The Golden Road to CRISPR/Cas9

From The Enigmatic Sequence in Escherichia coli to Human Genome Editing

This essay by Sonali Pal was submitted for the 2019 NxG Science Communicator Competition.

The advancements in the field of science in the late 1970s marked a novel frontier in what we call genetic engineering today. Here, DNA is inserted, deleted and modified/replaced in the genome of a living organism. This field has demonstrated a commitment to game-changing discoveries while adhering to a careful set of moral principles and convention. Homologous recombination is the most primitive methods of gene modification. It is broadly used in mouse embryonic stem cells to generate germline knock-out or knock-in mice. A major setback of using this standard approach is that it takes more than a year to generate a genetically modified mouse. Furthermore, this approach has been extended to human cells that appear to be far more challenging. A range of alternative approaches available that rely on the principle of site-specific recognition of  DNA sequences by antisense oligonucleotides, small molecules or short interfering RNAs that function to knockdown gene expression. These approaches often affect off-target genes and offer transient gene expression. To address these scientific issues, genome-editing technology emerged, which amalgamates the work of a plethora of scientists across the world. This technology imparts the ability to specifically and proficiently introduce an array of genetic alterations into mammalian cells, ranging from knock-in of single nucleotide variants to insertions of genes to deletion of chromosomal regions.

The past decade has witnessed some of the rapid innovations in genome-editing technology marked by the birth of popular gene-editing tools using engineered nucleases, i.e. zinc finger nuclease (ZFN) in 1985, transcription activator-like nucleases (TALEN) in 2011 and engineered meganucleases. These methods were awarded as methods of the year in 2011 by Nature methods. However, difficulties like the design of the protein, its synthesis, and validation remained a barrier to widespread adoption of these methods for routine use. The advent of clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 CRISPR/Cas9 triggered a revolution in the scientific community because it was cheaper, faster, and more efficient than other existing genome editing methods. The CRISPR/Cas9 system was awarded as the breakthrough of the year in 2015 by Science. It is the most reliable and versatile technology that provides the ability to add or remove DNA in the genome in a sequence-specific manner.

The journey of the CRISPR/Cas9 system ranges from its initial discovery in 1987 when a mysterious repetitive DNA sequence was discovered in E.coli to the first manifestation of CRISPR-mediated genome editing in eukaryotic cells in  2013. It is a collective contribution of several scientists across the globe that pushed this discipline forward. The CRISPR/Cas9 discovered by Francisco Mojica is a prokaryotic, adaptive immune system in nature whereby pieces of DNA from invading viruses are snipped off by a bacterial nuclease, CRISPR associated protein (Cas 9). The DNA fragment that is sliced off is saved as a memory for fighting future infections. In 2012, Jennifer Doudna and Emmanuelle Charpentier, revealed the biochemical mechanism of CRISPR technology. The CRISPR-Cas9 system acts on target DNA and can be engineered to edit eukaryotic DNA by designing guide RNA complementary to the target sequence. In 2013, Feng Zhang was the first to successfully adapt CRISPR-Cas9 for genome editing in eukaryotic cells.

During the phase when the CRISPR/Cas9 technique was in its infancy and the data was still emerging. A huge revelation of CRISPR/Cas9 application was the birth of the genetically-modified human babies. On 26 Nov 2018, a scientist in China named He Jiankui created Lulu and Nana in order to impair the gene for CCR5, which codes for a receptor that HIV uses to enter into the cells. The twins still carried both the functional and the disabled copies of CCR5 and were still vulnerable to HIV. However, facing controversies about ethics and safety of the work, it was widely condemned that germline cell and embryo genome editing is against the law.

The landmark work done on CRISPR/cas9 opens the door to a new era of its potential applications. It is used in the prevention and treatment of human diseases including single-gene disorders like cystic fibrosis, Duchenne’s muscular dystrophy (DMD) and in haemoglobinopathies like haemophilia, and sickle cell disease to more complex disease such as HIV and cancer. Nevertheless, significant challenges could be the choice of the delivery system, its efficacy, and the possibility of off-target effects. Recently, a new DNA editing tool more precise than CRISPR surfaced named, ‘prime editing’. It has raised fresh hopes in the treatment of genetic disorders. It can mend about 89% of the 75,000 or so harmful mutations.

CRISPR/Cas9 system has diversified stem cell research. Earlier, conventional genetic methods such as homologous recombination to study stem cells were time-consuming and costly but the arrival of the CRISPR/Cas9 system offered a remarkable plan of action to perform efficient and convenient genome engineering.

CRISPR-Cas biology has played an important role in the advancement and development of quick and precise infectious disease diagnostics. For instance, nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique combined with CRISPR-Cas9 can be used to differentiate between closely related Zika virus strains in vitro and in a macaque model.

CRISPR/Cas9 techniques leave no stone unturned, it has provided ample opportunities for plant biotechnologists to work on crop improvement. It has also been used to improve biotic and abiotic stress tolerance which results in the development of non-genetically modified (Non-GMO) crops with the desired trait. Moreover, notable areas for development will be the production of crop with enhanced yield, nutritional value, disease resistance and other traits.

In 2018, there were more than 17,000 papers with refinements to CRISPR, new manoeuvres for manipulating genes, improvements in precision and more. It has revolutionized everything from medicine to agriculture. CRISPR/Cas9 has been an important scientific issue in the past decade and will be the biggest science story for the next few years.

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