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General Social Survey

The coronavirus pandemic is a catastrophe. Certain emergency measures were taken to low the spread, and scientists have been working hard to expedite their researches of developing vaccines, reducing symptoms, and improving prognosis for patients. Many biological, epidemiological and medical concepts have entered the public’s field of vision. Gene-editing technology is a tool that may be widely used in precision medical treatments in the future. In this case, we concern about whether people’s understanding of gene-editing techniques has changed from the past in China. We are curious about public’s acceptance of it as well.

To compare public attitudes towards gene-editing technology, we picked some items from last year’s questionnaire and planned to ask those questions again. More new items were added as well. After rounds of modifications, the questionnaire was sent out in February and much data was collected by the time we stopped collecting, which is around October. By comparing them with the data collected in the same period in 2019, we were able to let public’s awareness and acceptance of gene-editing technology guides and constraints our project.

Handing out the questionnaire online

We divided the survey respondents into three groups according to their social attributes, and the differences in their attitudes towards certain given statements.

Analysis of the results

First off, we compared two groups of the respondents, and delightfully found out that there are no significant differences in either age distribution, gender, or educational background. That is to say, both of the respondents surveyed in 2019 and 2020 are representative of the general public in China. (The p-value for the difference in gender ratio is 0.583; the p-value for the difference in the proportion of people under 25, over 60 and other people participating in the survey is 0.211).

Public awareness

We observed and analyzed the changes in public attitudes before and after the outbreak of COVID-19 in four following aspects: understanding of gene-editing technology and related concepts, insight into hot topics related to gene-editing technology, acceptance of gene-editing applications, and recognition of theories related to gene-editing technology.

Difference in the understanding of gene-editing technology and related concepts from 2019 to 2020

After the COVID-19 outbreak, Chinese people understand more biological concepts and technologies. For example, the proportion of people who is familiar with gene-editing technology, gene targeted therapy and the CRISPR system is significantly larger in 2020 than in 2019. (p value<1e-6, chi-square test, and continuity correction).

Difference in the understanding of gene-editing technology and related concepts from 2019 to 2020, big blue dots has the greatest significance.

We also measured respondents' knowledge of coronavirus and virus detection technologies before and after the outbreak, and it is also clear that more and more people in China are becoming aware of and familiar with coronavirus and cutting-edge technologies such as gene-editing.

Difference in the public awareness of some gene-editing news from 2019 to 2020.

We examined the shift in respondents' attention to several gene-editing-related hotspots before and after the epidemic. Unlike in 2019 when 'the He Jiankui Affair' is the only news heard by many, people in 2020 know better about gene-editing tools, especially the CRSIPR system. Gene-editing technologies used in breeding and therapies are gaining public attention.

Public acceptance

Difference in the public acceptance of some gene-editing applications from 2019 to 2020.

In terms of the acceptance of gene-editing technology, ethical considerations favor either refining gene editing in microbiological experiments or supporting its application in human medicine, with few people being receptive to experimentation in the human embryo. It is noteworthy that, except for the proportion of people who disagree with gene editing for human embryo modification, the support for and acceptance of this application has declined since 2019, most likely because people are more cautious about the technical concepts of biology when it comes to the spread of COVID-19, in case some mutations evolve to produce undesirable products that could harm people, or because they are influenced by the epidemic.

Difference in the public's opinion on some gene-editing statements from 2019 to 2020.

We then listed four statements to the respondents and asked for their views. The four statements are mainly related to the prospects and significance of gene-editing technology. Although the public still find it relatively hard to accept gene-editing technology, they are optimistic about the prospects of gene-editing techniques when they become safe enough. We have to point out that we set two contradictory statements, that 'Parents cannot choose to modify their child's genes, even if they believe the modification is beneficial to the child', and that 'Children with congenital defects can be gene-edited with parental consent'. However, by slightly changes turn of phrase, the outcome was totally messed up. It's reasonable for us to doubt the actual public acceptance of gene-editing technology.

Grouping of respondents

To measure the awareness and acceptance of gene-editing technology by a representative sample of individuals, the qualitative options were converted into quantitative scores, which were standardized for all respondents, demonstrated in the blue-red color indicator (knowledge of the concept, concern about gene-editing related news, acceptance of the use of gene-editing technology, and views on its prospects). We then used hierarchical clustering to classify the respondents into three groups, before we analyzed the characteristics of their attitudes towards gene-editing technology through heat map visualization.

Clustering analysis of the survey respondents.

The clustering results demonstrate the high correlation within four issues investigated, which are acceptance of, degree of interest, awareness of, and opinion on gene-editing technology. All respondents could be break down into three groups: People in the first group are familiar with gene-editing related concepts, but lack enough attention to news in this field, however, they are very optimistic and supportive of the application of gene-editing technology. People in the second group are quite accepting of the application and development of gene-editing technology while fully grasping its relevant background. People in the last group, however, are also familiar with the background knowledge and follow news in this field, yet they refuse to apply gene-editing technology to experiments or to therapies, and are not optimistic about gene-editing prospects.

Finally, we added information on respondents' social attributes and visualized them on a heat map in an attempt to analyze the differences in age and gender among those holding the three types of views.

We finally used Generalized Linear Model and multinomial distribution to approximate the likelihood of different individuals belonging to the three groups of people. The assessment is considered based on the influence of gender, age, education, profession, and religion. The results show that Group 3 has less male than Group 2 (log odds ratio -0.76, p-value 0.017), and has a greater proportion of non-biology majors (log odds ratio 2.42, p-value 0.022). Group 1 has more young individuals rather than middle-aged ones than Group 2 (log odds ratio 0.938, p-value 0.001). We also observed a significantly higher proportion of people who bare religious beliefs in Group 2 (p-value 0.0017 and < 1e-6, respectively).

We can therefore infer that middle-aged males who don’t major in biology-related subjects, and religious populations are very likely not to accept or approve of gene-editing technology even if they are knowledgeable about it.

In conclusion, the COVID-19 pandemic has brought us endless mistress and trouble, and the only good thing is that we can learn a lesson from it. We might know a bit more about some basic biology and might learn to pay more attention to public health and to protect ourselves. We have observed a shift in the awareness and acceptance of gene-editing technology among the Chinese public before and after the COVID-19 outbreak. People are more familiar with gene-editing technologies, and are more cautious about it, which is a better trend. We are positive that gene editing technology will be optimized as people's views change, and eventually, we believe it can, and will be widely used.

The full questionnaire is attached below.







Interviews

This year, we were lucky enough to get useful suggestions from professors whose research is highly related to our project LUCAS. All are not strictly speaking interviews, but more like chatting, which were a lot more relaxed. We talked about the current state of society, technology application, experimental adjustment, project application, industry outlook, project evaluation, etc. We are so grateful to those professors for their tolerant attitudes, enthusiasm, useful advice and precious time.

Tianlin Cheng

Professor Cheng is a young researcher at Institute for Translational Brain Research in Fudan University. His field of research fouses on developing new gene-editing tools.

"The feasibility of your design is undoubted. Fluorescent signals can make a relatively quantitative judgment on the frequency of off-target events."

"Your project is of great significance in translational medicine. It is worthy to establish a system to reprogram a highly specific Cas9 for each target with potential drug-making potential. "

Project Background

Q: What are the main challenges in the development of CRISPR-Cas9 technology research?

A: One is the safety problem, which is whether the precise modification of one site affects or harms other sites; the other thing is the omnipotence of the modification tool, that is to say, whether the editing tool suits most editing sites.

Q: Why does the off-target effect exist in the CRISPR system?

A: Because guide RNA we used in gene-editing are derived from prokaryotes, whose genome is smaller than eukaryotes and doesn’t have so many similar sequences. Given the situation, if Cas protein, the enzyme that recognizes PAM has not evolved so well during the evolution process, it will miss the target.

Q: Are there any technologies or methods that are currently being studied or have already published that can improve the off-target effect?

A: Existing technical research has put relatively more attention on improving the universal off-target effect. If you start with the editing efficiency of a specific site, I don’t know if you can reduce the frequency of off-target events of other sites at the same time.

Q: If we try to optimize the CRISPR system in another way, say, randomly mutate the Cas9 protein and then let them go through many rounds of selection, will this method of our work?

A: It’s feasible, but such Cas9 protein usually has a much higher frequency of off-target events than wild-type Cas9.

Project Design

We briefly introduced our project design to Professor Cheng.

Q: Our understanding is that different Cas9 proteins can undergo different directed evolution processes to suit different targets, thus improving the off-target effect. Do you agree with this point of view?

A: Your method is feasible to reduce the frequency of off-target events by optimizing Cas9 proteins that recognize different PAM sequences. For different targets and PAM sequences that recognize these targets, Cas9 mutants with different PAM recognition sequences should be designed.

Q: In directed evolution experiments, antibiotics and lethal gene screens are mostly used. Is fluorescence screening reasonable and feasible?

A: The feasibility of your design is undoubted. Fluorescent signals can make a relatively quantitative judgment on the frequency of off-target events. Although antibiotics have higher screening efficiency and the screening process might be faster, their specificity is questionable. Maybe you can try using two screening systems and make comparisons.

Q: Are there any latest researches (published/unpublished work) on directed evolution to transform Cas protein?

A: You are on the track of precision medicine. The current technology will not be so intensively studied unless the drug is developed for a specific site, in which case your design surely has certain advantages and is competitive.

Application prospects

Q: Does your laboratory or any laboratory/clinical research requires and has expectations for highly selective proteins?

A: The biggest problem in the application of CRISPR is the off-target events, especially for editing in humans. Different laboratories are interested in different target sites, but their requirements for highly selective (high editing efficiency) protein is very high. To be honest, my lab is very much interested in highly accurate gene-editing techniques.

Q: After you’ve assessed our project, do we have a positive application prospect? Is it possible for us to put LUCAS into use on other occasions, such as a system for assessing the off-target effect of Cas9 protein?

A: Your project is of great significance in translational medicine. It is worthy to establish a system to reprogram a highly specific Cas9 for each target with potential drug-making potential. It is also feasible as an assessment system.

Q: We are currently optimizing the corresponding Cas9 protein for PDCD1, a target related to human non-small cell lung cancer. Are there other sites that can be our future targets?

A: Yes, such as PCSK9. Knocking out this gene can reduce LDL levels in the blood, which is beneficial to cardiovascular health.

Q: We feel that our system has great application value for disease models, however, does basic research have low expectations for highly specific proteins?

A: Basic research can eliminate failed samples through other screening methods, so there is indeed not such high demand, but your system can still be applied to fusion proteins and other mutants. For example, using fluorescent signals to screen Cas9 fusion protein that only modifies a single base, optimizing other Cas protein mutants. The sequence recognized by ordinary Cas9 is NGG. The latest research this year has discovered many universal protein mutants that can recognize other sequences such as NG and NYN. These mutants have low requirements for the identification of PAM sequences and a high risk of off-target effect. These mutants have a large room for optimization.

Q: Is it possible that even if LUCAS managed to optimize Cas9 protein, will it be difficult to implement in the clinical application of the human body? Is it true that current researches lack practicing on the human body?

A: I’m afraid yes. unless the therapy is a hundred percent sure to be safe, people are still very cautious when it comes to gene-editing, especially the editing will be integrated into the genome, the concept alone is hard for people to accept. I know two human body editing studies conducted right now, one is about the bcl11a, a gene associated with thalassemia, the other is something related to genetic blindness.

Feng Gu

A researcher at The 2nd School of Medicine, WMU. His field of research fouses on genetic diseases and new gene-editing tools.

Workload is our main concern when we decided not to use the directed evolution method.

Q: Was there only one mutation in your SaCas9 protein?

A: Yes. A series of mutants were constructed earlier, and they're not adopted.

Q: Was the method of directed evolution considered at that time?

A: There were such considerations, but there were also concerns that too much work should be done in eukaryotic cells, so we didn't do that.

Q: Did you use the model to predict outcomes?

A: In terms of modeling and computing, we collaborated with NH in the United States, who provided simulation on structures to assist screening, and many previous studies have attempted to apply structural computing.

Q: Is it true that directed evolution requires a lot of time and labor?

A: Not necessarily. For the success of continuous evolution methods like PACE, the system needs to be perfect. At present times, I mainly do the screening work, not the directed evolution in the narrow sense.

Q: Is it true that there are many in vitro experiments on Cas9 now, but few in vivo experiments?

A: Yes, and it’s mainly due to the problem of delivering and transporting, Cas9 protein is too big to transport.

Q: Why do you think the acceptability of gene-editing technology is still not high?

A: There are still many problems in gene-editing, such as legal regulations and ethical issues. It has better prospects in cancer research. Transportation strategy, just like off-target effect, is also a key issue (mainly in gene therapy), efficient targeted delivery.

Q: Can the background leakage effect of screening by antibiotics be ignored?

A: Generally, antibiotic screening (with high fidelity) can kill all the unwanted mutants.

Do you prefer dCas9 or Cas9 protein?

A: The main choice is to use Cas9 protein. dCas9 protein is still used, mainly because the results of the laboratory regulation are not so believable.

Ruilin Sun

Deputy general manager of Shanghai Model Organisms, Sun is experienced in the development and the use Cre-loxP mouse strain.

"Your project might be of great use when applying to certain editing sites that have strong off-target effects."

Q: In your opinion, what are the main obstacles encountered in the development of gene-editing tools, such as CRISPR-Cas9 system?

A: There are two main issues: ethical issue and off-target issue. In fact, the editing efficiency of the existing technology is already very high. If compared with TALEN and other technologies, the main problem is the off-target problem. Current mainstream optimization scheme, one is to optimize the gRNA sequence to improve the off-target effect; the other is to design a smaller Cas protein. The existing Cas protein is around 200kD, which is still too big for its function execution.

Q: Our project design is based on the principle of CRISPR-Cas9, and we hope to complete the optimization of the Cas9 protein in off-target problem improvement and significantly enhanced specificity through directed evolution. Do you have more suggestions and comments on our project design?

A: The existing technology can basically support the needs of all kinds of gene knockout techniques, because even if it is off-target, it can be screened or eliminated after editing. But if it is applied to disease models, especially human disease models, it will be different, in that case, extremely low frequency of off-target events is needed to ensure safety. Your project might be of great use when applying to certain editing sites that have strong off-target effects

Xiajing Tong

Professor and researcher at Shanghai University of Science and Technology. One of her previous works was to use C. elegans as model animal to select certain genes.

In response to the goal of our project to lower the off-target rate of Cas9 protein, Professor Tong first pointed out that this goal is hard to realize. For protein editing, if we want to make the phenotype stand out from the population, first thing needed to be done is giving selective pressure. For off-target effect, the most reliable detection strategy is sequencing. Therefore, the experiment requires a lot of sequencing to test the Cas9 off-target rate. And even if fluorescent screening is used, since directed evolution will produce a large amount of proteins, it will further increase the workload. Another problem is that if we only consider dCas editing, that is, cleavage efficiency, we only need to detect whether a specific site is cleaved. But if we want to detect off-target event, we need to screen the whole genome.

Prof. Tong then suggested that we can focus on editing efficiency and choose easy-to-observe phenotypes, such as the flight ability of Drosophila or the movement pattern of nematodes.

Next, we asked about the popularity of directed evolution. Prof.Tong said that the idea of applying random mutation to select target phenotype has long existed, but it has gradually become systemic in recent years. The general public might have heard of the word evolution, but the technical details are not popularized.

As for the screening criteria we are more concerned about, Pro.Tong said that both fluoroscent selection and antibiotics selection can be put into use. The only requirement is that the phenotype is obvious.

Another problem is, "The process of directed evolution cannot guarantee a completely optimized result, but in most cases it can only be partially optimized. It is only advantageous in the current state. The term 'best protein' itself is a paradox.” Pro.Tong agreed with this view. " There is no best protein, but only the dominant protein in the current environment", She said.

Yi Xiao

Special researchers at School of Lige Sciences and Biotechnology, SJTU. His field of research fouses on deveoping CRISPR-Cas9 system and biomolecular sesor- based biotechnology for gene-editing, transcriptiong regulation, screening, dynamic control, etc.

We briefly introduced our project design to Professor Cheng.

Q: What additional conditions does the CRISPR system need to work properly?

A: Extra conditions are not needed for CRISPR system other than Cas9 protein, sgRNA and the binding site. When sgRNA is transcribed, it undergoes self-folding and turns into functional structure.

Q: Are there any special requirements for temperature and other culture conditions while conducting experiments?

A: Refer to the literature and follow the protocol should be fine.

Q: Our experimental results of the transcription activation system is worrying. Could you please give us some advice?

A: Our lab repeated Feng Zhang's experiment but was unable to get transcription activation effect. We then tried the transcription suppression system. You might want to have a try as well.

Professor Xiao also helped check our plasmid construction as well as our overall design. Our plasmid has low copy number of dcas9 protein and high copy number of sgRNA. He assured us that the design will perfectly work.

Aside from that, we were lucky to get precious technical suggestions on Gibson assembly from one of the students in Xiao’s lab.

"If you want to use Dpn1 to reduce the amount of original plasmid after PCR amplification, make sure to inactivate Dpn1 at 80$^{\circ}$C for 20mins before adding Gibson mix. And run Gibson mix reaction for 3 hrs. 30 mins (10ul volume of total Gibson reaction)”

Elongate the homologous arm can effectively elevate positive rate of Gibson assembly. If you cannot obtain positive colony using your 20bp homologous arm, please try extending it to 40bp.

He also recommended some useful websites for Gibson assembly and PCR primer design and was of great use.

Di Chen

A medical graduate at Shanghai Jiao Tong University School of Medicine.

“ Gene editing therapy is of great potential. There has been a successful example of using CRISPR-Cas9 to edit PD-1 gene in vitro T cells, and then transplanting to non-small cell lung cancer (NSCLC) patients. This method can significantly improve the mid-term survival time of patients.”

Q: For non-small cell lung cancer patients, what are their states of living like and how about their quality of live?

A: As far as I know, lung cancer is a malignant tumor with one of the highest incidence and mortality rates in the world at present. And non-small cell lung cancer(NSCLC) is the most common subtype of lung cancer worldwide. However, most of the patients with NSCLC are confirmed at III stage and their 5-year survival rate is even less than 25%. For these patients, their quality of life remains dismal. Because of anesthesia, stress and trauma that the patients may encounter in cancer treatment, they often have problems with pain, nausea and insomnia, which greatly affect their daily life.

Q: Given that the patients live a dismal life, does the pain of disease and treatment put a burden on their mental state?

A: Chemotherapy is widely used to treat NSCLC patients clinically. However, on the one hand, chemotherapy can effectively kill cancer cells. On the other hand, the patients suffer from strong chemotherapy-related toxic, side effects, thus inducing their poor emotion such as fear and depression. Some clinical evidence shows that most patients, especially frail elderly, tend to generate negative emotions including fear for death, loneliness and powerlessnesss in chemotherapy.

Q: Considering such a severe condition in non-small cell lung cancer, does current treatment work for the patients? And how about their 5-year survival after treatment?

A: As you all know, NSCLC has become the leading cause of death in China. Its characteristics of high incidence, high mortality and poor prognosis has long been a deadlock in effective treatment. Chemotherapy, which is mostly used in clinical practice, however, can only modestly improve quality of patients' life and extend their lifespan instead of curing the cancer. And median survival time of many conventional treatment is even less than five years. Polluted environment and unhealthy lifestyle has led a higher and higher incidence recent years.

3. Q: So what are the disadvantages of conventional treatment? Can gene editing provide a promising aspect for treating NSCLC?

A: Honestly speaking, gene editing therapy has a great potential when conventional treatment reaches a bottleneck. In clinical practice, surgery is the primary choice for early-stage lung cancer while chemotherapy is more used in small cell cancer. However, for NSCLC, the prognosis of radiotherapy is very poor, and the five-year survival rate is less than 10%, while surgical treatment seriously affects the life quality of NSCLC patients with advanced cancer. The prognosis of these patients is extremely inferior because of the inability to resect the potential lesions. The median survival time is only 5-6 months. Emerging targeted meidications and immunotherapy may have a greater impact on patients with early undetectable metastatic diseases, but these treatments are still in the stage of accumulating clinical experience. If we can edit the important targets through gene editing, it may have a significant impact on the prevention of early stage tumors and the prevention of tumor recurrence in the future. Some research teams have already used CRISPR-Cas9 to edit PD-1 gene in vitro T cells, and then transplanted to non-small cell lung cancer (NSCLC) patients. This method can significantly improve the mid-term survival time of patients.

Q: So what are the disadvantages of conventional treatment? Can gene editing provide a promising aspect for treating NSCLC?

A: Honestly speaking, gene editing therapy has a great potential when conventional treatment reaches a bottleneck. In clinical practice, surgery is the primary choice for early-stage lung cancer while chemotherapy is more used in small cell cancer. However, for NSCLC, the prognosis of radiotherapy is very poor, and the five-year survival rate is less than 10%, while surgical treatment seriously affects the life quality of NSCLC patients with advanced cancer. The prognosis of these patients is extremely inferior because of the inability to resect the potential lesions. The median survival time is only 5-6 months. Emerging targeted meidications and immunotherapy may have a greater impact on patients with early undetectable metastatic diseases, but these treatments are still in the stage of accumulating clinical experience. If we can edit the important targets through gene editing, it may have a significant impact on the prevention of early stage tumors and the prevention of tumor recurrence in the future. Some research teams have already used CRISPR-Cas9 to edit PD-1 gene in vitro T cells, and then transplanted to non-small cell lung cancer (NSCLC) patients. This method can significantly improve the mid-term survival time of patients.