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Recent progress in using Drosophila as a platform for human genetic disease research
Journal of Genetic Medicine 2023;20:39-45
Published online December 31, 2023;  https://doi.org/10.5734/JGM.2023.20.2.39
© 2023 Korean Society of Medical Genetics and Genomics.

Wan Hee Yoon*

Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA
Wan Hee Yoon, Ph.D. https://orcid.org/0000-0002-9459-3139
Aging and Metabolism Research Program, Oklahoma Medical Research Foundation, 825 NE 13th St, Oklahoma City, OK 73104, USA.
Tel: +1-405-271-1574, Fax: +1-405-271-3765, E-mail: wanhee-yoon@omrf.org
Received November 19, 2023; Revised December 1, 2023; Accepted December 4, 2023.
cc This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
As advanced sequencing technologies continue to uncover an increasing number of variants in genes associated with human genetic diseases, there is a growing demand for systematic approaches to assess the impact of these variants on human development, health, and disease. While in silico analyses have provided valuable insights, it is essential to complement these findings with model organism studies to determine the functional consequences of genetic variants in vivo. Drosophila melanogaster is an excellent genetic model for such functional studies due to its efficient genetic technologies, high gene conservation with humans, accessibility to mutant fly resources, short life cycles, and cost-effectiveness. The traditional GAL4-UAS system, allowing precise control of gene expression through binary regulation, is frequently employed to assess the effects of monoallelic variants. Recombinase medicated cassette exchange or CRISPR-Cas9-mediated GAL4 insertion within coding introns or substitution of gene body with Kozak-Gal4 result in the loss-of-function of the target gene. This GAL4 insertion strategy also enables the expression of reference complementary DNA (cDNA) or cDNA carrying genetic variants under the control of endogenous regulatory cis elements. Furthermore, the CRISPR-Cas9-directed tissue-specific knockout and cDNA rescue system provides the flexibility to investigate candidate variants in a tissue-specific and/or developmental-timing dependent manner. In this review, we will delve into the diverse genetic techniques available in Drosophila and their applications in diagnosing and studying numerous undiagnosed diseases over the past decade.
Keywords : Genetic variation, Genetic diseases, Drosophila melanogaster, Genetic techniques, Gene editing, Clustered regularly interspaced short palindromic repeats.
Introduction

Since the publication of Mendelian Inheritance in Man (MIM) by Dr. Victor A. McKusick in 1966, a landmark work from a former Johns Hopkins University School of Medicine professor, more than 25,000 human genetic diseases have been documented [1]. Initially released as a book, MIM evolved into an online database, the Online Mendelian Inheritance in Man (OMIM), made available in 1987 [2]. The past decade has seen a remarkable increase in the number of cataloged genetic diseases, primarily due to advancements in human genetic research and sequencing techniques. In the latter half of the 20th century, the study of human genetic diseases largely relied on extensive pedigree studies, linkage analyses, and association studies using single-nucleotide polymorphism markers [3]. Despite these efforts, numerous diseases caused by rare genetic variants remained unidentified. The introduction of advanced next-generation sequencing techniques, including whole-genome and whole-genome sequencing (WGS) in the last two decades, has significantly enhanced the identification of these genetic variants [4].

The inherent nature of rare variants prevents statistical analysis and patients often present multiple candidate variants across various genes. Computational analysis tools such as PolyPhen and CADD have been developed to predict the impact of the patient variants [5,6]. However, these prediction programs have limitations. Utilizing genetic model organisms for testing variants has proven to be an efficient and reliable method to ascertain the pathogenicity of variants found in undiagnosed patients [7]. These models include Saccharomyces cerevisiae (yeast), Caenorhabditis elegans, Danio rerio (zebrafish), and Drosophila melanogaster (fruit flies). This review will focus on the utility of Drosophila model for studying human genetic diseases and recent advancements in Drosophila genetic technologies for determining the pathogenicity of genetic variants from rare diseases.

Advantages of Using Drosophila As a Genetic Model for Studying Human Diseases

Since Thomas Hunt Morgan pioneered the use of Drosophila in genetics in the early 1900s, studies using these fruit flies have revealed key principles of genetics and biology [8]. Efforts to validate Mendel's genetic findings with various organisms found success particularly with Drosophila, unlike early attempts using chickens.

The advantages of Drosophila as a genetic model are notable. Its four chromosomes simplify genetic studies, and its short life cycle, coupled with high fecundity, enhances efficiency. The low cost of fruit fly cultures, requiring basic ingredients like corn meal, sugar, yeast extract, and agar, makes it more economical than other systems. These attributes make Drosophila valuable for human genetic research, especially in identifying numerous variants/genes in rare diseases through WGS and exome sequencing. While mouse models are feasible, especially with CRISPR-Cas9 genome editing, testing multiple variants is impractical due to cost, life cycle length, and progeny number for analysis. Opposite to intuition, Drosophila shares many genetic similarities with humans; recent studies show that fruit flies share 57.9% of human genes and 73.1% of genes linked to rare diseases [9,10].

The accumulation of genetic technologies over the last century, including balancers, various mutagenesis methods (e.g., diverse transposable elements), and binary gene expression systems (such as GAL4-UAS), has made it easier to introduce patient variants into Drosophila. This positions the fruit fly as a preferred model for studying human genetic diseases. In the past decade, there have been significant advancements in Drosophila genetic technologies applicable to human genetic disease research. The following sections will discuss the challenges and troubleshooting strategies in selecting optimal genetic tools for studying various cases of human pathogenic variants.

Determine the Impact of Monoallelic Variants Using GAL4-UAS System

To determine the effects of heterozygous variants from patient in Drosophila, the GAL4-UAS system would be the ideal choice [11]. This approach, as exemplified by our investigation into ATAD3A (ATPase family, AAA domain containing 3A), provides a framework for analyzing heterozygous variants. The de novo p.Arg528Trp variant in ATAD3A, identified in five unrelated patients, is associated with developmental delay, peripheral neuropathy, and/or optic atrophy and cardiomyopathy [12]. Dominance in heterozygous variants is known to arise from haploinsufficiency, a dominant-negative function, or gain of function (hypermorph or neomorph). Haploinsufficiency was ruled out, as the individuals with a heterozygous deletion of ATAD3A are normal. Similarly, flies that are deficient for one copy of the gene do not display obvious phenotype.

For investigating dominant-negative function and gain of function, we took advantage of GAL4-UAS system [11], a binary gene expression genetic tool in Drosophila. Using the system we can express transgenes, including complementary DNA (cDNA), under the control of GAL4-UAS. Numerous tissue/cells or developmental timing specific Gal4 drivers have been available [13,14]. Ubiquitous overexpression of wild-type copy of the gene (Tub or Act-Gal4>UAS-dAtad3a [WT]) did not affect viability but led to expanded mitochondria, indicating that the gain of function phenotype is mild and promotes mitochondrial fusion. In contrast, ubiquitous or neuronal expression of dAtad3a carrying the homologous mutation to the patient variant (UAS-dAtad3a [R534W]) causes lethality and mitophagy and reduced numbers of mitochondria, suggesting that this variant is dominant-negative mutation [12]. These data are also consistent with the observation that ATAD3 proteins have been proposed to form oligomers [15]. Collectively, this study on ATAD3A variant identified a new neurological syndrome, later named Harel-Yoon syndrome (HYOS, MIM: 617183), but also demonstrated the effectiveness of the GAL4-UAS system in studying dominant diseases.

Use of Null Alleles Created by Chemical and Transposable Element-Mediated Mutagenesis

Over the past century, Drosophila researchers have generated a vast collection of mutant flies, many of which are well-characterized and available at public stock centers like the Bloomington Drosophila Stock Center (https://bdsc.indiana.edu) [16]. This resource allows researchers to access well-defined null mutant alleles for studying biallelic and recessive aspects of rare diseases. Traditional practice in Drosophila research involves performing rescue experiments to confirm if observed mutant phenotypes result from the loss of the gene of interest. These rescue experiments typically involve introduction of genomic DNA covering the gene [17] or UAS-cDNA (Drosophila reference cDNA) for rescue. For human disease studies, UAS-cDNA expression is preferred for its ease in testing multiple variants and the tissue specificity offered by the GAL4-UAS system [18].

One case in point is the study of the NRD1 (Nardilysin) gene, identified from a forward genetic screen (EMS mutagenesis on X chromosome) to isolate genes essential for neuronal maintenance and neurodevelopment [10,19]. From this screen, four null mutant alleles of Drosophila Nrd1 (dNrd1) were isolated, all resulting in late-onset neurodegeneration in photoreceptor neurons [19]. To assess the gene's relevance to human genetic diseases, collaboration was sought with human geneticists via GeneMatcher (https://genematcher.org), a web tool for connecting physicians, geneticists, and researchers [20]. The UCLA Clinical Genomics Center identified an individual with a homozygous truncating variant in NRD1. The patient exhibited severe global developmental delay, ataxia, and seizures. To assess the functionality of this variant, we first tested whether expression of human wild-type NRD1 cDNA could rescue dNrd1 mutant phenotypes. Subsequently, we tested the patient’s NRD1 variant in the same context. The results demonstrated that ubiquitous expression of the mutant NRD1 cDNA with the variant failed to rescue the loss of dNrd1, suggesting that the variant is a loss-of-function (LoF) allele [19]. Hence, this study demonstrated that combination of classical null alleles with GAL4-UAS system enables functional studies of recessive variants identified in human patients.

Combination of RNA Interference with UASHuman cDNA Strategy

In situations where mutant stocks are not available in public stock centers, RNA interference (RNAi) serves as a robust method to study LoF of a target gene [21]. However, a common limitation of RNAi is its tendency for off-target effects, meaning it may inadvertently knockdown mRNAs other than its intended target. Therefore, it's crucial to validate the phenotypes resulting from RNAi expression by rescuing them with a reference sequence, such as cDNA from another species. Drosophila cDNA is typically unsuitable for rescue because RNAi can target both endogenous mRNA and mRNA transcribed from UAS transgenes. Thus, while using RNAi is straightforward, it requires that human cDNA be available to counteract the loss of gene function or that cDNAs from other Drosophila species (e.g., Drosophila persimilis) are accessible [22].

An illustrative example is the study of ATP5F1D (ATP synthase, H+ transporting, mitochondrial F1 complex, δ subunit), where two patients with homozygous missense variants presented with episodic lethargy, metabolic acidosis, 3-methyglutaconic aciduria, and hyperammonemia [23]. Knockdown of Drosophila ATPsynδ, the homolog of human ATP5F1D, in the developing brain and eyes led to complete loss of the fly head. This phenotype was fully rescued by expressing wild type human ATP5F1D cDNA. However, expression of cDNA with the patient variants only rescued head-size phenotypes, resulting in abnormal eyes and antennae, akin to defects seen in other mutants affecting mitochondrial electron transport chain components [23]. This study demonstrated the importance of RNAi-based genetic studies and the crucial role of cross-species genetic tools in understanding the functional impact of human genetic variants.

Generation of T2A-Gal4 Null Mutant Allele Using Recombinase Medicated Cassette Exchange (RMCE)

As previously discussed, using null mutants or RNAi reagents can be simple and effective for studying biallelic variants in human genes. However, these strategies are not universally applicable, as some genes require specific expression in certain cells and tissues, and existing GAL4 drivers, like ubiquitous and pan-neuronal Gal4, may not accurately replicate the expression patterns of the gene of interest. Furthermore, the expression of some human wild type cDNAs using existing GAL4 drivers can be lethal. This necessitates a different approach to study human variants.

To illustrate, I would like to share an episode from my research on the OGDH (2-oxoglutarate dehydrogenase) and OGDHL (OGDH-like) genes [19]. OGDH is a crucial enzyme in the Krebs cycle within mitochondria. In spring 2015, human genetic collaborators informed me a patient with a recessive missense variant in OGDHL presented with severe developmental delays and other symptoms [24]. Initial attempts using RNAi for Drosophila Ogdh (dOgdh) were reliable for metabolic phenotypes, but expressing human OGDH or OGDHL cDNA did not rescue the lethality or metabolic issues caused by dOgdh knockdown. In fact, ubiquitous or tissue-specific expression of OGDH cDNA itself induced developmental lethality [19], suggesting that OGDH expression could be toxic if its expression is not correctly regulated temporally and spatially.

A breakthrough idea emerged from a different project on which I was collaborating with Dr. Pei-Tseng Lee, a lab mate who was researching learning and memory. We were creating a dOgdh-T2A-GAL4 line to define the neural circuit where dOgdh is expressed. The T2A-Gal4 toolkit, developed by Ben White's group, allows for efficient substitution of a MiMIC (Minos mediated integration cassette) transposable element located in a coding intron (an intron flanked by two coding exons) with an artificial exon containing SA (splicing acceptor)-T2A-GAL4-polyA (abbreviated as T2A-Gal4) [25]. MiMIC is a versatile tool for functional gene annotation in Drosophila and has a Swappable Integration Cassette (SIC) [26]. MiMIC SICs located between two attP sites can be exchanged with any DNA sequence flanked with attB sites through RMCE by fC31 integrase [26]. We took advantage of the presence of a MiMIC transposon inserted in a coding intron of dOgdh and created dOgdh-T2A-Gal4 line through RMCE.

This T2A-Gal4 insertion into the coding intron of dOgdh genomic locus results in truncating the protein at the insertion site, leading to the generation of a null mutant. In the other hand, the viral T2A sequence causes the truncation of the nascent dOgdh polypeptide and reinitiating of translation of the downstream GAL4 as an independent protein. The expression of Gal4 is under the control of the endogenous dOgdh regulatory control [19]. During the challenging period with the dOgdh RNAi, the generation of the dOgdh-T2A-GAL4 line happened concurrently. I realized that I had already created a null mutant for dOgdh that could express Gal4 under endogenous control. Using the dOgdh-T2A-GAL4 line, I could test null mutant phenotypes and simultaneously express UAS-human OGDH cDNA for rescue. Eureka!!! I ran to Dr. Hugo Bellen, my postdoctoral mentor, and shared this idea with him, who encouraged the approach and suggested using deficiency flies lacking the dOgdh genomic region to confirm the LoF phenotypes. This approach proved successful. Homozygous dOgdh-T2A-GAL4 flies exhibited embryonic lethality, which was rescued by expression of human OGDH cDNA, but not by the cDNA with the patient's variant [19]. Hence, the results indicate that the missense variant in OGDHL is a LoF allele and underlie the manifestation in patient. This study highlighted the utility of the T2A-Gal4 approach for studying human diseases. Furthermore, this study exemplified an approach of humanizing Drosophila to determine variants’ function in vivo [7].

Generation of T2A-Gal4 Alleles Using CRIMIC

MiMIC is a versatile tool for functional gene annotation in Drosophila and features a SIC located between two attP sites [26]. These SICs can be exchanged with any DNA sequence flanked with attB sites through RMCE by fC31 integrase [26]. In the previous section, we mentioned that Drosophila mutant alleles created by replacing ISC in MiMICs with an artificial exon encoding a SA-T2A-GAL4-polyA signal (T2A-Gal4) are valuable for studying patient variant studies, as it enables replacing the gene of interest in Drosophila with Drosophila or human UAS-cDNA. Hence, MiMIC alleles of Drosophila are invaluable resources for human disease studies.

The Drosophila Gene Disruption project (http://flypush.imgen.bcm.tmc.edu/pscreen), led by Dr. Hugo Bellen laboratory at Baylor College of Medicine, generated 17,500 MiMIC insertion lines [26,27]. Of these, approximately 1,860 genes have MiMIC insertions within introns, which can be converted into a T2A-GAL4 or GFP-tagged protein traps [28]. However, with around 13,600 Drosophila genes [29], a complementary strategy was needed to generate Drosophila alleles for genes that do not have a MiMIC insertion within a coding intron. Bellen laboratory developed a CRIMIC (CRISPR mediated integration cassette) strategy. This involves integrating a modified SIC (attP-FRT-SA-T2A-GAL4-polyA-FRT-attP) into a coding intron of gene of interest via CRISPR/Cas9-mediated homology directed repair (HDR). This strategy expanded the number of genes with MiMICs from 1,860 to approximately 6,000 [30], significant increasing Drosophila genetic resources and thus providing useful resources for human genetic studies.

Kozak-Gal4 Null Mutant Generation via CRISPR-Cas9 Editing

The MiMIC and CRIMIC-based T2A-Gal4 resources have been instrumental for numerous human disease studies [18,19,31-35]. However, their application is limited as a large coding intron, typically over 100 nt, is required for the genomic integration of an artificial exon, leaving over half of Drosophila genes untargetable by CRIMIC [28]. About 58% of Drosophila genes lack an optimal coding intron for the integration of artificial exons in all annotated splicing isoforms.

To address this gap, an alternative and efficient strategy is needed to create null mutant alleles that express GAL4 under the control of endogenous regulatory elements. Traditionally, Drosophila researchers have created GAL4 knock-in lines using the pw35GAL4 construct-mediated transgenesis and homologous recombination [36]. This process involves cloning a 3 kb homologous sequence for both arms into the vector. However, this cloning method is notoriously inefficient, and the selection of GAL4 knock-in flies typically requires an extended period of 4 to 5 months.

CRISPR/Cas9-mediated genome editing, which has been proven effective in Drosophila, offers a solution [30,37]. One approach involves using CRISPR/Cas9 to integrate a KozakGAL4 cassette that replaces the entire gene body [38]. This strategy employs creating double-stranded breaks at the 5’ and 3’UTR of the target gene, using two sgRNAs, and then replaces the coding sequence of genes with a Kozak sequence-GAL4-polyA-FRT-3xP3EGFP-polyA-FRT (KozakGal4) cassette [38]. In addition, for HDR-mediated integration, it uses short homology arms (100-200 bps) for HDR-mediated integration, effectively inserting large DNA segments (>5 kb) into the genome [39]. Kanca et al. [38] successfully applied this method to approximately 200 genes and found that the efficiency of transgenesis is about 80%. The KozakGal4 method is expected to be highly useful for studying human genetic diseases in genes where MiMIC and CRIMIC are not applicable.

CRISPR/Cas9-Mediated Tissue-Specific Knockout and cDNA Rescue

CRISPR/Cas9 genome editing significantly expanded the tool kit for Drosophila genetics [40]. It particularly when combined with classical genetic tools like the GAL4-UAS system, enabling tissue or cell-specific knockout of target genes [41]. Port et al. [41,42] created a large-scale transgenic short guide (sg) RNA library that enabled efficient CRISPR/Cas9-based disruption of target genes in a constitutive or conditional manner. This technique also allows for the introduction of pathogenic variants into the Drosophila genome.

Functional studies of variants from human patients in Drosophila often require a tissue-specific approaches. In cases where human genes have multiple paralogs with tissue-specific expression, while Drosophila has a single, ubiquitously expressed orthologs, tissue-specific expression of UAS-Cas9 along with ubiquitous sgRNA expression can be an effective strategy. However, the majority of sgRNAs in the Transgenic RNAi project and the Weizmann Knock Out project target common coding exons [41,42], limiting their use in studies requiring genomic knockout alongside rescue by GAL4-directed UAS-wild type cDNA expression.

To address this, a novel strategy was developed involving sgRNAs targeting exon-intron junctions, allowing selective targeting of genomic loci while sparing UAS-cDNA transgenes [31,35,43]. This approach, termed “CRISPR/Cas9-mediated tissue-specific knockout and cDNA rescue,” was exemplified in a study of OGDHL variants. In our study of OGDHL variants, we identified nine individuals from eight unrelated families with biallelic variants presenting diverse neurodevelopmental symptoms, including epilepsy and microcephaly [31]. The variants include nine missense mutations. Using the dOgdh-T2A-Gal4 allele, we found that dOgdh cDNA carrying mutations analogous to the human variants failed to rescue lethality resulting from dOgdh loss. However, this finding has limitations, as OGDHL is brain-specific in humans [44,45], whereas dOgdh is ubiquitously expressed in Drosophila [46]. Employing the new CRISPR knockout with cDNA rescue system, we demonstrated that neuron-specific knockout of dOgdh led to lethality, which was fully rescued by wild type dOgdh cDNA expression. Interestingly, while using the dOgdh-T2A-Gal4 allele showed all nine variants failing to rescue lethality, the new CRISPR system revealed that three variants failed to rescue, whereas the other six did, albeit with behavioral defects observed in the flies [31]. This result from the CRISPR-Cas9-mediated knockout with cDNA rescue approach allowed us to differentiate the allelic strengths of OGDHL variants in a tissue-specific manner, thereby enhancing our understanding of their impact and expanding the capabilities of Drosophila genetic tools. This approach is expected to be widely used for in-depth studies of human disease-associated variants and their specific roles in tissues or cells, as well as for broader genetic research in Drosophila.

Conclusion

The recent progress in Drosophila genetic technologies, particularly through the integration of CRISPR/Cas9 genome editing and classic genetic tools, has markedly enhanced the study of human genetic diseases. The development of innovative methods such as T2A-Gal4 alleles using CRIMIC, Kozak-Gal4 via CRISPR-Cas9 editing, and tissue-specific knockout and cDNA rescue strategies, has expanded our ability to model and understand complex genetic disorders. These advancements underscore Drosophila’s value in functional genomics, enabling precise dissection of variant pathogenicity and providing novel insights into disease mechanisms. This progress not only enhances our understanding of disease biology but also opens up potential therapeutic avenues, highlighting Drosophila’s continuing relevance in the era of personalized medicine.

Acknowledgements

None.

Funding

W.H.Y. is supported by Oklahoma Medical Research Foundation (OMRF), the National Institute of Neurological Disorders and Stroke (5R01 NS121298-03) of the National Institutes of Health (NIH), and Oklahoma Center for Adult Stem Cell Research (241006).

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