Every year there are more than 7,00,000 deaths from diseases such as Malaria, Dengue, Schistosomiasis, Human African Trypanosomiasis, Leishmaniasis, Chagas disease, Yellow Fever, Japanese Encephalitis & Onchocerciasis according to the World Health Organization, Mar’20. With our modern sciences that are so evolving these days, can’t we get rid of this life threatening vector, the mosquitoes, totally vanishing off or eliminating from the world or by a similar kind of an approach that can prevent a mosquito withdrawing transmission of such diseases?
I think it will be possible one day if we can use gene editing tools more, like scientists used the CRISPR/Cas9 system in order to produce fertile offspring or an offspring which doesn't have the ability to spread diseases & can actually counter a lot of mosquito spread diseases. This could ultimately prevent millions of people and animals from falling ill because of this invasive mosquito species.
More recently, the development of the CRISPR-Cas9 [clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system 4,5,6] has radically simplified the process of engineering nucleases that can cleave specific genomic sequences. A guide RNA (gRNA) complementary to a DNA target site directs the activity of the Cas9 endonuclease to that sequence, providing a means to edit almost any chosen DNA sequence without the need to undertake complex protein engineering and selection procedures. In addition to applications in genome editing, the specificity and the flexibility of the CRISPR-Cas9 system offers unprecedented opportunities to expedite the development of gene drive systems for the control of insect vectors of disease.
What is CRISPR-Cas9?
CRISPR-Cas9 is a method of genome editing that exploits a natural DNA-snipping enzyme in bacteria, called Cas9 (CRISPR-associated protein 9) to target and edit particular genes. CRISPR stands for Clustered regularly interspaced short palindromic repeats, which are segments of DNA of a particular structure found widely in bacteria and archaea (prokaryotes). In the wild, the CRISPR-Cas9 system is part of the prokaryotic immune system, which can snip out of the genome DNA acquired from foreign sources such as phages (bacterial viruses). The same molecular machinery is now being used to enable genetic material to be cut from and pasted into the genomes of other organisms, including eukaryotes such as humans. It might offer a tool for curing genetically based diseases.
THE ASCENT OF CRISPR
The latest possibilities for editing DNA with pinpoint accuracy are transforming. DNA has become a versatile polymeric substrate for making nanotechnological structures and artificial molecular-scale machinery for computation, pattern formation, and nanoscale assembly. For several decades now, these efforts have drawn on methods developed in and for biotechnology, and similarly they are likely to find ways of exploiting the advantages of the new technique called CRISPR/Cas9 for manipulating DNA.
BACKGROUND TO THE PROBLEM
Already mentioned in the title of problem, we know a mosquito along with all its breeds in respect of its sexual orientation, is known to spread more than hundreds of diseases among human individuals, as well as some domestic animals. The World Health Organization estimates that India has 15 million cases of malaria with 19,500–20,000 deaths annually & only 2 million cases and 1,000 deaths reported. Even in a small place like Kolkata, in the last 10 years, it was reported about two emerging & re-emerging mosquito spread diseases such as Japanese Encephalitis & Dengue. Changing their sequences of genes, nucleotide changes and thereby becoming more functional to attract the vectors of certain diseases & causing a lifelong harm on the human population.
EXPERIMENT TO ADDRESS THE ISSUE
1. ReMOT Control Delivery of CRISPR-Cas9 Ribonucleoprotein Complex to induce germline mutagenesis in the disease vector mosquitoes Culex pipiens pallens.
– Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption can be a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for non-specialist laboratories. Here, we can adapt the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing is as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species.
2. Digital droplet PCR & IDAA for the detection of CRISPR indel edits in the malaria species Anopheles stephensi.
– In April 2020, Rebeca Carballar-Lejarazu et al. demonstrated and compare the insertions or deletions (indels) detection capabilities of two techniques in the malaria vector mosquito Anopheles stephensi : Indel Detection by Amplicon Analysis (IDAA) and Droplet Digital PCR (ddPCR). Both techniques showed accuracy and reproducibility for indel frequencies across mosquito samples containing different ratios of indels of various sizes. Moreover, these techniques have advantages that make them potentially better suited for high-throughput nonhomologous end joining analysis and contained field testing of gene-drive mosquitoes.
3. Highly efficient site-specific Mutagenesis in Malaria mosquitoes using CRISPR.
– Application of the CRISPR-Cas9 system for highly efficient, site-specific mutagenesis in the diverse malaria vectors like Anopheles albimanus, when guide RNAs (gRNAs) & Cas9 protein are injected at high concentration, germline mutations are common & usually biallelic.
4. Targeted delivery of CRISPR-Cas9 ribonucleoprotein into their ovaries for heritable germline gene editing.
– We know, as of now, current approaches rely upon delivering Cas9 RNP by embryonic microinjection, which is quite challenging & are very limited to a small number of species & sometimes even it is inefficient. As mentioned earlier, we can develop ReMOT control to deliver Cas9 RNP to their germline by injecting into female mosquitoes. Already described by Duverney Chaverra-Rodrigues et al. in 2018, that they identified a peptide P2C that mediates transduction of Cas9 RNP from the female hemolymph to the developing mosquito oocytes, resulting inheritable gene editing of the offspring as high as 0.3 mutants per injected mosquito.
Can DNA nanotechnology enhance CRISPR?
DNA nanotechnology expert Hao Yan of Arizona State University thinks that the benefits of CRISPR/Cas9 could be two-way: DNA nanotechnology, he says, might be harnessed to provide molecular machinery that enhances the performance of CRISPR/Cas itself. “I envision that the marriage of DNA nanotechnology with CRISPR might overcome some shortcomings of the CRISPR/Cas9 technology, such as the high off-target rates”—the tendency of Cas9 to hit the wrong sequence, which seems to be a challenge for safe use in humans. “DNA technology-mediated delivery of CRISPR/Cas9 might also provide new ways to probe cellular pathways by allowing specific knockin or knockout of target genes or regulatory elements,” Yan says.
Given that living organisms are currently being re-engineered in biotechnology and synthetic biology as “living factories” for making materials, there could also be indirect impacts of the new high-precision gene editing in materials technologies. Chemist Yamuna Krishnan of The University of Chicago sees a possibility to use CRISPR/Cas9 to “tailor organisms to metabolize inorganics and produce inorganic materials”—for example, retooling existing organisms that already process inorganics, such as magnetotactic bacteria and fungi, “to produce a panoply of inorganic nanoparticles in a truly ‘green’ way.” Just as synthetic biologists are hoping to rewire microorganisms to make biofuels, hydrogen gas, and other useful chemical commodities, microorganisms might also be designed for constructing complex inorganic and composite functional materials. CRISPR/Cas9 seems sure to facilitate such efforts.
In short, it seems that the collaboration of CRISPR/Cas editing and artificial DNA-based nanosystems and materials will be limited only by our imaginations. As with any tool, the challenge is to figure out creative ways of using it.
APPROACH
Gene disruption by homology-directed repair (HDR) at three separate loci causes recessive female fertility. AGAP005958, AGAP011377 & AGAP007280 - these three genes confer a recessive female sterility phenotype upon disruption & if inserted into each locus CRISPR-Cas9 gene drive constructs designed to target & edit each gene. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission. (A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.
EXPECTED OUTCOMES & ALTERNATIVE STRATEGY
Although this is a very costly technique & can only be performed by scientists, researchers & professionals, this can be a gamechanger in modern science. We know already many workers had actually started working on it & to a certain extent, they are successful, but the real problem rises here i.e. across the globe there are over 3,500 different species of mosquitoes & approximately 10 to 15 billion of mosquitoes are parturated daily & some undergoing mutations in their genome on a large basis & then to make infertile of each & every species of them are quite tough to be honest.
Note
This is not a research article. This is just a demonstration of CRISPR-Cas9 system & it's implications that can be useful in future.
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