CRISPR, an emerging scientific tool allows one to edit the genome (genetic makeup) of an organism, in a bid to produce more desirable characteristics, suppress detrimental features or study genetic mutations in the organism. Although, it is still being explored for the seemingly endless possibilities it claims to offer, scientists have great hope and optimism in the health potential of this tool, having obtained favourable results from most studies carried out. Beyond its health-based benefits, the CRISPR tool also finds useful applications in agriculture and industry.
Understanding how scientists put this tool to use – in diagnosing, treating and unveiling the genetic basis of a wide range of diseases in humans – is like watching a seamstress go through her daily routine of designing, cutting and sewing. As a matter of fact, the CRISPR tool is more or less ‘the science fashion tool’ of the twenty-first (21st) century; researchers now rely heavily on and employ its pristine ability to study, design, edit, cut, sew, merge and fix whole genomes and organsims. With its advent, transforming and re-creating the human race has never been more realistic.
The genome is the special coded language that carries information responsible for the features and functions of an organism. It is found in all living things, often as a single-stranded (RNA) or double-stranded (DNA) string of nucleotides that code for amino acids. Each amino acid is encoded by a codon (a group of three nucleotides). Amino acids are the building blocks of proteins – macromolecules that play structural, functional and regulatory roles in living things. A change to the nucleotide sequence of the genome of an organism (mutation) may have far-reaching consequences that affect the health and survival of the organism.
CRISPR stands for ‘clustered regularly-interspersed (interspaced) short palindromic repeats’. It was first discovered in the 1980’s by serendipity, and was later deduced to be a bacterial antiviral ‘immune memory-recognition system’, used in the storage and recognition of viral genetic information (RNA). If a virus (bacteriophage) infects a bacteria, a portion of the viral RNA is stored in the bacterial genome (as CRISPR), and subsequently used to identify attacks from similar viruses. The CRISPR-associated protein (Cas9) is a bacterial enzyme that complements the CRISPR system. It attacks the invading virus by cleaving (cutting) its RNA, using information stored in the CRISPR region of the bacteria. Consequently, the CRISPR tool is better christened, a CRISPR-Cas9 ‘immune system’ tool in bacteria; well, in humans, it means even more.
The CRISPR-Cas9 revolution that birthed its application in gene editing was kick-started in 2012 by researcher, Jennifer Doudna, who first proposed its apparently precise genome-editing ability, as opposed to the then existing time-consuming, labour-intensive and error-ladened techniques. For the past seven (7) years, it has evolved by leaps and bounds, with its most recent derivative being, the ‘Prime Editing’ technique. So far, eager, inquisitive researchers have sacrificed several sacred cows in the world of science and medicine, going above and beyond what was termed acceptable or possible a few years or decades ago.
Before taking you through this episode and its sequel, it would not be so bad an idea to get a little bit more familiar with the themes and terms that punctuate the CRISPR terrain.
| GENOME: The genome is another term for the genetic material of an organism. It is often composed of DNA, except in some viruses with an RNA genetic material. The genome is more or less a lengthy string of ‘coded’ beads found in every cell of all living things. It carries the information – transferable from parents to offsprings – responsible for the features and functions of an organism.
| DNA: DNA stands for DeoxyriboNucleic Acid. It is the major genetic material in humans and most organisms. The DNA may be likened to a long string of four (4) beads – Adenine (A), Thymine (T) Guanine (G) and Cytosine (C) – paired (A – T; G – C) in a double-stranded helix. A, T, G and C are the ‘alphabets’ of the DNA code.
| RNA: RNA (RiboNucleic Acid) is an analog of the DNA. Apart from being the major genetic material in some viruses, it is also present in most other organisms as a DNA derivative. As in the DNA, the ‘RNA code’ also boasts of four (4) beads; however, it is single-stranded, and substitutes Uracil (U) for the Thymine (T) of DNA.
| NUCLEOTIDE: Nucleotides are the basic structural units of nucleic acids (DNA and RNA). They are derivatives of the hypothetical beads (A, T, G, C and U) in DNA and RNA. A typical nucleotide is made up of a nitrogenous base (A, G, C, T or U), a sugar moiety (Deoxyribose – DNA; Ribose – RNA) and a phosphate group. Take away the phosphate, and it becomes a ‘NUCLEOSIDE’. Nucleotides often associate in triplet combinations to form ‘CODONS’; each codon encodes a signal (Stop) or an amino acid. There are twenty (20) of such amino acids that link up (just like the nucleotide ‘beads’ of DNA or RNA) in long, sometimes branched string-like sequences to yield a ‘POLYPEPTIDE’, the final product of ‘GENE EXPRESSION’.
| GENE: A gene is the basic functional unit of the genome. Genes are made up of several nucleotide codons, which can be ‘expressed’ into an array of functional, structural and regulatory products (usually RNA or polypeptides).
| GENE EXPRESSION: The process of decoding the nucleotide sequence of a gene is known as ‘gene expression’. Gene expression proceeds in two phases: TRANSCRIPTION (the conversion of the nucleotide sequence in DNA to RNA) and TRANSLATION (the conversion of the nucleotide sequence in RNA to its final product, the amino acid sequence of a polypeptide). It is highly regulated and coordinated to allow for little or no errors (mutations), especially when such errors could compromise the normal functions and features in an organism.
| MUTATION: This is a permanent change to the nucleotide sequence of the DNA or RNA of an organism. Mutations may result from ‘mutagenic’ extrinsic factors such as radiation and certain chemical substances; or from ‘intrinsic’ factors while the cell makes copies of its DNA. Because of the high degree of specificity and accuracy required for an ideal gene expression, most organisms have an imbuilt machinery for identifying, correcting and reversing mutations to their DNA. Although most mutations are harmless, some beneficial modifications allow for slight variations in a family of organisms, sometimes conferring favourable features in these organisms. Cancers and other genetic diseases represent some of the detrimental effects of those mutations that were never identified, corrected or reversed. Substitution (point mutation), Deletion and Insertion of nucleotides are the major types of mutation. Most genetic diseases result from point mutations, and there are twelve (12) possible forms of such mutations.
| GENE-EDITING: Gene-editing is more or less the act of introducing deliberate mutations into the genome of an organism. Unlike conventional spontaneous mutations, these modifications to an organism’s genome are meticulously controlled to yield a desired result. By editing the genome, scientists and researchers are able to study the exact functions of various genes; understand the effects (both detrimental and beneficial) of mutations; and reverse or bypass an unfavourable mutation in order to produce improved features or functions in an organism.
Well, we could try to connect all these terms, strange as they may appear, in a sturdy framework that lightens the whole weight of information. The GENOME, composed of DNA (or RNA), is a long string of NUCLEOTIDES that carry information for the overall characteristics of an organism. Certain regions of the genome (GENES) encode products (often POLYPEPTIDES – long strings of amino acids) with a variety of roles. Errors in the genome are known as MUTATIONS, and may be corrected via GENE-EDITING tools and techniques.
Building upon this framework, one can readily picture the genome as a complex design of myriad threads, colours and styles; and CRISPR-Cas9 as an editing tool that can be employed to preserve the integrity of this design. For instance; if a ‘mutant’ (mutated, error-ladened) form of this design were reproduced, the CRISPR-Cas9 tool acting like a ‘scissors’ identifies and makes a cut at the point of mutation, so that relevant corrections could be effected. The Cas9 (CRISPR-associated protein) component of the CRISPR tool is a nuclease (a kind of biological scissors); it cuts nucleic acids, DNA and RNA at specific sites, guided by a special short RNA sequence (the guide RNA, gRNA). It is this ability for specificity especially, that has brought the CRISPR tool to its current position of relevance in genetics-based research.
Nevertheless, neither the CRISPR-Cas9 tool, nor its gene-editing technique is void of downsides. The error-prone, off-target and double-strand cuts, and somewhat limited gene-editing capacity of the CRISPR tool has however, been bypassed in the recently modified ‘Prime Editing’ technique .
Apart from its improved ability for on-target and single-strand cuts, ‘prime editing’ boasts of a wider gene-editing spectrum (covers all 12 forms of genomic point mutations and up to 89% of all disease-causing genetic mutations), with greater versatility, specificity and accuracy than CRISPR-Cas9. Other outstanding features of the prime editing technique are; a special prime editing guide RNA (pegRNA) – identifies the target site and encodes the edit(s) to be effected – and a reverse transcriptase.
LEARN A NEW WORD
MUTATION: This is a permanent change to the nucleotide sequence of the DNA of an organism. It usually results from insertion, deletion, modification or substitution of nucleotides in the DNA during replication or as a result of external agents such as radiation. Most organisms have inbuilt mechanisms for identifying, correcting and reversing mutations to their DNA. However, certain favourable mutations that help an organism adapt better to and survive in its habitat may be left unmodified. The often lifelong ‘genetic diseases’ result from unfavourable mutations that affect the functional or structural features of an organism. Some of the most common genetic diseases in humans are; Sickle Cell disease, Huntington’s disease, Cystic Fibrosis, Phenylketonuria, etc. Of all the available treatment and management options for dealing with genetic diseases, replacing or repairing an abnormal gene via the CRISPR-Cas9 gene editing technique, remains one of the most promising and formidable.
GUIDE RNA (gRNA): The guide RNA (gRNA) is a mini-RNA sequence of about 20 nucleotides that, as its name implies, ‘guides’ the CRISPR-Cas9 tool to the correct cut site on the DNA. It is often designed to be complementary in sequence to the target cut site, such that it is able to bind the DNA at this site. Off-target cuts in CRISPR has been linked to this sequence complementarity between gRNA and DNA, since gRNA may bind to any complementary sequence on the DNA outside the target cut site. This limitation however, has been improved upon in the prime-editing guide RNA (pegRNA) which both targets and encodes modifications to the cut site.
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