Scientists from the CSIR-Institute of Genomics and Integrative Biology, New Delhi, have developed an enhanced genome-editing system that can modify DNA more precisely and more efficiently than existing CRISPR-based technologies.

CRISPR occurs naturally in some bacteria, as a part of their immune system that limits infections by recognising and destroying viral DNA. In Nobel-prize winning work, scientists repurposed this bacterial defence mechanism to develop a novel approach for editing the genomes of higher-order organisms.

CRISPR’s off-target problem

Today, using CRISPR-Cas9, researchers can add, remove or alter specific DNA sequences in the genome of animals. This system has been used in various fields, including in agriculture — to improve the nutritional value of plants and increase the yield — and in healthcare to diagnose several diseases and treat genetic disorders.

The CRISPR-Cas9 gene editing tool uses a guide-RNA (gRNA) designed to find and bind to a specific part of the target genome. The gRNA directs an enzyme, Cas9, to the target site, which is followed by a short DNA sequence called protospacer adjacent motif (PAM). Cas9 recognises and binds to the PAM sequence, and acts as a molecular scissor that snips some damaged DNA. This automatically triggers the cell’s DNA repair system, which repairs the snipped part to insert the correct DNA sequence.

But the CRISPR-Cas9 system can also recognise and cut parts of the genome other than the intended portion. Such “off-target” effects are more common when using the SpCas9 enzyme derived from Streptococcus pyogenes bacteria. Scientists have been able to engineer versions of SpCas9 with higher fidelity but only at the cost of editing efficiency.

Switching SpCas9 with FnCas9

To overcome these issues, researchers are exploring Cas9 enzymes from Francisella novicida bacteria. While this Cas9, called FnCas9, is highly precise, it has a low efficiency as well.

To enhance it without compromising its specificity, researchers led by Debojyoti Chakraborty at CSIR-IGIB modified and engineered new versions of FnCas9.

The researchers tinkered with amino acids in FnCas9 that recognise and interact with the PAM sequence on the host genome. “By doing this, we increase the binding affinity of the Cas protein with the PAM sequence,” Dr. Chakraborty said. “The Cas9 can then sit on the DNA in a stronger configuration, and your gene editing becomes much more effective.”

The researchers also engineered the enhanced FnCas9 to be more flexible and edit regions of the genome that are otherwise harder to access. “This opens up more avenues for gene editing,” Dr. Chakraborty said.

Juicing the enzyme

Experiments to measure enzyme activity showed that enhanced FnCas9 cut target DNA at a higher rate compared to unmodified FnCas9.

CRISPR-based tools for diagnostics and therapeutics rely on the ability of the system to recognise specific single-nucleotide changes in the DNA. Nucleotides are the building blocks of DNA and RNA. Each nucleotide consists of a nucleobase, a phosphate group, and a sugar. Each nucleotide in DNA has one of four nucleobases: adenosine, thymine, guanine, and cytosine. A single-nucleotide change is when just one nucleotide in the genome needs to be ‘repaired’.

When the researchers tested the ability of enhanced FnCas9 to identify such changes in the genome, they found enFnCas9 outperformed unmodified FnCas9. An enhanced FnCas9-based diagnostic could target almost twice the number of changes compared to FnCas9, increasing the scope of detecting more disease-causing genetic changes.

Testing against an inherited blindness

Once Dr. Chakraborty’s team had shown the increased efficiency and activity of the enhanced FnCas9 enzyme, a team led by Indumathi Mariappan at the L.V. Prasad Eye Institute in Hyderabad explored the enzyme’s suitability for therapeutic applications.

The researchers used enhanced FnCas9 to edit the genome of human kidney and eye cells grown in lab dishes. It not only edited genes in these cells at a better rate than did SpCas9, it also showed negligible off-target effects.

The team finally sought to understand whether enhanced FnCas9 is a viable option for treating genetic disorders. They tested the enzyme’s efficiency at correcting a genetic mutation that causes Leber congenital amaurosis type 2 (LCA2), a form of inherited blindness. A single mutation in the RPE65 gene results in the loss of expression of a protein called retinal pigment epithelial-specific (RPE65), resulting in severe vision loss.

A surprising efficiency

The team isolated skin cells from an individual with LCA2 carrying the RPE65 mutation, and reprogrammed these cells to become induced pluripotent stem cells (iPSCs). Such cells can be made to grow into any cell type in the human body. When the researchers differentiated the iPSCs into cells of the eye’s retina, the cells expressed negligible levels of RPE65 protein.

The researchers delivered a CRISPR system with the enhanced FnCas9 enzyme into the individual’s iPSCs to correct the mutation responsible for low levels of this protein. When they sequenced the edited cells, they found that the CRISPR tool had corrected the mutation. The edited iPSCs when differentiated into retinal cells also showed normal levels of the RPE65 protein.

Dr. Mariappan said the team was taken aback by the efficiency of the editing. Most of the iPSCs carried the edits, and when the researchers grew colonies from individual edited iPSCs, they found that two colonies showed 100% mutation correction.

“We also examined the whole genome for off-target interactions and found only a few, of no major concern, as compared to several hits seen with other Cas9 proteins [we] examined,” she added.

What the research community needed

Previous reports have suggested that such corrected (person-specific) retinal cells can be transplanted back into a person to treat inherited blindness conditions like LCA2.

A group of U.S. researchers reported on May 6, 2024, in the New England Journal of Medicine that CRISPR injected directly into the eyes of people suffering from LCA2 has shown success in early clinical trials. But editing patient-specific stem cells and transplanting the mutation-corrected cells into patients is a safer option, according to Dr. Mariappan, “because this allows us to screen and confirm precise edits.”

“The research community needed this precision” in the CRISPR system, Shailja Singh, a researcher at Jawaharlal Nehru University, New Delhi, who uses CRISPR-based tools to model and study genetic diseases like sickle cell anaemia and β-thalassaemia, said. Such reduced off-target effects are critical for those who use CRISPR-based therapy to correct mutations, “so this is a very welcome approach.”

Dr. Singh added that while a precise enzyme without any off-target effects is much-needed, the delivery system must also be equally proficient. According to her, researchers should next focus on precisely delivering this tool into the nuclei of the target cells.

Making therapies for India

Dr. Chakraborty said the team is working on adapting the system to different delivery methods as well as reducing the size of the enFnCas9. “All these will come in the following studies,” he said.

The team is also in contact with some Indian companies to patent the technology. “This opens up the doors for not licensing from a foreign entity, which could be very, very expensive.”

Dr. Mariappan agreed: “With an indigenous patent for such a high precision editor, we are now in a better position to develop newer therapeutics at affordable costs for people in low- and middle-income countries like ours.”

Sneha Khedkar is a biologist turned freelance science journalist.

A study by researchers at the L.V. Prasad Eye Institute (LVPEI), Hyderabad, has found that around 70-80% of people who visit an eye hospital can benefit from teleconsultations because their problems aren’t serious enough to require attention at a hospital. The study was published in the journal Eye.

Telemedicine has emerged as a viable alternative to in-person consultations with doctors in many contexts because it saves patients time and expenses, which can be considerable if they are located in remote areas and/or are not well to do. But as more people pick this option, another advantage is coming to the fore: lower emissions.

Carbon footprint of healthcare delivery

Studies in high-income countries have shown that telemedicine is both a patient- and an environment-friendly means of healthcare service delivery. In India, where 70% of the population lives in villages, a hospital visit often requires expensive long-distance travel to urban centres, which imposes its own considerable carbon footprint.

Vehicular emissions are a major contributor to local pollution and global warming. In India, about 88% of the carbon dioxide emissions come from road traffic. Across cities alone, over a three-month period, the study found that teleconsultation led to 1,666 fewer kilometres of travel for patients and an average reduction of 176.6 kg of carbon dioxide emissions – figures the healthcare sector can’t afford to ignore.

According to one analysis, India’s healthcare sector emitted 74 million tonnes of carbon dioxide in 2014, around 3% of India’s total emissions of the gas that year. It is likely to have increased since: as the demand for health services increases, so too will the paradoxical harm to health due to their emissions.

“Every healthcare system should work towards carbon neutrality,” Padmaja Kumari Rani, the lead author of the study and network head of teleophthalmology at LVPEI, said. “Teleophthalmology is an efficient and effective tool that can help the eye health sector to achieve that goal.”

(Note: The author is affiliated with LVPEI.)

The teleophthalmology process

For the study, LVPEI researchers evaluated teleophthalmology, a specialised form of telemedicine that is customised for eye care.

In a teleophthalmology session, a patient remotely consults with an ophthalmologist over internet-based video chat. The teleconsultation is mediated through a smartphone app or facilitated by a technician at a primary healthcare centre. If the patient uses an app, they can book an appointment with a doctor, have an online consultation, and receive an e-prescription through the app.

“This system is primarily designed for follow-up patients, so they do not have to travel to a tertiary hospital for subsequent visits after treatment,” Dr. Rani said. “A lot of new patients tend to use teleconsultations for a second opinion before committing to a treatment plan at a hospital.”

If the teleconsultation is facilitated by an eye-care technician, the technician will first perform a comprehensive examination and take good-quality pictures of the eye, and upload the data to a server in the cloud. Many kilometres away, a doctor will download the data from the server, study them, and prescribe treatment or refer the patient to a higher-level hospital for additional diagnostics or treatment.

“Most Indians live in rural areas while most doctors operate from urban locations. This leads to a gap in health care access. Teleconsultations bridge this gap,” Dr. Rani continued. “By helping to defer travel, we can also save a significant amount of carbon emissions. All we need is a stable internet connection.”

Economic impact of teleophthalmology

The study involved 324 patients who received teleconsultations within a three-month period. This included 173 patients who visited LVPEI’s rural primary eye centres and 151 that visited urban tertiary hospitals. The researchers assessed their carbon footprint based on the type of transport the patients used to commute to the clinic. They also evaluated the economic impact using estimated cost savings from travel, food, and lost wages.

Patients at rural centres were tagged ‘green,’ ‘yellow’ or ‘red’ based on the severity and urgency of medical intervention required. Around 70% of such patients were tagged ‘green’ because they could benefit from a teleconsultation alone. The remaining 30% travelled to a hospital. Their travel and emission costs were used to validate the emissions and costs avoided by those tagged ‘green’. Patients in urban centres were classified as ‘new’ or ‘follow-up’; their modes of travel and costs were also evaluated and included in the study.

Half of the patients in rural areas (49.5%) said they would have travelled by bus, while 38.7% would have used a two-wheeler to access care. The researchers estimated that teleophthalmology saved 80 km of travel and reduced 2.89 kg of carbon dioxide emissions per rural-area patient on average. That translated to around 1.2 litres of petrol saved per person over three months (with an emission factor of 0.1135 kg of carbon dioxide per passenger per km).

The numbers were more pronounced for urban tertiary-care hospitals. Care-seekers from around India came to LVPEI’s tertiary centres in four southern Indian cities. Some 41% of them travelled by train; 19% flew; and 11% took buses. Each deferred patient visit saved an average of 1,666 km of travel and reduced carbon dioxide emissions by 176.6 kg over three months. Each decision to defer also saved around 76 litres of fuel.

Similarly, on average, each rural patient saved Rs 370 and each urban patient Rs 8,339 on travel expenses alone. When the researchers factored in indirect costs like food and lost wages, total savings among rural patients ballooned to Rs 29,100 and Rs 3.45 lakh among their urban counterparts.

“Patients with minor eye problems like mild refractive errors or regular preventive eye check-ups are the target demographic for teleconsultations,” Dr. Rani said.

Sayantan Mitra is a science writer at the L.V. Prasad Eye Institute.