Applications of the CRISPR-Cas-based technology to correct aneuploid and segmental aneuploid cells: The road from lab to clinic

 Corresponding author: AHMAD MOHAMMAD KHALIL Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Applications of the CRISPR-Cas-based technology to correct aneuploid and segmental aneuploid cells: The road from lab to clinic AHMAD MOHAMMAD KHALIL * Department of Biological Sciences, Faculty of Science, Yarmouk University, Irbid, Jordan. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 Publication history: Received on 12 October 2025; revised on 18 November 2025; accepted on 20 November 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.24.2.0999 Abstract Genetic disorders pose an enormous health threat to human public health. There are presently no disorder-reducing therapy options. The purpose of this article is to review the latest applications and prospects of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR-Cas) system in the treatment of human chromosomal disorders. One of the reasons for choosing this topic is because of the lack of related literature. In this review, we first briefly describe a human chromosomal disorder and then outline the molecular architecture and mechanistic basis of CRISPR-Cas9 for its treatment. Then, successful stories in this field are described. The process begins with identifying and cleaving particular sequences on the extra copy of a chromosome. This activates a repair response that guides the cell to eradicate the chromosome without harming the other healthy chromosomes. Thus, instead of editing single genes, scientists can effectively remove a chromosomal abnormality entirely, restoring normal gene expression and cellular function. Although the CRISPR-Cas method has operated well for curing chromosomal disorders in lab settings, the clinical applications are years away because several mechanistic questions have to be answered. Before clinical applications, the methods could be tested in vivo in animal models and adapted to become a keystone for future cell-replacement therapies. Challenges and limitations have to be addressed by geneticists, bioethicists, and policymakers before human translation. Keywords: Aneuploidy; Chromosomal Disorders; Chromosome Elimination; CRISPR-Cas Therapy; Segmental Aneuploid; Trisomy World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 510 Graphical Abstract 1. Introduction 1.1. What are CRISPR/Cas Systems? In 2013, the CRISPR-Cas system’s multi-genome editing abilities made possible powerful applications in basic science, biotechnology, and medicine.[1] By using a single effector protein and a customizable single-guide RNA (sgRNA), CRISPR-Cas9 surpasses former engineered nucleases such as zinc-finger nucleases (ZFNs) and transcription activatorlike effector nucleases (TALENs) in simplicity and flexibility.[2] Several studies have shown that the CRISPR system, as a gene programming instrument, can increase the gene diagnosis capability.[3][4] A variety of the CRISPR-Cas effectors (Figure 1 and Table 1) have been innovated as technology advances and developments enable researchers to join computation and experiments.[5] The Cas 9 protein consists of two functional lobes: the recognition (REC) lobe and the nuclease (NUC) lobe.[5] Attached to the REC lobe is the NUC lobe, which shelters and protects the dual nuclease domains, human umbilical vein endothelium cells (HuvC) and HNH (named for its conserved histidine-asparagine-histidine catalytic triad), which regulate the endonucleolytic cleavage of the DNA strands.[6] The HNH domain shows target-dependent activation (i.e., remains catalytically inactive until accurate base pairing between the gRNA and target DNA is formed] [7] and cleaves the complementary strand of the DNA duplex. Together, the HNH domain aligns with the target strand and cuts the DNA in a coordinated manner with RuvC, leading to a blunt-ended DSB. The REC lobe domain facilitates gRNA binding and conformational regulation, allowing the surveillance complex to accomplish high-fidelity recognition of the DNA substrate. A key turning point came about when researchers revealed that crRNA and trans-activating crRNA (tracrRNA) could be joined into a sgRNA, producing a simplified but highly effective system for leading Cas9 to specific DNA sequences.[8] The precise targeting of Cas9 is further imposed by the protospacer adjacent motif (PAM), a small conserved DNA sequence, located directly downstream of the target site.[9] World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 511 Figure 1 Comparison of the structural properties and the target of CRISPR/Cas9, CRISPR/Cas12, and CRISPR/Cas13. Abbreviations: dsDNA: Double-Stranded DNA; ssDNA: Single-Stranded DNA; ssRNA: Single-Stranded RNA. Source: [10, 11] Table 1 Comparison of the functional properties of CRISPR/Cas9, CRISPR/Cas12, and CRISPR/Cas13. * Aspect CEISPR-Cas9 CRISPR-Cas12 CRISPR-Cas13 Target dsDNA ssDNA, dsDNA ssRNA only Protospacer restrictions PAM PAM PFS Spacer size 16-24 nt 16-24 nt 22-35 nt Pre-crRNA processing No Yes No DNA recognition sgRNA (crRNA in complex with tracrRNA) crRNA crRNA tracrRNA Yes No No Cleavage pattern Blunt-ended DSB Sticky-ended DSB Degraded RNA Characteristics -No collateral cleavage -No secondary structure restrictions -Collateral cleavage -No secondary structure restrictions -Collateral cleavage -Secondary structure restrictions Application Gene editing nucleic acid detection Gene editing nucleic acid detection RNA knockout nucleic acid detection Active clinical trials Yes Yes No * Abbreviations: crRNA: CRISPR RNA; DSB: Double Strand Breaks; dsDNA: Double-Stranded DNA; nt: Nucleotide; PAM: Protospacer Adjacent Motif; PFS: Protospacer Flanking Site; ssDNA: Single-Stranded DNA; ssRNA: Single-Stranded RNA; tracrRNA: Transactivating RNA. Data extracted from:[10][11] One of the landmark events was the introduction of nucleases like Cas12 and Cas13 (Figure 1), which bind to crRNA targets, into nucleic acid detection systems. [11][12][13] These nucleases also differ in the cleavage pattern and PAM range. The Cas12 nuclease possesses unique enzymological features and can detect nucleic acids by cutting of surrounding single-stranded DNA (ssDNA).[14] On the other hand, Cas13 is an RNA-guided RNA-targeting endonuclease. Cas13a′s specificity for RNA, not including DNA changes, makes it a fascinating tool for transient knockout (KO) in research and possible therapeutic applications for diseases involving RNA.[5] The discovery of the trans-cleavage mechanism of these enzymes made them a promising and powerful proposal for the next generation CRISPR-based diagnostics (CRISPR-Dx). While Cas13’s RNA specificity is perfect for transient KO and diagnostics, its collateral cleavage can sometimes lead to off-target RNA degradation, restraining its therapeutic potential.[15][16] The main functions of the CRISPR-Cas could be summarized as follows: Targeting a definite sequence and cutting the relevant site, producing double-strand breaks (DSBs). The generated breaks are resolved by alternative and competing repair routes, largely error-prone non-homologous end joining (NHEJ) and high-reliability homology-directed repair (HDR).[17] The NHEJ-facilitated insertion or deletion incidences for gene disruption have proven invaluable. However, World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 512 the therapeutic goal of precise gene correction dictates the enhancement of HDR efficacy in mammalian cells, where this pathway is essentially limited by cell-cycle restrictions and enzymatic competition. The HDR is predominantly active during the S and G2 phases of the cell cycle once a sister chromatid is available as a template.[18] Despite its precision, HDR is inherently less effective in somatic cells owing to cell cycle limitations and pathway competition, restricting its usefulness in therapeutic and research contexts that require accuracy.[17] To overcome these limitations, several approaches have emerged to encourage HDR over NHEJ, thus improving the efficacy and fidelity of CRISPRCas9-based genome editing (GE). Many of these strategies, though potent, may cause unwanted genome-wide perturbations or increase the risk of chromosomal rearrangements because of excessive end resection or long repair activity.[19] Nevertheless, these approaches hold huge promise for applications in gene therapy, particularly when limited to ex vivo contexts such as patient-derived stem cells [20]. Recent advances in HDR-stimulating and NHEJovercoming strategies, describing developing solutions to CRISPR-Cas9’s systemic drawbacks, can be found in recent reviews.[8][11] 1.2. What are the Genetic Disorders? Genetic disorders include a broad spectrum of diseases. Mutations that occur in the genome of an organism can be classified into two major classes: gene mutations and chromosomal mutations. [21] The gene mutation is a change of the nucleotide sequence of a gene in the DNA, whereas a chromosomal mutation is a change of the structure or number of chromosomes. The effect of chromosomal mutations is greater than that of gene mutations because the degree of mutation in chromosomal mutations is high; only one gene is affected in the first, while several genes are affected in the second,[21] leading to carcinogenesis, morbidity, or mortality.[22] The influence of genetic chromosomal mutations on human health can be great on many things throughout the body, including growth and development, and physical appearance. Chromosomal mutations mostly result from errors in mitosis or meiosis.[21] There are four forms of structural chromosomal alterations: deletions, duplications, inversions, and translocations. Translocations involve the interchange of pieces of chromosomes between non-homologous chromosomes. Duplication is the generation of extra copies of a chromosome segment. Broken chromosomal portions are rotated 180ο and again inserted into the same site of the chromosome during inversions. Pericentric inversions contain a centromere. In the paracentric inversions, the inverting segment does not involve the centromere. The numerical chromosome alterations are called aneuploidy, defined as having in a single cell one or more extra or missing chromosomes. Lacking one of the chromosomes is known as monosomy (2n-1), while gaining an extra chromosome is called trisomy (2n+1). 1.3. CRISPR/Cas9-Based Therapy Genetic diseases often lead to substantial medical and economic burdens on patients and healthcare systems. Major significant advances in GE approaches to correct single-gene errors of rare monogenic disorders have been reported in the last decade, starting with in vitro experiments and advancing to in vivo studies and clinical trials.[23] In comparison, only a few efforts have been devoted to genetically correcting the improper dosage of genes for a whole chromosome in aneuploid cells.[23] This is because each chromosome carries hundreds of genes, and the addition or deletion of even a single chromosome disrupts the delicate balance of gene products in cells. Earlier reported methods to precisely delete a whole particular human chromosome or remove most of a chromosome were laborious and extremely inefficient. These methods can be classified into two main classes: nuclease-based and nuclease-dCas9 strategies to tether a definite protein to a chromosome of desire.[24] Nuclease-mediated strategies include the Cas9 system with one or several Chromosome-specific sgRNAs, where one or many DNA DSBs are produced in the arm,[23][24][25] or the (pericentromeric) region of the targeted chromosome,[26] causing total or partial loss of the targeted chromosome. Other such strategies include a mixture of centromere-proximal and centromere-distal DSBs [27] or artificial telomere sequence integration into the peri-centromere.[27][28] Targeted entire chromosome deletion could be accomplished by four main approaches: • Introducing an oppositely oriented locus of X over P (loxP) sites, which are specific 34 bp DNA sequences utilized in genetic engineering, into the intended chromosome, followed by a site-specific recombinase technology.[29] Incorporation of the inverted loxP into the chromosome was carried out by traditional gene targeting, CRISPR/Cas9 nickase system, and TALEN technology.[29][30] Chromosomes with inverted loxP locations can be converted to unstable acentric and dicentric chromosomes in a Cre recombinase-dependent World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 513 fashion. The unstable chromosome will be eliminated during cell division, thus enhancing the exclusion of the target chromosome having the inverted loxP sites. [22] • Introducing a Thymidine Kinase and Neomycin (TKNEO) resistant transgene, a specific fusion gene utilized in genetic engineering for positive or negative antibiotic selection, into one copy of a chromosome of interest, followed by drug selection of chromosome-elimination clones via spontaneous chromosome deletion.[31] • Both of these strategies are conventional methods for GE that demand two-step manipulation and give low yields of chromosome-deleted cells, and are therefore inappropriate for in vivo studies.[23] • Using ZFN-based knock-in (KI) of the X-inactive specific transcript (XIST) gene, located on the human X chromosome, to silence one copy of chromosome 21.[32] This procedure added another potential chromosomal therapy for Down Syndrome (DS), allowing the accurate and efficient insertion of the XIST gene into chromosome 21 in Induced Pluripotent Stem Cells (iPSCs). • Using multiple gRNAs of the CRISPR/Cas9 system CRISPR/Cas9 system for initiating multiple DNA DSBs into the target location are designed for a whole region of the target and consequently eliminate extra chromosomes.[23][24] By suppressing the cell’s intrinsic DNA repair mechanisms, scientists expanded the efficacy of removing the additional chromosome. Their findings indicated that this process restored natural gene expression and cellular function in edited cells. Genome editing utilizing CRISPR/Cas9 reportedly brings great loss of entire chromosomes.[33] Whole chromosome loss is usually discussed in the context of the severe and potentially fatal side effects of CRISPR/Cas9mediated gene therapy attempts.[34] “Trisomic rescue” was the first time the elimination of a whole chromosome was achieved using CRISPR. Although research directed toward getting rid of supernumerary chromosomes from trisomic cells is limited,[35] several success stories have been reported in the literature. The emerging biomedical application of CRISPR systems in this field is outlined in Figure 2 and will be summarized in the sections that follow. 2. Methods This review article used the major databases, including Google Scholar, PubMed, Scopus, and Web of Science to search literature published from 2005 to 2025. The keywords used in different combinations were “Aneuploidy”, “CRISPR”, “chromosome disorder”, “chromosome syndrome”, “genome editing”, and “therapy”. Following PRISMA guidelines, 281 papers were vigorously evaluated. Articles published in None-English-language as well those with insufficient data were excluded. Only 121 articles fulfilled the inclusion criteria following application of the exclusion criteria (Articles published in None-English-language or having insufficient data). The selected studies were organized into thematic groupings covering specific chromosome syndrome. 3. Results 3.1. Down Syndrome Human trisomy 21, responsible for DS, is the most frequent genetic cause of cognitive impairment and continues to be a key focus for researchers. In about 95% of the cases, it occurs when an individual has an extra copy of chromosome 21 in every cell of the body, where there should normally only be two copies. Down syndrome is the only survivable trisomy condition. It is one of the most frequent genetic disorders in humans, affecting about 1 in 700 live births.[36] Although this condition can be easily diagnosed early in development by genetic testing for aneuploidy, many treatments or modifying practices are symptomic interferences.[37] World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 514 Figure 2 Therapeutic gene editing using CRISPR/Cas9 offers theoretically promising avenues for treating chromosomal disorders by correcting the underlying genetic defect. Source: Author’s design In general, the exact set of genes causing the clinical phenotypes in DS, which is influenced by the increase in chromosome copy number, is mostly unknown. But it has been reported [22] that three copies of the RUNX1, ETS2, and ERG genes on chromosome 21 co-activate with somatic GATA1 mutations on the X chromosome elevate the risk of leukemia in DS. TALEN-based elimination of the GATA1 mutation in DS patient-stemmed human iPSCs (hiPSCs) restored the right hematopoiesis even in the presence of trisomy 21.[38] For clinical applications of this procedure, further improvements are needed for the complete silencing of the desired chromosome 21. In HeLa cells with three copies of chromosome 21, integration of the GFP and Herpes Simplex Virus-thymidine kinase (HSV-tk) gene holder encased by two inverted loxP positions on the homologous arms of chromosome 21 by homologous recombination following CRISPR/Cas9 system-mediated DNA nicking, produced unstable acentric and dicentric chromosomes for chromosome 21 elimination and a final normal 46 genotype.[22] A similar result was obtained by applying a CRISPR/Cas9 approach-mediated chromosomal deletion using two sgRNAs targeting repetitive sequences on chromosome 21 in the DS-iPSCs, converting trisomy 21 to disomy at a frequency of approximately 15%.[22] It was found that even when recombination happens between homologous chromosomes, every copy of chromosome 21 typically has unique gRNA targets. This feature offers the advantage of targeting only one of the three chromosome copies. CRISPR/Cas9-based targeted chromosome deletion dramatically altered researchers’ ability to produce disease models in various organisms, such as non-human primates. Moreover, this strategy would provide a possible therapeutic strategy to cure aneuploidy diseases, including DS, Klinefelter syndrome (XXY), and Jacobs syndrome (XYY}. [31][32][39] Yet, the off-target effects (OTEs) of the CRISPR/Cas9 system and the efficiency of aneuploidy rescue applied in this approach should be assessed and developed for use in basic and clinical research on aneuploidy disorders. Furthermore, researchers applied a ZFN-mediated strategy to KI the XIST gene on the Dual-specificity Tyrosine Phosphorylation Regulated Kinase 1A (DYRK1A) gene locus of chromosome 21, causing Barr body development to silence the additional copy of chromosome 21 in DS-iPSCs.[22] Remarkably, the trisomy-biased chromosome loss through iPSC reprogramming was also enforced in DS iPS cells, which lost the additional copy of chromosome 21. Interestingly, the long-term path of DS-iPSCs with trisomy 21 also led to T-Cell Lymphoma (TCL), thereby correcting the karyotype.[22] However, the mechanisms that cause TCL remain unclear. An approach that combines CRISPR-Cas9-based chromosome tagging and Microcell-Mediated Chromosome Transfer (MMCT) from hiPSCs as chromosome donor cells directly to other hiPSCs as chromosome recipient cells.[40] This procedure allows the more straightforward production of hyperaneuploidy disease models, genetic chromosome disorder models, and cells containing familial chromosomes. This strategy included tagging chromosome 21 and chromosome Y by CRISPR-Cas9 and moving human/mouse Artificial chromosome, chromosome 21, chromosome X, and chromosome Y, for which no previous reports confirm full-length introduction. This approach enables the study of rare diseases and promises to offer new perceptions into early developmental mechanisms by establishing a comprehensive set of important chromosomes/regions in hiPSCs. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 515 It was found that an overdose of genes in aneuploid cells could be adjusted by the insertion of a bulky, inducible XIST transgene into the targeted chromosome to silence one copy of it.[32] However, the efficacy of the targeted insertion was very low, and some genes may have escaped the inactivation process. A new application of the CRISPR/Cas9 tool was reported, (23) which involves the selective removal of a single specific chromosome by multiple DNA cleavages on the intended chromosome in cultured cells, embryos, and in vivo tissues. The cleavages made by these researchers were produced by a single sgRNA or two sgRNAs that recognized multiple chromosome-specific locations, or by a mixture of 14 sgRNAs, with each recognizing one specific locus. Thus, the CRISPR/Cas9 approach proved its capacity to eliminate human chromosome 21 (hChr21) in hiPSCs with trisomy 21, offering a novel method to generate animal models and treatments for aneuploidy. It is known that Down patients with partial trisomy and/or mosaic form showed a 1.6 Mb vital region on chromosome 21q22 with maximum level of transcription in DS.[41] This region is called the DS Critical Region (DSCR). Most of the genes found in DSCR are linked to the development of the brain and play an important role in learning and cognitive activities, and loss of them causes neuropathological issues in DS. Even some recent investigations confined the DSCR region to a very short distance, duplicated in all DS patients.[42] Therefore, consideration of a smaller number of genes, which are more specific for DS phenotypes, appears to be more logical for alterations to correct adverse phenotypes. Based on previous studies about more principal genes that have a higher impact in generating DS clinical manifestations (Figure 4), a method that can significantly lessen the main influences and clinical features in DS was suggested.[34] Two different CRISPR/Cas9 systems were applied concurrently in a protein-based delivery technique to both the brain cell culture and the DS mouse model. The first is a gene deletion approach and includes a Cas9 protein + two distinct sgRNAs for cutting off two ends of the DSCR region in chromosome 21, and the second involves a Cas9 protein + a sgRNA + a template dsDNA to excise a nonfunctional region of the centromere-proximal half of 21q. After that, the latter region was replaced by a newly planned DNA construct, which included some main genes in chromatin remodeling and epigenetic effects for the inactivation of one additional chromosome 21 (Figure 3, Table 2). Figure 3 The devised DNA construct for substituting specific, nonfunctional locations on chromosome 21. This comprises an enhancer for brain cells special promoter (i.e., MECP2 promoter), MECP2 expression promoter, XIST gene as a major epigenetic modifier factor for causing inactivation on the related chromosome, and a long non-coding RNA gene like L1 gene on behalf of a specific element for enhancing Xist RNA function. Also, an RNA polymerase terminator gene would be required to regulate the transcription activities. * L1: Like1; MECP2: Methyl CpG binding protein 2; XIST: X-inactive specific transcript. Source: Adapted from [43] In this way, the extra chromosome 21 would be repressed or inactivated by omitting the DSCR region, followed by insertion or KI of the regulatory DNA construct. However, inactivation can be observed in other chromosomes too, since the female's X chromosome is not the only deactivated chromosome example in the human body. Research showed that the inactivation could also occur in males’ germ cells as meiotic sex chromosome inactivation (MSCI) form.[44] World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 516 Table 2 Expressed and silenced genes on extra chromosome 21 in Down Syndrome patients. Gene region* To be expressed To be silenced MECP2 enhancer Enhancer for transcription from the MECP2 promoter - MECP2 expression promoter Selective expression of desired genes in neurons - XIST Aggregates histone changes, which inhibit transcription - L1 Overexpression of this gene is implicated in Xist RNA dispersal along the chromosome. - RNA polymerase terminator Termination of transcription of desired genes - All other genes: DSCR1, RCAN1, PCP4, TTC3, MNB, SOD, ETS2, SIM2, DYRK1, DSCAM, Other DSCR genes, Genes within BCE1 and MX1 - Critical regions involved in DS * BCE1: Beta-Secretase 1; DSCAM: DS Cell Adhesion Molecule; DSCR: Down syndrome critical region; DYRK1: Dual-specificity tyrosine phosphorylation–regulated kinase 1; ETS2: Erythroblast Transformation Specific 2; L1: Like1; MECP2: Methyl CpG binding protein 2; MNB: Minibrain; PCP4: Purkinje cell protein 4; MX1: Myxoma 1; RCAN1: Regulator of Calcineurin 1; SIM2: single-minded 2; SOD: Superoxide dismutase; TTC3: Tetratricopeptide Repeat Domain 3; XIST: X-inactive specific transcript. Source: Data extracted from:[43]. As discussed above, CRISPR/Cas9 can specifically cleave assigned sites in a DNA sequence through the direction of sgRNA. CRISPR/Cas9 with specially designed sgRNAs can target repetitive sequences and break all the repeats in the chromosome. As a result, the chromosome would be damaged beyond the limits of the intrinsic cell repair. Consequently, the targeted chromosome cannot join in the DNA replication during cell division, and will vanish in the offspring. Using CRISPR-Cas9, scientists successfully removed extra copies of chromosome 21 in DS cell lines, reestablishing normal gene expression.[35] This “surgical” procedure is carried out in utero (in the uterus during embryonic life). What makes this breakthrough specifically groundbreaking is that researchers achieved this chromosomal correction not just in lab-grown PSCs, but also in skin fibroblasts derived from a child with DS. The technique was capable of identifying and precisely targeting the duplicated chromosome, making certain that after removal, each cell kept one copy from each parent rather than two identical copies. The treated cells reverted from trisomy (three chromosomes) to disomy (two chromosomes), restoring normal karyotypes. The results were certified using standard tools in cytogenetics, such as FISH, Short Tandem Repeat (STR) profiling, and the G-banding technique. Edited cells returned to normal patterns of protein production. They also displayed better survival levels in certain tests, suggesting that the surplus genetic burden was successfully alleviated. In follow-up tests, investigators [35] examined how gene activity differed after eliminating the extra chromosome. The corrected cells developed slightly faster and had a shorter doubling time compared to the untreated trisomy cells, suggesting that getting rid of the extra chromosome may relieve the biological stress that reduces cell growth. This idea is supported by the finding that the corrected cells also generated fewer reactive oxygen species, which are harmful byproducts associated with cell damage and aging, reflecting perfected mitochondrial function and an overall boost in cell fitness. Furthermore, the genes linked to nervous system development were dialed up, while those tied to metabolism became less active. This change in gene expression may perhaps explain how fixing the chromosomal imbalance influences the cell’s general behavior. It also strengthens earlier results that extra copies of chromosome 21 disturb brain growth during early fetal development. Although promising, the technique is not yet ready for use in living organisms and clinical applications, as it may also change the remaining chromosomes. But researchers suppose that similar strategies could eventually be used in neurons and glial cells or other tissues, paving the way for potential future treatments for persons with DS. Since the presence of a third copy of a chromosome has effects on many things in the body, including growth and development as well as physical appearance, the question remains, “Would a person who underwent this therapy begin changing physically?” World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 517 3.2. Edwards Syndrome Edwards syndrome (ES) or Trisomy 18, is the second most common autosomal trisomy abnormality after trisomy 21 (DS).[45] In rare situations, trisomy 18 may come from a biological parent (through balanced translocation). If a family already has had one child with triplicate chromosome 18, the doctor may suggest having genetic testing to evaluate the chances of having another child with a similar disorder.[46] Children born with ES require specialized care to deal with their unique symptoms soon after they are born. Medical science has not discovered a cure for ES.[45] However, in some cases, surgical mediation may be needed to address particular health issues linked with ES. Scientists are exploring conventional therapy strategies for ES that have concentrated on managing symptoms instead of directly targeting the main genetic cause. CRISPR-Cas9 gene editing technique holds great promise in dealing with ES at a molecular level. It enables researchers to exactly modify DNA sequences and possibly correct the genetic errors linked to the syndrome. Although still in the infancy stages, CRISPR-Cas9 gene editing sets up an era for the treatment and intervention with ES. By exactly modifying the defective genes responsible for the disorder, these therapies can provide more efficient and personalized treatment options. It has been determined that CRISPR/Cas9 can be utilized to target and remove an allele-specific chromosome in trisomy 18 embryos.[47] Recently, a Japanese research team at Kyoto University and the RIKEN Center used the same strategy employed for chromosome 21 elimination to delete the extra chromosome 18 trisomic cells.[48] Utilizing the intrinsic cellular repair mechanisms, specifically designed gRNAs in CRISPR-Cas9 to target, bind repetitive sequences unique to chromosome 18, triggering Cas9-induced breaks. The engineered CRISPR-Cas9 systems were delivered via viral vectors to remove the additional chromosome in in vitro cell cultures. Though still in the experimental stages, CRISPR-Cas9 GE opens up an era of potential for the treatment and prevention of ES. Most treatments such as these would be performed by 20 weeks of gestation since most duplications appear in the first stage of uterine development. 3.3. Klinefelter Syndrome Klinefelter syndrome (KS) is a common congenital disease that results when a male at birth has at least one extra X chromosome added to a normal male karyotype, 46, XY.[49] Around 90% of males with KS have the 47, XXY karyotype. The XXY aneuploidy is the most prevalent disorder of sex chromosomes in humans, with a prevalence of one in 600 males.[49] The additional X chromosome in 47, XXY, results occasionally from either meiotic nondisjunction when a chromosome fails to segregate during the first or second division of gametogenesis or from mitotic nondisjunction in the emerging zygote.[50] If the diagnosis is not performed prenatally, the 47, XXY males may display a variety of subtle clinical signs, such as smaller testicles, which are unable to produce normal levels of the hormone testosterone. There are presently no gene therapy options for KS. However, there are some ongoing or planned clinical trials searching for the use of GE technologies such as CRISPR-Cas9 to repair or delete genes on the extra X chromosome. It was reported [51] that the Y chromosome could be eliminated in embryonic stem cells (ESCs) and zygotes by CRISPR/Cas9-based GE. Similarly, the Y chromosome completely deleted by multiple CRISPR/Cas9-based DNA cuts on the targeted chromosome in ESCs, cells in vivo, and zygotes with high efficacy.[23] It was demonstrated that using the same strategy, one of two homologous X chromosomes, which is characteristic of KS (XXY), in mouse embryos with the XX karyotype could be effectively removed.[52] The majority of the chromosome-specific repeated sequences are found in non-coding regions, and thus these side effects could be reduced by targeting the non-coding DNA sequences within short regions (< 2 kb) without noticeable biological functions.[23] Alternatively, these mutations in the remaining X chromosome (KS) cells could be avoided by using sgRNAs that can target only one of the two or one of the three homologous chromosomes. The Y chromosome could be deleted by using 14 single-target sgRNAs. But, minimizing the number of sgRNAs and improving the efficacy of chromosome removal may make this strategy more applicable. These attempts are still in their experimental stages and have not yet been confirmed for their safety and effectiveness. During reprogramming KS-modeled XXY mouse primary fibroblasts into iPSCs, aneuploidy was corrected at a rate of approximately 20%.[53] The euploid XY iPSCs produced from the KS model mice differentiated into functional testicular sperm capable of producing the F1 and F2 generation pups. Unfortunately, GE using CRISPR/Cas9 has been reported to cause enormous chromosome deletions and, rarely, X and autosome entire chromosome elimination.[31][51] World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 524 Key Limitations Although the CRISPR/Cas toolbox for treating chromosomal disorders may become an important area of future investigation, it includes key challenges that must be addressed for clinical applications. These limitations can be summarized as follows: The first major challenge is how to selectively target one of two or three chromosomes, and the molecular and cellular characteristics, such as location, function, gene expression, etc. In accordance with these desirable process considerations, possible gene therapy for chromosomal disorders ranges from downregulation of inhibitory miRNAs and transcriptional activation by ZFNs, TALENs, or CRISPR-Cas9 technology. A critical practical limitation of the CRISPR/Cas9-mediated method is how to induce multiple DNA cuts efficiently. Also, the efficacy of chromosome deletion differs among diverse repeat sequences, and it is not easy to predict whether these repetitive sequences “work well”. In addition, only a few cell types tolerate huge chromosome-scale deletions. Cells from non-inbred species like humans may behave in unpredictable and unphysiological modes when a whole chromosome becomes haploid following a chromosomal deletion. Although the dCas9-based methods are attractive for evading DSBs, which may lead to unanticipated key genomic changes,[110] they cannot control whether to gain or lose the target chromosomes. Unanticipated endonuclease activity is noticed at the nontarget allele even after the target chromosome is lost. Thus, defending nontarget alleles from Cas9-induced DSBs.[35] These issues may be tackled by epigenomic methods that do not cause DSBs. When the target chromosome is not deleted by Cas9 treatment, alternatives are established in the remaining target chromosome, and critically, the target genome is substituted by a sequence no longer identifiable by the employed gRNA.[35] The failure of removal of the extra chromosome may result from off-target editing, which potentially affects unintended genomic loci. Although scientists did not detect evident off-target mutations or chromosome rearrangements in the chromosome-removed cell lines and mice,[23] it will be necessary to evaluate potential OTEs before CRISPR/Cas9-based chromosome deletion could be used clinically. The therapeutic translation of CRISPR-Cas9 relies on mitigating nuclease promiscuity through elevated-fidelity alternatives and chemical guide modifications.[111] To enhance the specificity of the target nucleic acid, the CRISPR protein is modified, and an efficient delivery mode of the CRISPR-Cas system is used to minimize the off-target problems.[112] Base editors (BEs) provide a way to edit single nucleotides without running the danger of causing DSBinduced toxicity. Still, base editing faces some of the similar challenges already mentioned for CRISPR systems, including OTEs, more so with Cytosine BEs than Adenine BEs.[113] Moreover, base editing is restricted by its packaging by viral vectors due to the large size of BEs.[114] As an alternative strategy that avoids the potential off-target problems, it has been stated that iPSC reprogramming possibly corrects structural and numerical chromosomal anomalies. [53] The previous studies [35] concentrated on subtelomere targeting and without insight into how focusing on other chromosomal zones and their combinations, such as peri-centromeric and centromeric regions, affects chromosome exclusion efficacy. Also, they were limited by the usage of only two cell types (iPS cell line and skin fibroblasts). The experiments provided proof-of-concept for the “Trisomic rescue” strategy. Assessing this approach in clinically related cell types, like neurons and glial cells, would significantly promote its ability for translational applications. In addition, the WGS analysis was not made on cells after all treatments.[35] Thus, comprehensive data on genome alterations across all cells utilized in these investigations may not be enough. The restrictions associated with the delivery of the editing components. Most of the Cas proteins are large molecular weight proteins (∼4.2 kb), limiting the CRISPRs to the size of the carrier viral genes.[115] To overcome this difficulty, researchers have to develop low molecular weight CRISPR orthologs or split-Cas systems, which usually compromise activity or specificity [111]. Researchers have responded to this obstacle by using nonviral inventions such as lipid nanoparticles (LNP), gold NPs, and polymeric carriers. These modalities offer promise for the transient delivery of Cas RNP complexes and reducing OTEs. However, attaining tissue-specific targeting, endosomal evasion, and effective cellular uptake remains an obstacle to robust in vivo editing.[116] Overcoming immunological barriers to bacterial nucleases and optimizing delivery means is important for clinicalgrade uses. Improvements of the CRISPR tool, the efficacy of the CRISPR-Cas system must be enhanced to reduce the immune response. It was indicated [117] that more than 50% of the human subjects had preexisting anti-Cas9 antibodies against the most commonly used Cas9 orthologs. The use of the immunogenic epitope-engineered Cas9 orthologs with decreased antigenicity and inducible anti-CRISPR modules reduces virus-associated immunogenic problems and progressively regulates editing.[118] To confront immunogenicity, researchers have investigated World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 525 humanized and engineered Cas orthologs with diminished antigenicity. For example, Cas12a from Acidaminococcus sp. shows distinct PAM requirements and reduced immunogenicity. Another potential use of the CRISPR technology would be to model evolutionary changes to chromosome. This is, in particular, a significant ethical issue regarding human genome alterations. With somatic edition, any possible risk would be enclosed within the individual after informed consent to participate in the therapy. These considerations have led to debate over both pros and cons, emphasizing the monetary cost, efficacy, access to this resource, and the need to create regulating legislation.[119] On one hand, some people believe that chromosomal disorders are quite life-threatening and also quality-of-lifedestroying. CRISPR is just a practice of splicing genetic code into a gene (or in the case of trisomy, cutting up an extra chromosome). This CRISPR treatment most likely would be a technique done in the womb (during fetal development) can’t change DNA after the child has been born. On the other hand, critics believe CRISPR is “evil” since embryonic editing not only eliminates autonomy in the decision-making process of the later-born people, but also allows unpredictable and permanent side effects to be passed down through generations. Other opponents argue that many people with DS wouldn’t consider it a “disease” or necessarily a death sentence; they need to be “fixed”. So, the prospect of human GE will probably become a conflict between the government and citizens about the issues of ethics, patient rights, and medical “freedom,”. Still, some other people imagine the CRISPR/Cas9-mediated chromosome elimination approach being applied in conjunction with gene drives, as deletion of the Y chromosome could result in the disappearance of males and collapse of mosquito populations that command global attention with remarkable consistency. Regulatory barriers (and bans) to achieving CRISPR’s hopes. The reported breakthrough in GE technology-mediated trisomy rescue was achieved in vitro (in the lab) and is still far from being approved for application in humans. Among these approaches, KaryoCreate a recently reported advanced chromosome eradication technique using the dCas9 system is incapable of targeting a chromosome; for example, chromosome 21, due to the absence of particular repetitive sequences in the pericentromeric region proper for gRNA design.[120] Multiple regulatory obstacles stand between the appealing technology and its widespread clinical application. Governmental organizations in many countries are forbidden from financing embryonic editing research, restricting researchers to private funding.[121] 5. Conclusion and Implications for Future Earlier studies focused on the outcomes of karyotype correction by chromosome deletion without sufficiently addressing the factors defining chromosome repair versus loss. More studies will be necessary to add a temporal dimension, i.e., comprehending how genomics change during development. The directions of the next investigations have to stress understanding how Cas9-handled chromosome s are lost and the results of Cas9-cured chromosome left in the genome. Previous work was restricted to the manipulations of only iPS cells and skin fibroblasts. In the future, research must intensify the study of clinically related cell types, like neurons and glial cells, before applying CRISPR in clinical trials. Also, future studies should investigate the effect of genes involved in the DSB repair machinery on the degree of chromosome excision. Another potential option in the future could be editing the disease-causing genes of an affected fetus in utero, where embryos are produced, and then CRISPR GE is deployed to eliminate the genetic mutation from affected preimplanted embryos. Following this, the edited embryos are implanted. But uses of CRISPR/Cas9 on human embryos are still largely hypothetical. Finally, prospective parents may in the future be able to choose CRISPR’s somatic therapeutic intervention for a diseased child postnatally (after birth). Procedures to achieve somatic GE applying CRISPR in children or adults are divided into two groups: ex vivo and in vivo. The ex vivo treatments are the most likely to become a significant option for expectant parents in the near future. Ex vivo GE involves separating a patient’s cells, correcting them in culture, and then transfusing edited cells back into the patient. Today, there are several labs around the world working with CRISPR on Usher's Syndrome, a condition that causes loss of vision and hearing. Applying such radical genetic interventions to living organisms imposes risks that need to be carefully examined. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 526 The human germline editing for therapeutic purposes continues to be highly controversial. It is ethically unfavorable in its current state, and its debates may not be considered until enough long-term research on the ongoing somatic CRISPR therapy clinical trials is conducted. Extensive research on chromosome editing in live rodents and nonhuman primates is needed to test potential “treatments” for all kinds of conditions in humans. While they may be years away from obtaining any kind of approval by the FDA or Ethics committees for examining human subjects Advocates for people with chromosomal syndromes emphasize that this revolution should not be viewed as a quest to “eradicate” individuals with these conditions, but rather as a means to offer families more options and improve quality of life. If some therapy for a genetic disorder is available, why not do it to prevent suffering? There is no human ugliness in using this technology. There is neither “eugenics” nor religious “evil” going on. Compliance with ethical standards Acknowledgments The author apologizes to those colleagues whose work is not cited due to restrictions on the number of references. Disclosure of conflict of interest The author declares no conflict of interest. Data availability statement No datasets were produced or analyzed during the current study. Funding This work received no funding from internal or external sources. Author Contributions The author confirms that he was solely responsible for the conception, design, analysis, interpretation, drafting, and final approval of the article. References [1] Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819–823. [2] Gupta RM, Musunuru K. Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9. The Journal of Clinical Investigation. 2014; 124:4154-4161. [3] Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, et al. RNA targeting with CRISPR-Cas13. Nature. 2017;550(7675):280–284. [4] Lin P, Qin S, Pu Q, Wang Z, Wu Q, Gao P, et al. CRISPR-Cas13 Inhibitors block RNA editing in bacteria and mammalian cells. Molecular Cell. 2020;78(5):850–861. [5] Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJJ, et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nature Reviews Microbiology. 2020;18(2):67–83. [6] Hidalgo-Cantabrana C, Barrangou R. Characterization and applications of Type I CRISPR-Cas systems. Biochemical Society Transactions. 2020;48(1):15–23. [7] Azeez SS, Hamad RS, Hamad BK, Shekha MS, Bergsten P. Advances in CRISPR-Cas technology and its applications: revolutionising precision medicine. Frontiers in Genome Editing. 2024; 6:1509924. [8] Park S-J, Lee GE, Cho SM, Choi E-H. Recent applications, future perspectives, and limitations of the CRISPR-Cas system. Molecular Therapy. 2025; 36(3):102634. [9] Vink JNA, Baijens JHL, Brouns SJJ. PAM-repeat associations and spacer selection preferences in single and cooccurring CRISPR-Cas systems. Genome Biology. 2021; 22:281. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 527 [10] Tyumentseva M, Tyumentsev A, Akimkin V. CRISPR/Cas9 landscape: Current state and future perspectives. International Journal of Molecular Sciences. 2023; 24(22):16077. [11] Khakil A. Insights from stem cell and CRISPR-based therapies for diabetes mellitus: A systematic review. Journal of Biotechnology and Biochemistry. 2025; (In Press) [12] Hillary VE, Ceasar SA. A Review on the mechanism and applications of CRISPR/Cas9/Cas12/Cas13/Cas14 proteins utilized for genome engineering. Molecular Biotechnology. 2023; 65:311-325. [13] Navarro C, Díaz MP, Duran P, Castro A, Díaz A, Cano C, et al. CRISPR-Cas Systems: A Functional perspective and innovations. International Journal of Molecular Sciences. 2025; 26(8):3645. [14] Liu L, Li X, Ma J, Li Z, You L, Wang J, et al. The molecular architecture for RNA-guided RNA cleavage by Cas13a. Cell. 2017;170(4):714–26e10. [15] Ai Y, Liang D, Wilusz JE. CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells. Nucleic Acids Research. 2022; 50:e65. [16] Li Y, Xu J, Guo X, Li Z, Cao L, Liu S, et al. The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model. Genome Biology. 2023; 24:20. [17] Liao H, Wu J, VanDusen NJ, Li Y, Zheng Y. CRISPR-Cas9-mediated homology-directed repair for precise gene editing. Molecular Therapy. 2024; 35:102344. [18] Li G, Yang X, Luo X, Wu Z, Yang H. Modulation of cell cycle increases CRISPR-mediated homology-directed DNA repair. Cell and Bioscience. 2023; 13:215. [19] Liu W, Li L, Jiang J, Wu M, Lin P. Applications and challenges of CRISPR-Cas gene-editing to disease treatment in clinics. Precision Clinical Medicine. 2021; 4:179-191. [20] Valenti MT, Serena M, Carbonare LD, Zipeto D. CRISPR/Cas system: An emerging technology in stem cell research. World Journal of Stem Cells. 2019; 11:937-956. [21] Merrick, L, Campbell A, Muenchrath D, Fei S, Suza W. Mutations and Variation. In: Suza, WP, Lamkey KR, (Eds). Crop Genetics. Iowa State University Digital Press. DOI: 10.31274/isudp.2023.130 [22] Akutsu SN, Fujita K, Tomioka K, Miyamoto T, Matsuura S. Applications of genome editing technology in research on chromosome aneuploidy disorders. Cells. 2020; 9(1):239. [23] Zuo E, Huo X, Yao X, Hu X, Sun Y, Yin J, et al. CRISPR/Cas9-mediated targeted chromosome elimination. Genome Biology. 2017;18(1):224. [24] Truong MA, Cané-Gasull P, Lens SMA. Modeling specific aneuploidies: from karyotype manipulations to biological insights. Chromosome Research. 2023; 31(3):25. [25] Nahmad AD, Reuveni E, Goldschmidt E, Tenne T, Liberman M, Horovitz-Fried M, et al. 2022. Frequent aneuploidy in primary human T cells after CRISPR-Cas9 cleavage. Nature Biotechnology. 40(12):1807–1813. [26] Girish V, Lakhani AA, Thompson SL, Scaduto CM, Brown LM, Hagenson RA, et al. 2023. Oncogene-like addiction to aneuploidy in human cancers. Science. 2023; 381(6660):eadg4521. [27] Eleveld TF, Bakali C, Eijk PP, Stathi P, Vriend LE, Poddighe PJ, et al. Engineering large-scale chromosomal deletions by CRISPR-Cas9. Nucleic Acids Research. 2021; 49 (21):12007–12016. [28] Shih J, Sarmashghi S, Zhakula-Kostadinova N, Zhang S, Georgis Y, Hoyt SH, et al. Cancer aneuploidies are shaped primarily by effects on tumour fitness. Nature. 2023; 619(7971):793–800. [29] Sato H, Kato H, Yamaza H, Masuda K, Nguyen HTN., Pham TTM, et al. Engineering of systematic elimination of a targeted chromosome in human cells. BioMed Research International. 2017; 2017:1–5. [30] Omori S, Tanabe H, Banno K, Tsuji A, Nawa N, Hirata K, et al. A Pair of maternal chromosomes derived from meiotic nondisjunction in Trisomy 21 affects nuclear architecture and transcriptional regulation. Scientific Reports. 2017; 7:764. [31] Li LB, Chang KH, Wang PR, Hirata RK, Papayannopoulou T, Russell DW. Trisomy correction in Down syndrome induced pluripotent stem cells. Cell Stem Cell. 2012; 11:615–9. [32] Jiang J, Jing Y, Cost GJ, Chiang JC, Kolpa HJ, Cotton AM, et al. Translating dosage compensation to trisomy 21. Nature. 2013; 500:296–300. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 528 [33] Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013; 154:1380–1389. [34] Turocy J, Adashi EY, Egli D. Heritable human genome editing: research progress, ethical considerations, and hurdles to clinical practice. Cell. 2021; 184(6):1561-1574. [35] Hashizume R, Wakita S, Sawada H, Takebayashi S-I, Kitabatake Y, Miyagawa Y, et al. Trisomic rescue via allelespecific multiple chromosome cleavage using CRISPR-Cas9 in trisomy 21 cells. Proceedings of the National Academy of Sciences (Nexus). 2025; 4, (2): pgaf022. [36] Alldred MJ, Martini AC, Patterson D, Hendrix J, Granholm AC. Aging with Down syndrome-where are we now and where are we going? Journal of Clinical Medicine. 2021; 10(20):4687. [37] Dahdouh EM. Preimplantation genetic testing for aneuploidy: a review of the evidence. Obstetrics and Gynecology. 2021; 137(3):528–534. [38] Chiang JC, Jiang J, Newburger PE, Lawrence JB. Trisomy silencing by XIST normalizes Down syndrome cell pathogenesis demonstrated for hematopoietic defects in vitro. Nature Communication. 2018; 9(1):5180. [39] Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, et al. Correction of a genetic disease in mouse via use of CRISPRCas9. Cell Stem Cell. 2013; 13:659–662. [40] Uno N, Miyamoto H, Yamazaki K, Egawa M, Kobayashi H, Kazuki K, et al. Microcell-mediated chromosome transfer between non-identical human iPSCs. Molecular Therapy. 2024; 5(4):102382. [41] Hamosh Ada SPM. Down syndrome critical region gene 4; DSCR4. Genomic Coordinates (GRCh38) 2010; 21:38. https://www.omim.org/entry/604829 [42] Pelleri MC, Cicchini E, Locatelli C, Vitale L, Caracausi M, Piovesan A, et al. (2016). Systematic reanalysis of partial trisomy 21 cases with or without Down syndrome suggests a small region on 21q22. 13 as critical to the phenotype. Human Molecular Genetics. 25:2525–2538. [43] Tafazoli A, Behjati F, Farhud DD, Abbaszadegan MR. Combination of genetics and nanotechnology for Down Syndrome modification: A Potential hypothesis and review of the Literature. Iranian Journal of Public Health. 2019; 48(3):371-378. [44] Song R, Ro S, Michaels JD, Park C, McCarrey JR, Yan W. Many X-linked microRNAs escape meiotic sex chromosome inactivation. Nature Genetics. 2009; 41:488–493. [45] Balasundaram P, Avulakunta ID. Edwards Syndrome. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing. 2025. [46] Dotters-Katz SK, Kuller JA, Grace MR, Laifer SA, Strauss RA. Management considerations for ongoing pregnancies complicated by Trisomy 13 and 18. Obstetrical and Gynecological Survey. 2016; 71(5):295-300. [47] Reshef EA, Marin D, Xu J, Treff NR, Egli D. Allele specific chromosome removal by CRISPR/Cas9 to correct Trisomy 18. Fertility and Sterility. 2023; 120(4):e284. [48] Kazuki Y. et al. Targeted elimination of chromosomes 13/18/21 using CRISPR-Cas9. Science Advances. 2024. [49] Butler G, Srirangalingam U, Faithfull J, Sangster P, Senniappan S, Mitchell R. Klinefelter syndrome: going beyond the diagnosis. Archives of Disease in Childhood. 2023; 108(3):166-171. [50] Zitzmann M, Aksglaede L, Corona G, Isidori AM, Juul A, T'Sjoen G, et al. European Academy of Andrology guidelines on Klinefelter syndrome. Endorsing organization: European Society of Endocrinology. Andrology. 2021; 9(1):145-167. [51] Adikusuma F, Williams N, Grutzner F, Hughes J, Thomas P. 2017. Targeted deletion of an entire chromosome using CRISPR/Cas9. Molecular Therapy. 2017; 25(8):1736–1738. [52] Xue C, Greene EC. DNA repair pathway choices in CRISPR-Cas9-mediated genome editing. Trends in Genetics. 2021; 37:639-656. [53] Hirota T, Ohta H, Powell BE, Mahadevaiah SK, Ojarikre OA, Saitou M, et al. Fertile offspring from sterile sex chromosome trisomic mice. Science. 2017; 357:932–935. [54] Tuke MA, Ruth KS, Wood AR, Beaumont RN, Tyrrell J, Jones SE, et al. Mosaic Turner syndrome shows reduced penetrance in an adult population study. Genetics in Medicine. 2019; 21(4): 877–886. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 529 [55] Gravholt CH, Ferlin A, Gromoll J, Juul A, Raznahan A, van Rijn S, et al. New developments and future trajectories in supernumerary sex chromosome abnormalities: a summary of the 2022 3rd International Workshop on Klinefelter Syndrome, Trisomy X, and XYY. Endocrine Connections. 2023; 12(3): https://doi.org/10.1530/EC22-0500. [56] Luo Y, Zhu D, Du R, Gong Y, Xie C, Xu X, et al. Uniparental disomy of the entire X chromosome in Turner syndrome patient-specific induced pluripotent stem cells. Cell Discovery. 2015; 1:15022. [57] Steiner M, Saenger P. Turner Syndrome: An Update. Advances in Pediatrics. 2022; 69(1):177-202. [58] Sheppard O, Wiseman FK, Ruparelia A, Tybulewicz VL, Fisher EM. Mouse models of aneuploidy. The Scientific World Journal. 2012; 2012:214078. [59] Lee HG, Imaichi S, Kraeutler E, Aguilar R, Lee YW, Sheridan SD, et al. Site-specific R-loops induce CGG repeat contraction and fragile X gene reactivation. Cell. 2023; 186(12):2593-2609. [60] Gadban A, Gunapala KM, Taylor V, Benvenisty N. Therapeutic strategies for fragile X syndrome and implications for other gene-silencing disorders. Nature Genetics. 2025; 57, 1812–1822. [61] Yrigollen CM, Davidson BL. CRISPR to the rescue: Advances in gene editing for the FMR1 Gene. Brain Sciences. 2019; 21; 9(1):17. [62] Protic DD, Aishworiya R, Salcedo-Arellano MJ, Tang SJ, Milisavljevic J, Mitrovic F, et al. Fragile X syndrome: From molecular aspect to clinical treatment. The International Journal of Molecular Sciences. 2022; 23(4):1935. [63] Tabolacci E, Pietrobono R, Moscato U, Oostra BA, Chiurazzi P, Neri G. Differential epigenetic modifications in the FMR1 gene of the fragile X syndrome after reactivating pharmacological treatments. European Journal of Human Genetics. 2005;13(5):641-648. [64] Gunapala KM, Gadban A, Noreen F, Schär P, Benvenisty N, Taylor V. Ascorbic Acid ameliorates molecular and developmental defects in human-induced pluripotent stem cell and cerebral organoid models of Fragile X syndrome. International Journal of Molecular Sciences. 2024; 25(23):12718. [65] Bak RO, Gomez-Ospina N, Porteus MH. Gene editing on center stage. Trends in Genetics. 2018; 34:600–611. [66] Shah S, Sharp KJ, Ponny SR, Lee J, Watts JK, Elizabeth Berry-Kravis E, et al. Antisense oligonucleotide rescue of CGG expansion–dependent FMR1 mis-splicing in fragile X syndrome restores FMRP. The Proceedings of the National Academy of Sciences. 2023; 120 (27):e2302534120 [67] Park CY, Halevy T, Lee DR, Sung JJ, Lee J.S., Yanuka O, et al. Reversion of fmr1 methylation and silencing by editing the triplet repeats in fragile x ipsc-derived neurons. Cell Reports. 2015; 13:234–241. [68] Xie N, Gong H, Suhl JA, Chopra P, Wang T, Warren ST. Reactivation of fmr1 by crispr/cas9-mediated deletion of the expanded cgg-repeat of the fragile x chromosome. PLoS ONE. 2016; 11:e0165499. [69] Liu XS, Wu H, Krzisch M, Wu X, Graef J, Muffat J, et al. Rescue of fragile x syndrome neurons by DNA methylation editing of the fmr1 gene. Cell. 2018; 172:979–992 e976. [70] McCutcheon SR, Rohm D, Iglesias N, Gersbach CA. Epigenome editing technologies for discovery and medicine. Nature Biotechnology. 2024; 42:1199-1217. [71] Zafarullah M. Genomic editing of FMR1 premutation in human fibroblast cell lines via delivery of purified Cas9 ribonucleoproteins. Poster presented at 7th Genomic and Big Data Summit; 2017 September 26-27; San Diego California. USA. [72] Lee B, Lee K, Panda S, Gonzales-Rojas R, Chong A, Bugay V, et al. Nanoparticle delivery of crispr into the brain rescues a mouse model of fragile x syndrome from exaggerated repetitive behaviours. Nature Biomedical Engineering. 2018; 2:497–507. [73] Ferreira GK, Cardoso E, Vuolo FS, Galant LS, Michels M, Goncalves CL, et al. Effect of acute and long-term administration of gold nanoparticles on biochemical parameters in rat brain. Materials Science and Engineering C. Materials for Biological Applications. 2017; 79:748–755. [74] Rohm D, Black JB, McCutcheon SR, Barrera A, Berry SS, Morone DJ, et al. Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing. Cell Genomics. 2025; 5(2):100770. [75] Hingar S, Schneeberger Pané M, Romero MJO. Prader Willi syndrome: advances in genetics. Advanced Genetics. 2025; 113: 29-52. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 530 [76] Bieth E, Eddiry S, Gaston V, Lorenzini F, Buffet A, Conte Auriol F, et al. Highly restricted deletion of the SNORD116 region is implicated in Prader-Willi Syndrome European Journal of Human Genetics. 2015; 23:252-255. [77] Mendiola AJP, LaSalle JM. Epigenetics in Prader-Willi Syndrome. Frontiers in Genetics. 2021; 12:624581. [78] Crenshaw MM, Graw SL, Slavov D, Boyle TA, Piqué DG, Taylor M, et al. An Atypical 15q11.2 microdeletion not involving SNORD116 resulting in Prader-Willi s. Case Reports in Genetics. 2023; 2023: 4225092. [79] Wang SE, Jiang YH. Potential of epigenetic therapy for Prader-Willi Syndrome. Trends in Pharmacological Sciences. 2019; 40(9):605-608. [80] Nakamura M, Gao Y, Dominguez AA, Qi LS. CRISPR technologies for precise epigenome editing. Nature Cell Biology. 2021; 23:11-22. [81] O'Geen H, Ren C, Nicolet CM, Perez AA, Halmai J, Le VM, et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Research. 2017; 45:9901-9916. [82] Nuñez JK, Chen J, Pommier GC, Cogan JZ, Replogle JM, Adriaens C, et al. Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell. 2021; 184:2503-2519.e2517. [83] Klann TS, Black JB, Chellappan M, Safi A, Song L, Hilton IB, et al. CRISPR-Cas9 epigenome editing enables highthroughput screening for functional regulatory elements in the human genome. Nature Biotechnology. 2017; 35(6):561-568. [84] Simeonov DR, Gowen BG, Boontanrart M, Roth TL, Gagnon JD, Mumbach MR, rt al. Discovery of stimulationresponsive immune enhancers with CRISPR activation. Nature. 2017; 549(7670):111-115. Erratum in: Nature. 2018l; 559(7715):E13. [85] Diao Y, Li B, Meng Z, Jung I, Lee AY, Dixon J, et al. A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening. Genome Research. 2016; 26(3):397-405. [86] Korkmaz G, Lopes R, Ugalde AP, Nevedomskaya E, Han R, Myacheva K, et al. Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9. Nature Biotechnology. 2016; 34(2):192-198. [87] Langouët M, Gorka D, Orniacki C, Dupont-Thibert CM, Chung MS, et al. Specific ZNF274 binding interference at SNORD116 activates the maternal transcripts in Prader-Willi syndrome neurons. Human Molecular Genetics. 2020; 29(19):3285-3295. [88] Das D, Karthik N, Taneja R. Epigenetic small-molecule modulators targeting metabolic pathways in cancer. In: Kundu TK, Das C. (eds) Metabolism and Epigenetic Regulation: Implications in Cancer. Subcellular Biochemistry. Vol 100. Springer, Cham. 2022. pp 523–555. [89] Sabin S. Gene therapy and gene editing in human disease: CRISPR and beyond. Research Archives of Rising Scholars. 2023; DO10.58445/rars.484 [90] Morris CA, Braddock SR; COUNCIL ON GENETICS. Health care supervision for children with Williams syndrome. Pediatrics. 2020; 145(2):e20193761. [91] Gregory MD, Mervis CB, Elliott ML, Kippenhan JS, Nash T, Czarapata JB, et al. Williams syndrome hemideletion and LIMK1 variation both affect dorsal stream functional connectivity. Brain. 2019; 142:3963–3974. [92] Wrona D, Pastukhov O, Pritchard RS, Raimondi F, Tchinda J, Jinek M, et al. CRISPR-directed therapeutic correction at the NCF1 locus is challenged by frequent incidence of chromosomal deletions. Molecular Therapy. 2020; 17:936–943. [93] Decimi V, Fazio G, Dell'Acqua F, Maitz S, Galbiati M, Rizzari C, et al. Williams syndrome and mature B-Leukemia: A random association? The European Journal of Medical Genetics. 2016; 59:634-640. [94] Kimura R, Ishii Y, Tomiwa K, Awaya T, Nakata M, Kato T, et al. Williams-Beuren syndrome as a potential risk factor for Burkitt Lymphoma. Frontiers in Genetics. 2018; 9:368. [95] Sarnow K, Schwab SG, Rio O, Mukherjee J, Bjerkvig R, Johannessen TA. P15.02 loss of Williams Syndrome Transcription Factor (WSTF) leads to improved temozolomide sensitivity in human glioblastoma cells irrespective of MGMT expression. Neuro-Oncology. 2021; 23(Suppl 2):ii56. [96] Zhang P, Huang A, Ferruzzi J, Mecham RP, Starcher BC, Tellides G, et al. Inhibition of microRNA-29 enhances elastin levels in cells haploinsufficient for elastin and in bioengineered vessels--brief report. Arteriosclerosis, Thrombosis, and Vascular Biology. 2012; 32(3):756-759. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 531 [97] Li W, Li Q, Qin L, Ali R, Qyang Y, Tassabehji M, et al. Rapamycin inhibits smooth muscle cell proliferation and obstructive arteriopathy attributable to elastin deficiency. Arteriosclerosis, Thrombosis, and Vascular Biology. 2013; 33(5):1028-1035. [98] Misra A, Sheikh AQ, Kumar A, Luo J, Zhang J, Hinton RB, et al. Integrin β3 inhibition is a therapeutic strategy for supravalvular aortic stenosis. Journal of Experimental Medicine. 2016; 7;213(3):451-463. [99] Kinnear C, Agrawal R, Loo C, Pahnke A, Rodrigues DC, Thompson T, et al. Rescues the phenotype of elastin insufficiency in patient induced pluripotent stem cell-derived vascular smooth muscle cells. Arteriosclerosis, Thrombosis, and Vascular Biology. 2020; 40(5):1325-1339. [100] Xie Y, Zhou Yi, Chen Y, Chen B. When Cri du chat syndrome meets Edwards syndrome. Molecular Medicine Reports. 2015; 11:1933–1938. [101] Holland P, Wildhagen M, Istre M, Reiakvam OM, Dahl JA, Søraas A. Cri du chat syndrome patients have DNA methylation changes in genes linked to symptoms of the disease. Clinical Epigenetics. 2022;14(1):128. [102] Shen J, Wang Y, Liu Y, Lan J, Long S, Li Y, et al., Behavioral abnormalities, cognitive impairments, synaptic deficits, and gene replacement therapy in a CRISPR engineered rat model of 5p15.2 deletion associated with Cri du Chat syndrome. Advanced Science. 2025; 12(14):e2415224. [103] Wong PK, Cheah FC, Syafruddin SE, Mohtar MA, Azmi N, Ng PY, et al. CRISPR gene-editing models geared toward therapy for hereditary and developmental neurological disorders. Frontiers in Pediatrics. 2021; 9:592571. [104] Fink JJ, Robinson TM, Germain ND, Sirois CL, Bolduc KA, Ward AJ, et al. Disrupted neuronal maturation in Angelman syndrome-derived induced pluripotent stem cells. Nature Communication. 2017; 8:15038. [105] Gavril EC, Luca AC, Curpan AS, Popescu R, Resmerita I, Panzaru MC, et al. Wolf-Hirschhorn syndrome: Clinical and genetic study of 7 new cases, and mini review. Children. 2021; 8(9):751. [106] Jiang Y, Sun H, Lin Q, Wang Z, Wang G, Wang J, et al. De novo truncating variant in NSD2gene leading to atypical Wolf-Hirschhorn syndrome phenotype. BMC Medical Genetics. 2019; 20:134. [107] Kawai T, Kinoshita S, Takayama Y, Ohnishi E, Kamura H, Kojima K, et al. Loss of function in NSD2 causes DNA methylation signature similar to that in Wolf-Hirschhorn syndrome. Genetics in Medicine Open. 2024; 2:101838. [108] Lozier ER, Konovalov FA, Kanivets IV, Pyankov DV, Koshkin PA, Baleva LS, et al. De novo nonsense mutation in WHSC1 (NSD2) in patient with intellectual disability and dysmorphic features. Journal of Human Genetics. 2018;63(8):919-922. [109] Nevado J, Ho KS, Zollino M, Blanco R, Cobaleda C, Golzio C, et al. International meeting on Wolf-Hirschhorn syndrome: Update on the nosology and new insights on the pathogenic mechanisms for seizures and growth delay. Proceedings of The American Journal of Medical Genetics - Part A 2020; 182:257–267. [110] Leibowitz ML, Papathanasiou S, Doerfler PA, Blaine LJ, Sun L, Yao Y, et al. Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing. Nature Genetics. 2021; 53(6):895–905. [111] Wang Y, Jiang H, Li M, Xu Z, Xu H, Chen Y, et al. Delivery of CRISPR/Cas9 system by AAV as vectors for gene therapy. Gene. 2024; 927:148733. [112] Naeem M, Majeed S, Hoque MZ, Ahmad I. Latest developed strategies to minimize the off-target effects in CRISPRCas-mediated genome editing. Cells. 2020; 2;9(7):1608. [113] Zou S, Sun Y, Tang W. Charting the development and engineering of CRISPR base editors: lessons and inspirations. Cell Chemical Biology. 2025; 32 (6):789-808. [114] Rees HA, Liu DR. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet. 2018; 19:770–788. [115] Khalil AM. Cell delivery strategies: Review of the recent developments, Challenges, and Opportunities. International Journal of Applied Science-Research and Review. 2022; 9:60. [116] Taha EA, Lee J, Hotta A. Delivery of CRISPR-Cas tools for in vivo genome editing therapy: Trends and challenges. The Journal of Controlled Release. 2022; 342: 345-361. [117] Charlesworth CT, Deshpande PS, Dever DP, Camarena J, Lemgart VT, Cromer MK, et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nature Medicine. 2019; 25(2):249-254. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 509-532 532 [118] Jacobsen T, Liao C, Beisel CL. The Acidaminococcus sp. Cas12a nuclease recognizes GTTV and GCTV as noncanonical PAMs. FEMS Microbiology Letters. 2019; 366(8):fnz085. [119] van Dijke I, Bosch L, Bredenoord AL, Cornel M, Repping S, Hendriks S The ethics of clinical applications of germline genome modification: A Systematic review of reasons. Human Reproduction. 2018; 33:1777–1796. [120] Bosco N, Goldberg A, Zhao X, Mays JC, Cheng P, Johnson AF, et al. KaryoCreate: a CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres. Cell. 2023; 186(9):1985–2001.e19. [121] NASEM. Human Genome Editing: Science, Ethics, and Governance. Washington, DC. The National Academies Press; 2017.