**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)


*Role of CRISPR Technology in Gene Editing of Emerging and Re-emerging Vector Borne Disease DOI: http://dx.doi.org/10.5772/intechopen.104100*


**Table 3.**

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

*Role of CRISPR Technology in Gene Editing of Emerging and Re-emerging Vector Borne Disease DOI: http://dx.doi.org/10.5772/intechopen.104100*

**Figure 5.** *Showing application of sterile insect technique.*

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 [53–55]. 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 aegypti* in 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 aegypti* gustatory 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 gambiae* mosquitos, which transmit malaria. TEP1 has been identified as an immunity gene in *An. gambiae* against plasmodium infection [57]. The induced mutations lowered protein synthesis, and the resulting TEP1 mutants were more vulnerable to *Plasmodium berghei* infections. 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. aegypti* and *An. stephensi* using 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].
