Seven-month-old Kyle “KJ” Muldoon Jr was born with a life-threatening rare genetic disorder. This disorder was caused by a mutation in a gene called carbamoyl phosphate synthetase 1 (CPS1). This mutation impaired his ability to process proteins, leading to toxic ammonia buildup and brain damage. More than half of infants affected by this disorder die before their first birthday, and survivors often face severe developmental delays.
In response to KJ’s urgent case, precision genome editing medicine recently made history. Within seven months, a collaboration of 11 academic and industry teams of researchers developed the first customised genome-editing therapy tailored for KJ. They designed genome-editing agents, engineered delivery vehicles, and completed manufacturing, safety testing, and regulatory approvals.
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This prompts inquiry into how biological organisation works, from DNA to the whole body. Let’s understand.
Human body as an enormous architectural complex
The human body is like an enormous building composed of 37 trillion cells, each comparable to a room. These cells are organised spatially and functionally into tissues, organs, and finally, the body itself – just as rooms combine to form living units, corridors, and the entire building.
Each cell contains the genome: an approximately three-billion-letter sequence written in four alphabets (A, T, G, C) divided among 23 paired chromosomes, equivalent to a building’s design plan divided in 46 volumes (23 pairs) stored in every room of the building.
Now each cell is dedicated to a specific task and contains specialised proteins – the cell’s functional “devices”. It is analogous to each room containing particular equipment suited to its functions, just as cells contain distinct proteins to perform specific roles.
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The instructions for assembling each protein are encoded in genes, the functional units of DNA. One might imagine a computer monitor as a protein coded by a single gene, while a complex machine composed of multiple interacting parts would correspond to several coordinated genes.
Genetic diseases are in our DNA?
Proteins are polymers of 20 amino acids, which are assembled in specific gene-coded sequences of varying lengths to form their primary structure. Each amino acid is specified by contiguous three-letter words called codons (e.g., GCG codes for alanine). Codons are degenerate: multiple codons can encode the same amino acid. Of the 64 possible codons, 61 specify amino acids, while stop codons mark the end of a protein.
Point mutations – changes in any of the three letters within a codon – can have different consequences: some are silent, others substitute one amino acid for another (chemically equivalent or non-equivalent amino acid), and some introduce an early stop codon that truncates a protein. Insertions or deletions (INDELs) can further shift the reading frame, producing nonfunctional or even toxic proteins.
In addition, mutations may arise spontaneously or be induced by external factors like radiation, xenobiotics, or pollutants. Although cells’ repair systems constantly correct errors, the genome’s vast size makes mutations inevitable. Therefore, genetic diseases are an intrinsic consequence of our molecular structure.
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For example, spontaneous deamination of cytosine, which converts a C·G pair into an A·T pair, accounts for nearly half of all pathogenic human mutations. More than 96 per cent of observed human genetic variations are single-nucleotide variants (SNVs), and about 99 per cent of them still await clinical interpretation.
Key principles of genome editing for precision medicine
At the organismal level, correcting pathogenic mutations is challenging. Edits must occur in a sufficient proportion of cells for the corrected ones to dominate replication and replace unedited cells.
Only minimal, non-toxic doses of editing agents can be administered, and these must achieve high precision by minimising off-target effects and random insertions or deletions that could disrupt genes. Delivery vehicles must be engineered to reach the target organ safely, while ensuring that genome editors themselves are non-toxic and do not provoke immune reactions or other side effects.
Current genome-editing systems repurpose molecular tools that evolved in other organisms for unrelated functions. For example, the transcription activator-like effector (TAL) from the plant-infecting bacterium Xanthomonas has repeat-variable diresidues (RVDs) that can be reprogrammed to recognise specific DNA sequences. When fused to a non-specific nuclease that cuts DNA, it forms a programmable nuclease known as TALEN.
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Similarly, the CRISPR-Cas system originates from bacterial adaptive immunity against viruses. A Cas nuclease, guided by a programmable RNA sequence (gRNA), can target virtually any DNA locus for cleavage.
Mutated versions, such as dead-Cas9, retain DNA-binding ability but lack nuclease activity. When fused to enzymes like cytosine deaminase, they enable base editing, converting cytosine to uracil within a narrow single-stranded DNA window without generating double-strand breaks.
The backbone of genome editing
Advances in protein engineering and structural biology have been central to the development of genome-editing tools. Understanding recurring sequence patterns, or motifs, that produce conserved structural folds in proteins has allowed scientists to reprogramme natural protein complexes for new functions.
For instance, haemoglobin – the oxygen-carrying protein that gives blood its red colour – contains 574 amino acids arranged in four chains, each folded into an identical three-dimensional structure. This remarkable precision, repeated trillions of times in the human body, exemplifies the stability motifs can confer.
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Motif analysis also underpins the design of DNA-binding proteins, such as Cas nucleases, where specificity depends on the DNA sequence recognized, the binding length, tolerance to mismatches, and off-target propensity.
Modern genome-editing
Modern genome-editing systems apply these insights, and can be broadly categorised into two main types:
Base editors (BEs): They combine the CRISPR – Cas targeting system with enzymes that chemically convert one DNA base pair into another – either C.G to T.A base editors (CBEs) or A.T to G.C base editors (ABEs).
Classical CRISPR Editors: Cas nucleases make double-strand breaks (DSBs) in DNA. The cell repairs these breaks through non-homologous end joining (NHEJ) – an error-prone machinery that introduces insertions or deletions (INDELs) and knocks out unwanted, toxic genes. Alternatively, an additional DNA molecule can be provided to perform homology directed repair (HDR) to obtain precise genome editing. However, HDR is always outcompeted by NHEJ and is limited to specific periods of cell cycles.
Moreover, a representative CBE is a multiprotein fusion of Cas9 from bacteria streptococci, single strand specific cytidine deaminase from rat (rAPOBEC1) followed by to two tandem uracil glycosyl inhibitor (UGI)- a protein derived from bacterio-phage (a virus that attacks bacteria) connected through flexible 9 amino acid linkers.
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Bipartite nuclear localisation signal sequence (bpNLS) on both terminals targets the whole complex to nuclear compartment of the cell. ABEs similarly consists of mutant and wild type adenosine deaminase from human gut bacteria Escherichia coli, Cas protein and bpNLS attached through 32 amino acid linkers.
The engineered complexes are further refined by a method called directed evolution, where cloning the complex of interest in a bacterium and challenging it against selection pressures e.g. higher antibiotic resistance give rise to beneficial mutations. For example, adenine/adenosine deaminase exits naturally for RNA not for DNA- total seven rounds of directed evolution were performed to identify 14 mutations to obtain DNA specific adenine deaminase.
Compared to permanent nature of DNA editing, RNA editing has the flexibility of being reversible and can install epi-transcriptomic modifications. Rigorous research have led to 10-100-fold lower off-targeting by these complexes which otherwise is caused due to base editors themselves, 5-fold lower CRISPR off-targeting for CBEs, incorporation of circular permutant Cas proteins in CBE and ABE to address differential accessibility of genome locus by these complexes and the problem of by-stander mutation have been tackled by using a slow reacting deaminase.
How genome editing reaches cells and concerns it raises
Chemical delivery vehicles (cationic lipids and nanoparticles) and viral delivery vectors (Adenovirus, lentivirus, Sendai virus and retrovirus) are common delivery vehicles for genome editors, though some can trigger adverse immunogenic response and hepatotoxicity.
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High-voltage electroporation is also a powerful tool in genetic engineering. It is primarily used for ex-vivo (outside the body) applications. Here, cells are edited and prepared in a test-tube and subsequently transplanted onto recipient tissues.
Several in-vivo (inside the body) genome-editing therapies are in various stages of clinical trials for diseases such as familial hypercholesterolemia and other atherosclerotic cardiovascular diseases, glycogen storage disorders and α1-antitrypsin deficiency. Ex vivo and prime-editing trials are also underway for T-cell leukemia, sickle cell disease, and β-thalassemia.
While progress is underway, widespread access to genome editing also raises several concerns such germline modifications can permanently enter the natural gene pool, and the pursuit of enhanced traits or “designer” traits may introduce profound clinical, ecological and social risks.
Post read questions
What is precision genome editing medicine? What is the function of the CPS1 gene, and how does its mutation lead to ammonia buildup?
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What types of delivery vehicles are used for genome editors, and what are their relative advantages and limitations?
Genome editing technologies can blur the boundary between therapy and enhancement. Comment and suggest ways forward.
Analyze how insights from structural biology and protein motif analysis have contributed to engineering tools like Cas9 and TALENs.
How might social perceptions of “designer genetics” influence regulatory and policy decisions about genome editing?
(Dr. Arunangshu Das is the Principal Project Scientist at the Centre for Atmospheric Sciences, Indian Institute of Technology, Delhi.)
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