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Role of CRISPR Technology in Gene Editing of Emerging and Re-emerging Vector Borne Disease

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Kaushal Kumar Mahto, Pooja Prasad, Mohan Kumar, Harshita Dubey and Amar Ranjan

Submitted: January 29th, 2022Reviewed: March 1st, 2022Published: April 23rd, 2022

DOI: 10.5772/intechopen.104100

Mosquito Research - Recent Advances in Pathogen Interactions, Immunity, and Vector Control...Edited by Henry Puerta-Guardo

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Mosquito Research - Recent Advances in Pathogen Interactions, Immunity, and Vector Control Strategies [Working Title]

Dr. Henry Puerta-Guardo and Dr. Pablo Manrique-Saide

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Vector borne diseases are rampant across the world. Due to spread and establishment of vector species in different geographical areas, vector adaptation and resistance towards many insecticides the only option left is vector control for various vector borne diseases. Recent advancement in the field of genome editing have provided a variety of tools like, CRISPR, a novel genome editing techniques which can be applied for the control and prevention of many deadly diseases like dengue, chikungunya, filariasis, Japanese encephalitis and Zika. The present chapter is aimed to discuss the recent advancement in genome editing tools such as, their application, challenges, and limitations in vector control. Additionally, this chapter would potentially be advantageous to understand the hurdles, knowledge gaps in eliminating vector borne disease.


  • gene editing
  • vector borne disease
  • insecticide
  • resistance

1. Introduction

Vector-borne disease account for more than 17% of all infectious diseases, affecting approximately 700,000 people each year and dengue alone accounts for more than 3.9 billion people in 129 countries [1]. Mosquitoes are becoming increasingly resistant to insecticides and antimalarial drugs, necessitating the development of new methods to combat the disease as the gap between frequent outbreaks has been decreasing now. Children are very prone to malaria and children under the age of five account for the majority of fatalities. To combat vector borne diseases scientific communities have been working for decades and have successfully developed several techniques like sterile insect technique (SIT), precision-guided sterile insect technique (PgSIT), Zink Finger (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPER). CRISPR is a novel gene editing technique in the scientific community developed by Emmanuelle Charpentier of France and Jennifer A. Doudna of the United States who were awarded the Nobel Prize 2020 in the field of chemistry for discovering the CRISPR/Cas9 genetic scissors. CRISPER technique was initially introduced by Ishino et al. in the year 1987 [2] and has been widely used in genome editing in mosquito species for a number of years [3, 4, 5]. CRISPER-Cas9 based genome editing has emerged as one pf the most effective, diverse, and adaptive technique for gene editing.

Countries like Saudi Arabia, Turkey, Korea, Philippines, India, USA, Europe, China, and Japan are using crisper technique for combating much vector borne disease (Figure 1). Based on the burden of mosquito vector associated disease across various geographical areas there is an immediate requirement of advancement in these techniques (Table 1).

Figure 1.

Countries using crisper as gene editing tool in different areas.

Mosquito vector speciesDiseaseDistributionBurdenVaccine availabilityInterventionReferences
AedesChikungunya, Zika, dengue lymphatic filariasis, Rift Valley fever, yellow feverCentral and South America, Asia, Africa, South Eastern United State390 million (DEN), Avr 106,000 (CHK, ZK), Approx. 200,000 (YF), 859 million (LF)NODEET, IR3535 or icaridin, elimination of breeding site[6]
AnophelesLymphatic filariasis, malariaCountrywide859 million (LF), 241 million (Malaria)Malaria (yes)LLINs, ITNs, IRS[7, 8]
CulexJapanese encephalitis, lymphatic filariasis, West Nile feverMiddle East Europe, Asia, Japan, China, India3 billion (JE), 859 million (LF)Only JE (Yes)Mosquito repellents, vaporizers[8, 9]
MansoniaBrugian filariasis, yellow feverSouth East Asia AustraliaApp. 200,000 (YF)NoRepellents, elimination of breeding site[6]

Table 1.

Showing mosquito borne disease and their distribution, burden and intervention.


2. Brief history of CRISPER

Ishino and his colleagues identified CRISPER for the first time in E. coliin 1987, with a 1664-nucleotide sequence [2]. From 1993 until 2005, it was extensively discovered. CRISPER function were established in 2007 following the discovery of genes proximal to the CRISPER locus in 2002 and foreign viral DNA sequences in CRISPER space in 2005. CRISPER was discovered by two laboratories at the same time in 2013 to become the most powerful gene editing technique known (Figure 2).

Figure 2.

Flow chart showing history and discovery of CRISPER/Cas technique.

Arbo viral diseases such as dengue, chikungunya, Zika and malaria are a major public health problem across the world. To counteract the spread of mosquito-borne disease, researchers transformed CRISPER/Cas 9 into extremely effective “gene drive” systems capable of spreading disease resistance genes across whole populations. Researchers packaged disease resistance genes, CRISPER, gRNA, and Cas9 components into a single DNA construct to produce a gene drive [10]. After insertion, the gene drive replicates autonomously into both parental chromosomes and is inherited by around 99.5% of progeny. Advances in gene drive technology promises urgent alternatives for disease control. Two approaches for controlling arbo-viral diseases for controlling arbo-viral disease using gene-edited mosquitoes have recently gained significant attention. The first approach is “Population Replacement” where the wild mosquito population, which carry or transmits the pathogen, is replaced by the normal ones. The principle of gene drive underlies this strategy. Gene drive makes use of an inheritance quirk to pass on a trait to more than half of a mosquito’s offspring allowing it to spread rapidly over a population. Another strategy is “Population Suppression” which involves the reduction of mosquito population, resulting in less mosquito genic condition and fever mosquito capable of transmitting pathogens.


3. Gene drive

Gene drive are selfish genetic tools that can be re-designed to invade a population and they hold tremendous potential for the control of mosquitoes that transmit disease [10]. Targeting the mosquito vector in order to interrupt transmission has been the mainstay of successful malaria control programs over to interrupt transmission has been the mainstay of successful malaria control programs over the years. Gene drives represents a powerful tool to achieve this in a targeted way that is species-specific, requires minimal infrastructure and is self-sustaining (Figure 3). Moreover, if successful, the benefits of this type of intervention would be available to all, regardless of differential access to healthcare.

Figure 3.

Propagation of altered gene through gene drive mechanism.

Several CRISPER/Cas9 based clinical therapies have been described in the last 3 years. Many clinical trials have been completed in recent years, with some results reported; including the CRISPER/Cas9 based clinical treatment of acquired immunodeficiency syndrome (AIDS) [10], sickle cell disease (SCD) [11], thalassemia and various cancer [12, 13]. Emmanuelle Charpentier and Jennifer Doudna were given the Nobel Prize in 2020 in recognition of their accomplishment using CRISPER/Cas9 technology. The mechanism behind Crisper technique is shown in Figure 4. CRISPER gene editing tool in mosquito species mainly Aedes aegypti, Anopheles stephensi, Anopheles gambiaewith target site and its application are shown is Tables 2 and 3.

Figure 4.

Mechanism of Crisper/Cas9.

MosquitospeciesGenome editing toolTargeted genesApplicationReference
Ae. aegyptiCRISPR/Cas9ECFPFunctional genomics[14]
Ae. aegyptiCRISPR/Cas9NixConversion of females into harmless males[15]
Ae. aegyptiCRISPR/Cas9Aaeg-wtrwSite-specific mutations[16]
Ae. aegyptiCRISPR/Cas9Kmo, loqs, r2d2, ku70, lig4 and nix genesTransgenic strains and gene drive[17]
An. stephensiCRISPR/Cas9M1C3 and m2A10P. falciparumresistance strains[18]
An. gambiaeCRISPR/Cas9AGAP005958, AGAP007280 and AGAP011377An. gambiaepopulation suppression[19]
An. gambiaeCRISPR/Cas9X-linked rDNA sequenceSex-distortion in An. gambiae[20]

Table 2.

Crisper gene editing tool with target site and its application.

ECFP, enhanced cyan fluorescent protein; Nix, male-determining factor gene; Aaeg-wtrw, Ae. aegyptiwater witch locus; kmo, kynurenine 3-monoxygenase; loqs, loquacious; r2d2, r2d2 protein; ku70, ku heterodimer protein gene; lig4, ligase4; m1C3 and m2A10, anti-parasite effector genes; AGAP005958, AGAP007280 and AGAP011377, An. gambiaefemale-fertility genes.

Origin and biological basisThe method was first employed to remove screw-worms in 1950.Prior to 1970, yeast group I introns with greater than Mendelian inheritance proportions were identified.Zinc-binding first fused for site-specific DNA cleavage in 1996TALE proteins discovered in Xanthomonasspecies 2009 and conjugated to FokI endonucleaseChanges genes associated with male fertility (resulting in sterile progeny) and female flight in Aedes aegyptiS. pyogenesdestruction and memory of parasitic nucleic acid
Initial use for genome modificationCochliomyia(1954)E. coli(1998)Xenopus laevis(2001)S. cerevisiae(2010)D. melanogasterHuman cell lines (2012)
Year used in mosquitoes195520112013201320132015
MechanismRearing of mosquito followed by separation of males and sterilize them and releasing of irradiated males to mate with wild females resulting infertile egg.Endonuclease encoded by HEG detects and cleaves genomic DNA, allowing a gene cassette to be integrated by cell HDR machinery.ZFN domain recognizes and bind to a sequence of nucleotide triplets, and crate a dsDNA breaks in backbone.TALE domains recognize and bind to a sequence of nucleotides, and cut DNA backbone, together creating a double-stranded break.Strategy is to release an increasing number of sterile males based on SIT.Cas 9 protein make complex with guide RNA and binds to genomic DNA and produce a double stranded and with insertion of programmed DNA.
Gene driveNoYesNoNoYesYes
  • Produced offspring’s were infertile

  • Insertion into a known site

  • High efficiency

  • First gene editing technique for reverse genetics.

  • Site-specific editing.

  • Site-specific editing

  • Efficient mutagenesis.

  • Production of infertile male.

  • Flightless female

  • Site-specific highly efficient for mutagenesis and as a drive.

  • Mass rearing and irradiation require precision processes.

  • Radiation can reduce male mating fitness

  • Requires pre-existing target-sites.

  • Re-engineering of the HEG, or transgenesis for insertion of target sites.

  • Expensive, requires in vitro optimization

  • Requires protein engineering

  • Radiation can reduce male mating fitness

  • Drive mechanism generates drive-resistant alleles

References[21, 22, 23][24, 25, 26, 27, 28, 29][30, 31, 32, 33, 34][35, 36, 37, 38, 39, 40][41][2, 42, 43, 44, 45, 46, 47]

Table 3.

Showing a brief comparison of Crisper and other gene editing techniques.

3.1 How crisper work?

A detailed description of each technique is given below:

3.2 Sterile insect technique (SIT)

SIT is a proven ecologically safe method of controlling wild populations. Since the 1950s, SITs have been employed to manage insect pests in the United States and across the world. SIT is a species-specific, non-polluting insect management approach that depends on the release of a large number of sterile insect [21, 22, 23]. Throughout the twentieth century, large-scale vector control initiative effectively reduced disease transmission levels to zero or near zero levels over areas. Countries which were close to elimination of malaria and yellow fever, include Cuba, Panama, Brazil and in Pan America [46, 47, 48, 49] and Singapore (very low level of dengue incidence) [50, 51, 52].

Sterile insect technique involves following steps:

  • Mass production of mosquitoes.

  • Separation and sterilization of males.

  • Mass release of male mosquito by drone, jeep, helicopter, etc.

  • Sterile male compete with wild males to mate with wild females.

  • Production of infertile egg.

SIT may be a preferable method of reducing mosquito populations in places where the use of insecticides is not practical, is not accepted by the community, or where pesticide resistance has diminished insecticide efficacy (Figure 5).

Figure 5.

Showing application of sterile insect technique.

3.3 Precision-guided sterile insect technique (PgSIT)

A revolutionary scalable genetic control technology uses a CRISPR-based technique to design deployable mosquitoes capable of population suppression (41). Males do not transmit diseases; hence, the strategy is to release an increasing number of sterile males. Mosquito populations can be reduced without the use of toxic chemicals and pesticides. It affects genes associated with male fertility (resulting in sterile progeny) and female flight in Aedes aegypti, the mosquito species responsible for the transmission of illnesses such as dengue fever, chikungunya, and Zika.

PgSIT is based on a dominant genetic technique that permits simultaneous sexing and sterilization, allowing eggs to be released into the environment while assuring only sterile adult males emerge. The system is self-limiting and is not expected to persist or spread in the environment, which are two safety factors that should allow this technology to be accepted. PgSIT eggs can be delivered to a place threatened by mosquito-borne disease or generated at an on-site laboratory that can manufacture the eggs for nearby deployment. Once the PgSIT eggs are released into the wild, infertile PgSIT males will develop and eventually mate with females, reducing the natural population as desired.

3.4 Zink Finger (ZFN)

A few researchers have used ZFN to custom-edit the genomes of vector mosquitoes. Zinc-finger domains identify the shapes of nucleotide triplets in the major groove of a DNA double-helix and may be engineered to recognize a specific 18-nucleotide sequence, allowing a large number of protein effectors to be recruited to a specific place in the genome [53, 54, 55]. Zinc-finger domains are conjugated to a FokI type II restriction endonuclease and designed in pairs to recognize sequences flanking a target-site, resulting in a double-stranded break at a particular genomic locus [37, 55].

Inspite of the high cost and the low success rate of a ZFN, it was still being used by most laboratories for biological studies. For example, DeGennaro et al. [32] investigated the involvement of the odorant receptor coreceptor (orco) gene and the odorant receptor pathway in host identification and susceptibility to the chemical repellent N,N-diethyl-meta-toluamide (DEET) in Aedes aegypti[35]. The developed ZFN was injected into embryos of Aedes aegyptiin this experiment. When compared to the wild type, the orco mutants developed in this work had lower spontaneous activity and odor-evoked responses. In the absence of CO2, orco mutant mosquitoes did not respond to human odor.

In an another set of experiment McMeniman et al. injected ZFNs into pre-blastoderm stage embryos to mutate the Aedes aegyptigustatory receptors (AaegGr3) gene, a subunit of the heteromeric CO2 receptor, and found that the Gr3 mutant lacked electrophysiological and behavioral responses to CO2 [54].

3.5 Transcription activator-like effector nuclease (TALEN)

Finally, in 2010, a low-cost technology that could be developed in-house made targeted mutagenesis available to molecular biology laboratories: transcription activator-like effector (TALE) nucleases, or TALENs. TALENs, like ZFNs, are modular, can be encoded on a plasmid via cloning, and are relatively efficient [31]. Finally, the recognition of each nucleotide on a DNA target was encoded in the 12th and 13th amino acids of each 34 amino-acid repeat; a peptide stretch of 18 or 19 repeats could be engineered to recognize any nucleotide sequence and could induce site-specific DNA cleavage when conjugate to the FokI domain [30, 35, 56]. Smidler et al. reported the targeted disruption of the thioester containing protein1 (TEP1) gene using TALEN in Anopheles gambiaemosquitos, which transmit malaria. TEP1 has been identified as an immunity gene in An. gambiaeagainst plasmodium infection [57]. The induced mutations lowered protein synthesis, and the resulting TEP1 mutants were more vulnerable to Plasmodium bergheiinfections. In addition to the previously described ZFN, TALEN has been employed as a powerful genome editing technique to alter the targeted genes in disease-causing mosquitoes. Gene-editing in Ae. aegyptiand An. stephensiusing ZFNs and TALENs were reported in 2013 [32, 33, 57]. As there is a less difficulty to construct TALENs in the lab, therefore, TALENs were more accessible than previous gene-editing approaches. However, the timing of TALEN development was almost concurrent with the leveraging of CRISPR/Cas9 biology for gene-editing, meaning that TALENs usefulness was short-lived.

3.6 Meganucleases

Usefulness HEs, also known as meganucleases, can cleave double-stranded DNA at specific recognition site of 14–40 bp in length [58]. HEG-induced dsDNA break and activate the cell’s recombination repair system, which uses the HEG-containing homologous chromosome as a template for repair. As a result, in a process known as ‘homing’ the HEG is copied to the broken chromosome. HEGs spread through populations by using this transmission distortion mechanism [59]. In An. gambiae, HEs have recently been proposed as a method of genetic sterilization or sex-ratio distortion [58, 59].


4. Ethical issues

CRISPR/Cas9 offers a wide range of uses and enormous life-changing potential, but it will take decades to develop. The proper use of biotechnology necessitates meticulous planning and strict control, both of which are far from reality. Altering a gene might have unforeseeable and unfavorable repercussions in the genetically edited species, as well as in other species, and can result in the emergence of new and undiscovered animal and human disease. CRISPR/Cas9 has a wide range of potential immediate public health benefits: it can be used to treat vector borne disease, but there are, nevertheless, significant dangers. Gene editing should be implemented with caution and followed by more research. Gene editing techniques have the potential to diminish biodiversity and harm ecosystems. The UNESCO Declaration on Bioethics and Human Rights recognizes humans as essential members of the ecosystem. How and under what conditions do we have the authority to alter biological beings, and how far do we have that authority? Is it possible to foresee the effects of modifying or removing a particular animal species from its ecosystem? Is there a method to limit the negative consequences as well? The variety of prospective uses for gene drive technology (particularly the manufacturing of bioweapons) and their consequences in this predicted situation are certainly unexpected. Besides this, there are environmental concerns; for example, what will be the impact on predatory fish and insects that consume mosquito larvae, is still under debate. Therefore, in order to environment interest, it should be thoroughly investigated.


5. Discussion

Insects are a highly diversified group that inhabits many biological niches, has specific habitat adaptations, and performs diverse behaviors. CRISPR system is a very successful tool for precision genome editing in the mosquito compared to the relatively low throughput and high cost of ZFN- and TALEN-mediated mutagenesis. Precision genome engineering in mosquitos holds a lot of potential for studying the genetic basis of behavior and developing genetic strategies to control vector populations [56]. The development of new CRISPR tools and platforms for molecular diagnostics has the potential to revolutionize health care and enhance global epidemiological management. SIT approach has various limitations, adopting innovative methods like PgSIT to control mosquito-borne diseases is a suitable way to implement population control measures.


6. Conclusion

CRISPER technology is a new approach in the field of vector-transmitted disease that is causing dispute among government, non-government, and policymakers. Scientists all around the world, however, have the opportunity to implement this technology for the benefit of society, notably for the control of the vector mosquitoes. However, it is more crucial to adhere to all the rule and regulations and take stringent biosafety precautions to avoid unintended and unwanted outcomes from genome editing. Existing vector control strategies are unprepared to deal with arboviral disease’s exceptional development and reemergence. The tremendous successes in localized eradication, as well as the final inability to eradicate malaria globally, aroused interest in genetic methods to mosquito control. In contrast to several application of Crisper technique it might be disused and scientific discussion around CRISPR is crucial. Despite the risks, CRISPR represents a tremendous potential for humanity, and the precise gene editing will bring in a bright future.



Authors are gratefully acknowledging ICMR-National Institute of Malaria Research, New Delhi, India, for providing the suitable environment facilities that help us to draft the proposed study.


Conflict of interest

The authors declare that they have no known competing financial interests or personal ties that would seem to have influenced the work presented in this study.


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Kaushal Kumar Mahto, Pooja Prasad, Mohan Kumar, Harshita Dubey and Amar Ranjan

Submitted: January 29th, 2022Reviewed: March 1st, 2022Published: April 23rd, 2022