Genomics and Applied Biology 2024, Vol.15, No.2, 107-119 http://bioscipublisher.com/index.php/gab 110 3.3 Success stories and case studies Several successful case studies highlight the potential of CRISPR-Cas9 in mosquito research. One notable example is the use of CRISPR-Cas9 to create targeted mutations in the malaria vector Anopheles stephensi using the ReMOT Control technique, which bypasses the need for embryo injections and makes the technology accessible to more laboratories (Macias et al., 2019). Another success story involves the precise integration of a marker gene into the odorant receptor co-receptor (Orco) in Anopheles sinensis, significantly impairing the mosquito's ability to locate and discriminate human hosts (Wang et al., 2022). These studies demonstrate the versatility and effectiveness of CRISPR-Cas9 in advancing mosquito research and vector control. 3.4 Limitations and challenges Despite its advantages, the CRISPR-Cas9 system faces several limitations and challenges. One major concern is off-target effects, where unintended genomic sites are edited, potentially leading to adverse outcomes (Gupta et al., 2019; Guo et al., 2023). Efforts to mitigate these effects include the development of high-fidelity Cas9 variants and paired nickases that reduce off-target activity (Gupta et al., 2019). Another challenge is the efficient delivery of CRISPR components into mosquito cells. Traditional methods like embryo injections are technically demanding and inefficient, prompting the exploration of alternative delivery systems such as non-viral vectors and ReMOT Control (Li et al., 2018; Macias et al., 2019). Additionally, the ethical and ecological implications of releasing genetically modified mosquitoes into the environment require careful consideration and regulatory oversight (Wang and Doudna, 2023). 4 Other Emerging Gene Editing Technologies 4.1 TALENs (transcription activator-like effector nucleases) Transcription Activator-Like Effector Nucleases (TALENs) are engineered nucleases that can create double-strand breaks (DSBs) at specific locations in the genome. TALENs are composed of a DNA-binding domain derived from transcription activator-like effectors (TALEs) and a FokI nuclease domain. The DNA-binding domain can be customized to target specific DNA sequences, making TALENs highly versatile for genome editing applications. TALENs have been successfully used in various fields, including the creation of animal models and gene therapy development (Bak et al., 2018; Li et al., 2020; Castro et al., 2021). 4.2 ZFNs (zinc finger nucleases) Zinc Finger Nucleases (ZFNs) are another class of engineered nucleases that facilitate targeted genome editing by creating DSBs at specific genomic locations. ZFNs consist of a DNA-binding domain composed of zinc finger proteins and a FokI nuclease domain. The zinc finger proteins can be engineered to recognize specific DNA sequences, allowing precise targeting. ZFNs have been utilized in both basic research and clinical applications, including the development of disease models and potential therapies for genetic disorders (Khan et al., 2019; Li et al., 2020; Castro et al., 2021). 4.3 Base editing and prime editing Base editing and prime editing are newer gene editing technologies that allow for precise modifications at the single-nucleotide level without creating DSBs. Base editing uses a modified CRISPR-Cas9 system to convert one DNA base pair into another, enabling targeted point mutations. Prime editing, on the other hand, uses a reverse transcriptase enzyme fused to a Cas9 nickase to directly write new genetic information into a target DNA site. These technologies offer higher precision and reduced off-target effects compared to traditional genome editing methods (Naeem et al., 2020). 4.4 Comparative advantages and disadvantages Each gene editing technology has its own set of advantages and disadvantages, making them suitable for different applications: TALENs: TALENs offer high specificity and can target a wide range of DNA sequences. However, they are relatively complex to design and produce, which can limit their widespread use (Li et al., 2020; Castro et al., 2021).
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