Navigating the frontier: Crispr's evolution in animal science and the roadblocks ahead

Introduction

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are currently in the spotlight of active research in biology. (Makarova and Koonin., 2015) [52] . Y.

Structural and biochemical studies

Cas9 possesses a bilobed structure, with the guide RNA nestled between the nuclease and alpha-helical lobes. A solitary bridge helix connects these two lobes. The RuvC nuclease domain cleaves the non-target DNA strand, whereas the HNH nuclease domain cleaves the target DNA strand, both found in the multi-domain nuclease lobe. Sequentially dissimilar sites that interact in the tertiary structure to generate the RuvC cleavage domain encode the RuvC domain (Jinek et al., 2012; Doudna et al., 2014; Jinek et al., 2014). Target DNA must have a protospacer adjacent motif (PAM), made up of the three-nucleotide sequence NGG, which is an essential characteristic. The PAMinteracting domain (PI domain), situated close to Cas9's Cterminal end, is responsible for identifying this PAM. Cas9 transitions between the apo, guide RNA-bound, and guide RNA: DNA-bound states through discrete conformational changes. Cas9 can determine the CRISPR locus's intrinsic stem-loop topology, which is responsible for forming the crRNA-tracrRNA ribonucleoprotein complex (Sternberg et al., 2014; Jiang et al., 2015; Jinek et al., 2014). Cas9 further recognizes and breaks down the target dsDNA when it forms a complex with trans-activating crRNA (tracrRNA) and CRISPR RNA (crRNA). A chimeric single-guide RNA that has been shown to have the same function as the original RNA complex takes the place of the crRNA-tracrRNA complex.Cas9 anchors the sgRNA base in a Tshaped architecture coupled with the target ssDNA (Jinek et al., 2012; Jiang et al., 2017; Nishimasu et al., 2014). The DNA-bound Cas9 enzyme's crystal structure reveals the position of the HNH domain in addition to unique conformational changes in the alpha-helical lobe about the nuclease lobe. The protein consists of a recognition lobe (REC) and a nuclease lobe (Jiang et al., 2017; Nishimasu et al., 2014; Anders et al., 2014) Mechanisms of crispr/cas-9 genome editing CRISPR/Cas-9 genome editing involves three main steps: recognition, cleavage, and repair. (Jinek et al., 2012; Doudna et al., 2014; Jinek et al., 2014) [37,23,39] . The engineered sgRNA guides Cas-9 to the target gene, triggering a double-strand break (DSB) three base pairs upstream of the PAM sequence. Cas-9 then initiates local DNA melting, forming an RNA-DNA hybrid, followed by cleavage of the DNA strands. Repair occurs through nonhomologous end joining (NHEJ) or homology-directed repair (HDR), with NHEJ being error-prone and HDR requiring a homologous DNA template for precise gene editing (Sternberg et al., 2014; Jiang et al., 2015; Jinek et al., 2014) [39] .

Delivery of crispr-cas systems

Various methods exist for introducing CRISPR-Cas systems into cells, primarily differing in the delivery of the gRNA and Cas9 protein. The gRNA can be supplied as RNA or DNA cloned into a plasmid, while the Cas9 protein can be delivered as a protein, transcribed mRNA, or plasmid DNA (Yin et al., 2019; Staahl et al., 2017; Zuris et al., 2015) [97,75,102] . Delivery of the Cas9 protein as a ribo-protein complex offers advantages due to its transient presence and minimal off-target effects (Zuris et al., 2015; Ramakrishna et al., 2014; Staahl et al., 2017) [102,68,75] . Physical techniques like electroporation, microinjection, and mechanical cell deformation, as well as carriers such as viral vectors (e.g., lentiviruses, adenoviruses, adeno-associated viruses) and non-viral methods (e.g., lipid nanoparticles, polymer nanoparticles, DNA nanostructures), can be used to transfer gRNA and Cas9 into cells (Doudna & Charpentier, 2014; Ramakrishna et al., 2014; Wang et al., 2019) [23,68,85] . Both viral and non-viral carriers protect gRNA and Cas proteins, enhancing delivery reliability and efficacy (Wang et al., 2016) [87] .

Application of CRISPR-Cas9

Role in Gene Therapy Gene therapy, a leading advancement in medical biotechnology, involves modifying defective genes and replacing them with healthy DNA. CRISPR/Cas-9, alongside other techniques, has revolutionized gene therapy, with 22 treatments authorized between 1998 and 2019. Notably, CRISPR/Cas-9 holds promise for treating a range of genetic disorders, including sickle cell disease, cystic fibrosis, and Duchenne muscular dystrophy. In sickle cell disease, CRISPR/Cas-9 boosts fetal hemoglobin production by inhibiting the BCL11A gene (Brendel et al., 2016; Frangoul et al., 2021; Traxler et al., 2016; Canver et al., 2015) [9,24,81,14] . Similarly, it shows potential for correcting the CFTR gene mutation in cystic fibrosis (Schwank et [73,56,3,20] and restoring dystrophin expression in Duchenne muscular dystrophy (Ousterout et [65,62,77,58] . CRISPR-based therapies have also entered human trials for lung cancer treatment, demonstrating promising results in modifying T-cells to target cancer cells effectively (Nelson et al., 2016; Tabebordbar et al., 2016; Young et al., 2016) [62,77,99] .

Pipeline of CRISPR-Cas-assisted drug discovery

CRISPR-Cas tools have revolutionized genetic manipulation across various organisms, from human ESCs to the malaria parasite, previously deemed nearly impossible. These advancements accelerate functional genomics, aiding in the discovery and validation of therapeutic targets, particularly in mammalian models and human cells (Akcakaya et al., 2018; Joung et al., 2017) [1,40] . They offer potential in improving animal models, enhancing safety testing, and refining patient treatment plans. Moreover, CRISPR-Cas editing enables the development of personalized cellular therapies, such as cancer-targeting T cells and reprogrammed induced pluripotent stem cells (iPSCs), for both genetic and non-genetic disorders (Cai et al., 2020; Kosicki et al., 2018; Gaudelli et al., 2017) [13,45,25] . In drug development, CRISPR-Cas systems streamline discovery, validation, and safety testing processes without significant delivery or administrative hurdles. This paves the way for novel therapeutic approaches and paradigms, making CRISPR-Cas indispensable in advancing medicine (Tycko et [82,50,23] .

CRISPR-based assays for rapid detection of SARS-CoV-2

The COVID-19 pandemic escalated the urgency for widespread testing to detect and mitigate transmission. While qRT-PCR tests are the gold standard, they have limitations. Nucleic acid-based tests offer higher sensitivity, and efforts to enhance efficiency include integrating LAMP-based isothermal detection. Combining CRISPR-based techniques with isothermal technologies, as proposed by Broughton et al. (2020) [10] , Joung et al. (2020) [41] , Lalli et al. (2020) [46] , promises rapid and sensitive detection of SARS-CoV-2 nucleic acids.

Potential of CRISPR/Cas9 Gene Editing as a Treatment Strategy for Neurological disorders

The distinguishing feature of neurodegenerative illnesses, which include Huntington's disease (HD), Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD), is agedependent and selective neurodegeneration. These neurodegenerative diseases are more common as human life expectancy increases; the pathophysiology of most of these illnesses is still unknown, and there are currently no reliable treatments for these significant brain malfunctions ( [5,12,6] . Despite decades of understanding Alzheimer's disease (AD) at the molecular level, finding effective treatments has been challenging. Clinical trials targeting beta-amyloid have often fallen short, prompting a need to explore alternative therapies (Cummings et [17,34,31,72] . Cas9-mediated knock-in mutations can aid in creating animal models for diseases caused by mutant protein toxicity, such as Huntington's disease. ( [18,59,94] .

CRISPR/Cas9 in cancer immunotherapy

Current treatment medications often have high toxicity and low success rates, despite efforts in immunotherapy and chemotherapy to control cancer cell growth. Additionally, evolving mutations in proto-oncogenes and tumorsuppressive genes limit the effectiveness of multi-targeted therapies to a few carcinogenesis pathways. ( [7,84,57] . CRISPR/Cas9 corrects causal mutations with minimal toxicity and enhances immunotherapy. However, key drawbacks include ethical concerns, generic limitations upstream of the Protospacer Adjacent Motifs (PAM) leading to off-target alterations, and a lack of consensus on risk assessment. (Pickar-Oliver & Verma et al., 2020) [67] . Animal models are crucial in refining CRISPR/Cas9 experiments for improved genome editing specificity and enhanced anti-tumor responses. Clinical trials utilize the CRISPR/Cas9 system in immune cells for precise genome modifications. Recent advancements in error-free in vitro technologies aim to overcome limitations of this geneediting system. The article focuses on using CRISPR Cas9 technology to treat treatment-resistant cancers. ( [54,55,60] . Additionally, the use of CRISPR/Cas9 is aided as an emerging supplementation of immunotherapy, currently used in experimental oncology.

CRISPR and HIV: New technique in human blood unveils potential paths toward cure

CRISPR/Cas-9 gene-editing technology shows promise in treating microbially-induced infectious diseases, particularly HIV/AIDS. Temple University researchers demonstrated in animal models that deleting the HIV-1 genome using CRISPR/Cas-9 halted replication and removed the virus from infected cells (Yin et [96,87,42] . CRISPR/Cas-9 can also modify genes encoding the chemokine coreceptor type-5 (CCR5) in host cells, blocking HIV entry (Xu et [94,85,49,21] . T cells from human blood were genetically modified using CRISPR-Cas9, deleting multiple genes. When exposed to HIV, cells lacking essential viral replication genes showed reduced infection, while those lacking antiviral components showed increased infection (Wang et al., 2015; Zhang et al., 2021; D'Orso, 2017) [91,101] .

CRISPR-Cas system: A potential alternative tool to cope antibiotic resistance

Antimicrobial resistance (AMR) poses a global health threat, fueled by the transfer of AMR genes among bacterial pathogens via horizontal gene transfer (HGT). To combat this, new approaches are urgently needed (Tacconelli et al., 2018; Homes et al., 2016;). CRISPR/Cas systems, originally a prokaryotic immune mechanism, have emerged as powerful tools for combating AMR by targeting and cleaving DNA sequences encoding antibiotic resistance genes (Bikard et al., 2014; Citorik et al., 2014) [98,8] . Given the slow pace of antibiotic development compared to bacterial evolution, alternative strategies such as bacteriophage therapies, antibacterial peptides, bacteriocins, and anti-virulence chemicals are crucial in tackling antibiotic-resistant infections (Imai et al., 2019; Tacconelli et al., 2018) [33,[79][80] . The CRISPR-Cas system has been leveraged for molecular recording and selectively targeting antibiotic resistance genes, offering promise in combating AMR (Yosef et al., 2015; Bikard et al., 2014) [98,8] .

CRISPR/Cas9 technology on cardiac research: From disease modelling to therapeutic approaches

Genome-editing technology, particularly the CRISPR/Cas9 system, has been widely used to correct DNA mutations, ranging from single base pairs to large deletions, in both in vitro and In vivo models, enhancing our understanding of cardiovascular disorders like lipid metabolism and electrophysiology (Savarese & Lund, 2017; Wu, 2017; Kessler et al., 2015) [71,93,43] . CRISPR/Cas9 facilitates gene knockout or knockin in human cells, particularly in induced pluripotent stem cells (iPSCs), offering valuable insights into disease mechanisms (Savarese & Lund, 2017; Wu, 2017; Musunuru, 2013; Kessler et al., 2015) [71,93,43] . Despite its potential, challenges related to biology, technology, and ethics hinder its therapeutic application in cardiovascular diseases (Savarese & Lund, 2017; Wu, 2017; Kessler et al., 2015) [71,93,43] . Remarkably, CRISPR/Cas9 has demonstrated efficacy in correcting genetic defects in postnatal/adult mice, exemplified by its ability to edit the PCSK9 gene, resulting in reduced blood cholesterol levels and lowered risk of coronary heart disease (CHD) (Wang et al., 2017; Ran et al., 2015) [90,91] .

CRISPR in livestock and poultry: From editing to printing

Since the advent of the CRISPR revolution, precise genome editing of large animals has been achievable for application in biomedicine and cattle. The process is not necessarily a simple, quick, or safe path from editing to printing, or from genetic engineering to producing the required animals. Selecting the optimum method for genome editing, embryo generation, zygote microinjection or electroporation, cryopreservation, and embryo transfer is necessary when using CRISPR in large mammals. These procedures can be time-consuming and expensive (Hai et al., 2014; Whitworth et al., 2014) [29,92] . The primary technological advancements and frequently asked questions to enhance this revolutionary biotechnology in big animals. In light of the rising worldwide demand for food, CRISPR improves livestock production in several ways, including efficiency gains, a decrease in the environmental effect of farming, improved pest management, and improved animal welfare and health. It is no longer a technical challenge. For the first time in the CRISPR era, debates and agreements, chances and dangers, advantages and disadvantages, ethics and science should all be reexamined (Lassner & Peterson, 2015; Larson et al., 2013) [48,47] . Over time, CRISPR technology has evolved and improved, enabling it to create transgenic bird lines primarily for food purposes, especially for producing meat or eggs. The application of CRISPR technology may result in the sustainable and effective development of poultry products, so assisting in addressing issues related to global food security (Oishi et al., 2016; Van et al., 2020) [64,83] . Growth, feed conversion, digestibility, increased egg output, and general improved performance of chickens raised for meat and eggs could all be significantly impacted by CRISPR technology. Technological developments in CRISPR may also improve disease resistance, vaccination delivery, and immune response. This will lead to improvements in the health of the poultry, the safety of immunizations derived from chicken eggs, and the production and safety of food (Lillico et al., 2013; Guo et al,. 2021) [51,26] . Gene inactivation by indels introduction is known as knockout (KO). Efficiency of the CRISPR/Cas9 tool ranges from 10% to over 90% in a variety of animals and cell types, including human, sheep, goat, cattle, pig, and mouse. Prior to the commercial release of CRISPR/Cas9 ribonucleoprotein (RNP), KO efficiency was dependent on plasmid transfection. However, RNP delivery offers a greater KO efficiency and circumvents the drawbacks of using DNA plasmid delivery (Zhang et al., 2021; Wang et al., 2021) [101,88] . After CRISPR/Cas9 RNP delivery, the RNP activates quickly to perform DSB, and indels are visible very quickly. Since RNP is removed from the cells in less than a day, there is a lower chance of off-target mutations. Plasmid delivery, on the other hand, carries the danger of inadvertent off-target mutation and may even contribute to a vector's integration into the host genome (Kim et al., 2014; Richardson et al., 2016) [44,70] .

Anti-crispr proteins

Anti-CRISPR proteins, found in phages, hinder the normal function of the bacterial immune system CRISPR-Cas, effectively reversing its unintended effects. They act as blockers of Cas proteins, allowing phages to evade CRISPR-mediated defense mechanisms. Before the discovery of anti-CRISPR proteins, phages relied on acquiring mutations to reduce their binding affinity to CRISPR, but bacteria could counter this through "priming adaptation." Anti-CRISPR proteins are now recognized as the most effective strategy for ensuring phage survival during bacterial infections (Pawluk et al., 2018; Altae-Tran et al., 2023) [66,2] .

Ethical issues

CRISPR-Cas9 technology offers affordable, precise genome editing with applications spanning human health, agriculture, and environmental conservation. However, ethical concerns, especially regarding germline editing in humans and environmental impacts, must be addressed through global legislation. While regulations are necessary, they shouldn't hinder scientific progress. This article explores CRISPR-Cas9's uses and ethical dilemmas across various domains, emphasizing the need for comprehensive ethical deliberation.

Conclusion

CRISPR is becoming an indispensable tool in biological research. Once known as the CRISPR is rapidly developing into a vital tool for biological study. The programmable ability of the Cas9 enzyme, once recognized as the bacterial immune system against invasive viruses, is transforming an array of industries, including biotechnology, medical research, and the livestock industry. These days, CRISPR-Cas9 is used for more than only editing genes. Catalytically hindered inactive Cas9 has applications in chromatin engineering, imaging, gene regulation, and epigenetic editing. By learning about and comprehending these difficulties, we will be better equipped to assess the extent of their constraints and develop strategies for overcoming them. Undoubtedly, CRISPR-based technologies will continue to revolutionize biotechnological, clinical, and basic research. There are some challenges in store, though. The possible immunogenicity of CRISPR-Cas9 proteins is one such barrier. Therefore, further study must be done to deal with the safety and specificity of such technologies in tandem with the existing improvements. In addition, enough thought must be given to the ethical and social ramifications of these technologies for them to be useful to all societal strata and to benefit all of animal and humankind.

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