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Gene editing goes mainstream

IFM_ Gene editing
The idea of using gene editing to treat disease or alter traits dates back at least to the 1950s, when the double-helix structure of DNA was discovered

Medical innovations typically take 17 years, from the moment a lightbulb lights up in a scientist’s mind to the first person to benefit from it. But every once in a while, an idea is so powerful and profound that its effects are felt much more quickly. That was with CRISPR gene editing, which celebrated its 10th anniversary in May 2023. It has already had a major impact on laboratory science, improving precision and accelerating research, and leading to clinical trials for a handful of rare diseases and cancers. Scientists predict that over the next decade, CRISPR will spawn several approved medical treatments that will be used to modify crops, making them more productive and more resilient to disease and climate change.

What are Gene editing and CRISPR?

Gene editing is the ability to make highly specific changes in the DNA sequence of a living organism, essentially altering its genetic makeup. Gene editing is done using enzymes, specifically nucleases, which have been engineered to target a specific DNA sequence, where they make cuts in the DNA strands, allowing the removal of existing DNA and the insertion of replacement DNA. Key to the gene editing technologies is a molecular tool called CRISPR, a powerful technology discovered in 2012 by American scientist Jennifer Doudna, French scientist Emmanuelle Charpentier and refined by American scientist Feng Zhang and colleagues.

The CRISPR worked precisely, allowing researchers to remove and insert DNA at desired locations. Significant advances in gene-editing tools have lent renewed urgency to long-standing debates about the ethical and social implications of genetic engineering in humans. Many questions, such as whether genetic engineering should be used to treat human diseases or alter traits such as beauty or intelligence, have been asked in one form or another for decades. However, with the advent of simple and efficient gene editing technologies, notably CRISPR, these questions were no longer theoretical and the answers to them had very real implications for medicine and society.

The idea of using gene editing to treat disease or alter traits dates back at least to the 1950s, when the double-helix structure of DNA was discovered. In the mid-20th century, researchers realized that the base sequence in DNA is mostly passed from parent to offspring faithfully, and that small changes in sequence can mean the difference between health and disease. The realization of the latter led to the inescapable assumption that with the identification of molecular flaws that cause genetic diseases, there would be an opportunity to correct those flaws and thereby enable the prevention or reversal of diseases. This thought was the basic idea behind gene therapy and was considered the holy grail of molecular genetics from the 1980s onwards.

However, developing gene editing technology for gene therapy proved difficult. Many early advances did not focus on correcting genetic errors in DNA, but on attempting to minimize their consequences by providing a functional copy of the mutated gene, either inserted into the genome or as an extrachromosomal unit (outside the genome). While this approach was effective for some disorders, it was complicated and limited in scope. To truly correct genetic errors, researchers had to be able to create a double-stranded break in the DNA at precisely the desired location in the more than three billion strands of DNA that make up the human genome. Once the double-strand break was created, the cell could efficiently repair it using a template that directed the replacement of the defective sequence with the good sequence. However, making the initial break at precisely the desired location—and nowhere else—within the genome was not easy.

Breaking DNA at desired locations

Before the introduction of CRISPR, two approaches were used to generate site-specific double-strand breaks in DNA: one was based on zinc finger nucleases (ZFNs) and the other was based on transcription activator-like effector nucleases (TALENs). ZFNs are fusion proteins composed of DNA-binding domains that recognize and bind to specific three to four-base pair sequences. For example, to confer specificity on a nine-base pair target sequence, three back-to-back fused ZFN domains would be required. The desired arrangement of DNA-binding domains is also fused to a sequence encoding a subunit of the bacterial nuclease Fok1. To enable a double-stranded cut at a given site, two ZFN fusion proteins must be constructed, one binding to opposite strands of DNA on either side of the target site. When both ZFNs are bound, the neighbouring Fok1 subunits bind to each other and form an active dimer that cuts the target DNA on both strands.

TALEN fusion proteins are designed to bind to specific DNA sequences flanking a target site. But instead of zinc finger domains, TALENs use DNA-binding domains derived from proteins from a group of plant pathogens. For technical reasons, TALENs are easier to construct than ZFNs, especially for longer recognition sites. Similar to ZFNs, TALENs encode a Fok1 domain fused to the engineered DNA-binding region. Thus, once the target site is bound on both sides, the dimerized Fok1 nuclease can introduce a double-strand break at the desired DNA position.

Unlike ZFNs and TALENs, CRISPR uses RNA-DNA binding instead of protein-DNA binding to drive nuclease activity, simplifying design and allowing the application to a wide range of target sequences. CRISPR was derived from the adaptive immune system of bacteria. The acronym CRISPR refers to clustered, regularly spaced short palindromic repeats found in most bacterial genomes. Between the short palindromic repeats are sequence sections that clearly originate from the genomes of bacterial pathogens. Older spacers are located at the distal end of the cluster and newer spacers representing newer pathogens are located near the proximal end of the cluster.

Transcription of the CRISPR region results in the production of small guide RNAs containing hairpin formations from the palindromic repeats linked to spacer-derived sequences, allowing each to bind to its respective target. The formed RNA-DNA heteroduplex then binds to a nuclease called Cas9, directing it to catalyze the cleavage of double-stranded DNA at a position near the junction of the target-specific sequence and the palindromic repeat in the guide RNA. Because RNA-DNA heteroduplexes are stable and because designing an RNA sequence that specifically binds to a unique target DNA sequence requires only knowledge of the Watson-Crick base pairing rules (adenine binds to thymine [or uracil in RNA], and cytosine binds to guanine), the CRISPR system was preferable to the fusion protein designs required to use ZFNs or TALENs.

Another technical advance came in 2015 when Zhang and colleagues reported using Cpf-1 instead of Cas9 as a nuclease pairing with CRISPR for gene editing. Cpf-1 is a microbial nuclease that offers potential advantages over Cas9, including the need to only require a CRISPR guide RNA for specificity and to make staggered (rather than blunt) double-stranded DNA cuts. The altered nuclease properties may have allowed better control over the insertion of surrogate DNA sequences than was possible with Cas9, at least under certain circumstances. Researchers suspect that bacteria also harbour other genome-editing proteins.

CRISPR to treat cancer

CRISPR has the potential to improve cancer treatment, by boosting the immune system, It has been utilised in blood cancer patient trials since 2016 to alter the patients’ own immune cells outside of the body to start an immunological attack on the malignancy. Multiple forms of blood cancer have been successfully treated using this strategy, known as CAR-T. Until recently, CAR-Ts had to be manufactured specifically for each patient, which required resources that some patients may not have.

According to Rachel Haurwitz, CEO, president, and co-founder of the company alongside Doudna, Caribou Biosciences is working on an “off-the-shelf” version of the medication that will be available in a freezer for the following patient who requires it. Weeks of preparation time and possible costs would be reduced in this way. CRISPR has the potential to improve cancer treatment. It has been used in blood cancer patient trials since 2016 to alter the patients’ own immune cells outside of the body to start an immunological attack on the malignancy. Multiple forms of blood cancer have been successfully treated using this strategy, known as CAR-T. Until recently, CAR-Ts had to be manufactured specifically for each patient, which required resources that some patients may not have.

A single genetic “misspelling” is responsible for more than 6,000 uncommon inherited disorders. For these, CRISPR gives the option of eliminating the problematic gene, boosting an alternative gene, or changing the genetic “letters” that are creating issues. Later this year, the first CRISPR-based gene treatment for sickle cell disease is anticipated to receive approval, USA Today reported.

Applications and controversies

CRISPR has been used in a variety of ways. For example, it has been applied to early embryos to create genetically modified organisms and injected into the bloodstream of laboratory animals to achieve extensive gene editing in subsets of tissues. CRISPR-based approaches have been used to alter the genomes of crop plants, livestock, and laboratory model organisms, including mice, rats, and non-human primates. By modifying the genomes of bacteriophages (bacteria-killing viruses) with CRISPR technology, scientists have been able to develop methods to destroy antibiotic-resistant bacteria. CRISPR systems also enabled the creation of animal models of human diseases and the removal of HIV from infected cells. In a mouse model of human disease, CRISPR-Cas9 was successfully used to correct a genetic error, resulting in the clinical rescue of diseased mice.

In 2015, a group of scientists that included Doudna advocated caution in applying CRISPR-Cas9 technology to humans, at least until the safety and ethical implications of human gene editing could be properly considered. Other researchers advocated going full steam ahead, arguing that the new technology held the key to alleviating many human ailments and that it would be unethical to withhold it. Around the same time, reports from China indicated that gene-editing experiments had been carried out on human embryos. In late 2018, twin girls were born in China with an altered gene that lowers HIV infection risk, making them the world’s first genetically engineered human babies. The implications of this breakthrough were seen as potentially redefining the future of human genetics.

CRISPR gene editing is revolutionizing genetic engineering, with the potential for medical treatment and agriculture. Despite ethical debates, CRISPR has shown promise in laboratory science and clinical trials due to its precise DNA-editing capabilities. Expect significant impact in the future with approved medical treatments and advancements in various fields.

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