reinforced our idea. Thus began our project of developing a targeted RNA editing platform using dCas13d (also known as dCasRx) fused to the adenosine domain of ADAR2 (ADAR2_DD) that has minimal off-target activities. Of the different Cas13 RNA-binding enzymes, we chose CasRx due to its small size which would aid therapeutic delivery. More information on the mechanism of this platform may be found on our RNA Editing page.

Integrated Human Practices

Interview with Professionals

Besides understanding the views of the general public, we also contacted several doctors and academics to delve deeper into the technical insights behind RNA editing as well as the requirements for its future clinical adoption. With their knowledge and expertise, we obtained valuable information in progressing and branching out our RNA Editing project.

Dr Joanne Ngeow

Dr Joanne Ngeow is an Associate Professor in the Lee Kong Chian School of Medicine (LKCSoM). She is also a Senior Consultant in the Division of Medical Oncology at the National Cancer Centre Singapore (NCCS) and heads the NCCS Cancer Genetics Service.

From our discussion, we understood that the clinician's point of view on the adoption of RNA editing, or other gene therapies for the matter, depends on its safety. She mentioned that for our construct to be clinically useful, there is a need to prove that its specificity has to be high. This led us to think of ways to improve the specificity of the existing dCas13-ADAR2_DD RNA editing platform, as well as an important goal of achieving low off-target activities.

Team NTU-Singapore with Dr Ngeow at the LKCSoM

Assoc Prof Eric Yap

Associate Professor Eric Yap is a medical geneticist and a former Rhodes Scholar. He is one of the few local researchers with dual backgrounds in medicine and science.

We were encouraged by his positivity toward RNA editing, though he echoed the sentiments of the necessity for a safe and efficient product before clinical adoption may be achieved. There were also many obstacles to prove its functionality for therapeutic use in humans. He mentioned the attractiveness of RNA editing being transient and reversible, which is an advantage over DNA editing. Edited transcripts would also not be heritable, which may be less of an ethical issue compared to the permanence of DNA editing.

Team NTU-Singapore with Dr Yap at his lab in the LKCSoM!

Dr Wan Yue

Dr Wan Yue is a senior research scientist at the A*STAR Genome Institute of Singapore (GIS). Her research interests are in studying functional RNA structures and understanding their roles in regulating cellular biology.

As an expert in RNA structural biology, we approached Dr Wan Yue to understand the technicalities of our RNA editing platform. She reminded us that since RNA tends to form different secondary and tertiary structures in the cell, it may hinder the binding of our gRNA to the target. As such, we decided to investigate whether RNA structures would affect the editing efficiencies of our dCas13-ADAR2_DD construct. From her recommendation, we looked into the icSHAPE data to identify open (linear) and structured RNAs for characterisation of RNA editing. This led to the development of our Open/Structured project.

Our team leaders with Dr Wan Yue at GIS

Assoc Prof Xavier Roca

Assoc Prof Xavier Roca is a professor in the NTU School of Biological Sciences and the course coordinator of The RNA World (BS3015). His research interest is in studying the mechanisms of pre-messenger RNA splicing in human cells.

Our discussion with Assoc Prof Xavier Roca made us think deeper into our RNA editing project, and ways to analyse off-target effects. There were 3 ways of off-target editing which were brought up during the conversation. Firstly, there could be off-target A-to-I editing within the target region of our mRNA (i.e. cis off-targets). Secondly, there could be editing on unintended targets from gRNA mispairing (i.e. trans off-targets). Lastly, the duplex structures of certain RNAs may be recognised by ADAR and be unintentionally edited. We were also encouraged to look at the relationship between RNA structure and editing.

Our guys with Assoc Prof Xavier Roca!