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Gene & Protein in Disease Enhancing fertility with CRISPR

advancing scientific landscape, Cas9–RNA complexes can to the crRNA; and the RuvC-like Cas9 domain, which
be effectively employed as genome engineering agents breaks down the non-complementary strand of the target
in eukaryotic cells, encompassing those of plants and DNA. 37,39,40 This process induces DSBs at the targeted DNA
animals. 26-29 On the other hand, base editing through site. Following the cleavage and editing of the targeted
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CRISPR enables precise DNA modification without causing gene sequence, the DNA repair mechanism is activated, as
double-strand breaks. In this regard, Xie et al. efficiently illustrated in Figure 1. Two common repair methods are
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induced C-to-T conversions at multiple loci, producing homology-directed repair (HDR) and non-homologous
pigs with single or multiple point mutations in genes. Base end joining (NHEJ). While NHEJ is more prevalent in
Editor-Targeted and Template-free Expression Regulation mammals for DSB repair, it is less effective than HDR and
(BETTER), which utilizes CRISPR-guided base editors for may introduce unintended mutations. NHEJ simply ligates
diverse multigene expression without library construction, the two DNA strands with minimal processing. HDR
has shown promise and is thus useful for large-scale is favored in Cas9-involved gene editing due to its high
refinement of expressions of multiple genes. In addition, efficiency in repairing DSBs. 42-44
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Li et al. demonstrated a one-step approach for generating
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base-mutant mice using a third-generation base-editing 2.3. Advantages of CRISPR over traditional gene
system. The development of new base editor variants has editing technologies
expanded design possibilities, allowing recognition of CRISPR technology has surpassed traditional gene editing
various protospacer-adjacent motifs. For instance, Rosello tools such as TALENS and ZFN in numerous aspects,
et al. outlined experimental strategies for cytosine base presenting significant advantages. One such advantage lies
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editing in zebrafish, allowing precise substitution of in the high specificity of CRISPR technology for genome
interest. Moreover, co-selection with loss-of-function editing. Continuous technological advancements further
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mutations facilitates direct analysis of injected embryos, contribute to the reduction of off-target effects, with recent
revealing the phenotypic impact of targeted substitutions. innovations such as prime editing demonstrating even
Conclusively, current progress in CRISPR gene editing and greater precision and fewer off-site effects. Notably, the
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base editing suggests ongoing advancements in enhancing simplicity of creation and implementation sets CRISPR
specificity, delivery, and ethical considerations for broader apart from other gene editing technologies. Its versatility
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applications in disease management. and flexibility make CRISPR suitable for various genome-
related studies, encompassing gene knockin and knockout,
2.2. Components and mechanism of CRISPR regulation of gene expression, and the management of
The CRISPR and associated systems are categorized into hereditary conditions. Moreover, CRISPR facilitates
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two main classes, encompassing six significant types with more effective location-specific gene editing, addressing
33 subtypes and additional variants. 34,35 Class 1 comprises challenges posed by classical gene therapy. The capability
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type I, type III, and type IV, while Class 2 includes the of CRISPR to simultaneously activate multiple genes is a key
more prevalent type II, type IV, and type V. Among these advantage, overcoming limitations inherent in traditional
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types, type II stands out as the most commonly utilized for gene overexpression technologies by incorporating
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genome editing. Notably, type I and type II operate on numerous gRNAs in a single vector. Conclusively, CRISPR
the proteins Cas3 and Cas9, respectively. The Cas9 system technology offers numerous advantages compared to its
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derived from Streptococcus pyogenes is the preferred choice predecessors, emerging as a pivotal tool in gene editing
due to its convenience and superior efficacy among all with reduced off-site effects and effective applications in
CRISPR-associated systems. 38 gene therapy.
The fundamental principle of CRISPR and associated 3. Applications of CRISPR in reproductive
systems is elucidated through Type II CRISPR biology
technology. In Type II, the CRISPR system comprises
two main components: A gRNA, primarily consisting of The genetic revolution, coupled with advancements in
a 20-nucleotide sequence (crRNA) containing the target genetic editing and genome engineering techniques
sequence complementary to one strand of the target over the past 40 years, has bestowed on us a profound
DNA; and the transactivating crRNA that binds to the understanding of molecular mechanisms and the complete
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endonuclease Cas9 protein. The Cas9 endonuclease, a sequencing of our genomes. This transformative era
sizable protein, induces double-strand breaks (DSBs) has particularly empowered the application of gene-
at a specific site in the DNA. The CAS9–RNA complex editing technologies like CRISPR/Cas9 in the realm of
encompasses two nuclease domains: the His-Asn-His reproduction, a physiological phenomenon that stands as
domain, which cleaves the DNA strand complementary a prime target for intervention.

Volume 3 Issue 1 (2024) 3 https://doi.org/10.36922/gpd.2701