CRISPR-Cas9 Genome Editing Research Articles

Lymphocyte-Specific Protein 1 Regulates Expression and Stability of Endothelial Nitric Oxide Synthase.

Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), plays a critical role in blood pressure regulation. Genome-wide association studies have identified genetic susceptibility loci for hypertension in human lymphocyte-specific protein 1 (LSP1) gene. LSP1 is recognized as modulator of leukocyte extravasation, and endothelial permeability, however, the role of LSP1 in regulation of NO signaling within endothelial cells (ECs) remains unknown. The present study investigated the role of LSP1 in the regulation of eNOS expression and activity utilizing human macrovascular ECs in vitro and LSP1 knockout (KO) mice. In ECs, specific CRISPR-Cas9 genomic editing deleted LSP1 and caused downregulation of eNOS expression. LSP1 gain-of-function through adenovirus-mediated gene transfer was associated with enhanced expression of eNOS. Co-immunoprecipitation and confocal fluorescence microscopy revealed that eNOS and LSP1 formed a protein complex under basal conditions in ECs. Furthermore, LSP1 deficiency in mice promoted significant upregulation and instability of eNOS. Utilizing a mass-spectrometry-based bottom-up proteomics approach, we identified novel truncated forms of eNOS in immunoprecipitates from LSP1 KO aortae. Our experimental data suggest an important role of endothelial LSP1 in regulation of eNOS expression and activity within human ECs and murine vascular tissues.

Behavioral characterization of G-protein-coupled receptor 160 knockout mice.

Neuropathic pain is a devastating condition where current therapeutics offer little to no pain relief. Novel nonnarcotic therapeutic targets are needed to address this growing medical problem. Our work identified the G-protein-coupled receptor 160 (GPR160) as a potential target for therapeutic intervention. However, the lack of small-molecule ligands for GPR160 hampers our understanding of its role in health and disease. To address this void, we generated a global Gpr160 knockout (KO) mouse using CRISPR-Cas9 genome editing technology to validate the contributions of GPR160 in nociceptive behaviors in mice. Gpr160 KO mice are healthy and fertile, with no observable physical abnormalities. Gpr160 KO mice fail to develop behavioral hypersensitivities in a model of neuropathic pain caused by constriction of the sciatic nerve. On the other hand, responses of Gpr160 KO mice in the hot-plate and tail-flick assays are not affected. We recently deorphanized GPR160 and identified cocaine- and amphetamine-regulated transcript peptide (CARTp) as a potential ligand. Using Gpr160 KO mice, we now report that the development of behavioral hypersensitivities after intrathecal or intraplantar injections of CARTp are dependent on GPR160. Cocaine- and amphetamine-regulated transcript peptide plays a role in various affective behaviors, such as anxiety, depression, and cognition. There are no differences in learning, memory, and anxiety between Gpr160 KO mice and their age-matched and sex-matched control floxed mice. Results from these studies support the pronociceptive roles of CARTp/GPR160 and GPR160 as a potential therapeutic target for treatment of neuropathic pain.

In Vivo CRISPR/Cas9-Mediated Gene Ablation in Murine B Cells.

CRISPR-Cas9 genome editing is a powerful tool for assessing the functional role of candidate genes. In vitro CRISPR/Cas9 screens have been used to rapidly assess the role of thousands of genes in the differentiation and function of immune populations. However, the physiological relevance of a gene is often dependent on signals received in the tissue microenvironment, such as exposure to growth factors, chemokines, cytokines, and cell contact-dependent signals, which may not be recapitulated in an in vitro setting. Additionally, in vitro approaches are not sufficient to induce the differentiation of all cell populations limiting the cell types that can be screened. This has posed a major barrier to understanding the genes regulating the differentiation of germinal center B cells. Here, we describe an approach to perform an in vivo Crispr-Cas9 screen to specifically ablate genes in activated B cells. Using this approach, we have been able to reveal novel transcriptional regulators of germinal center B cell differentiation following viral infection.

Quantitative Measurement of Tau Aggregation in Genetically Modified Rats with Neurodegeneration.

Animal models of neurodegenerative diseases have helped us to better understand the pathogenesis of neurodegenerative diseases. However, recent failure to translate pre-clinical model studies to the clinic urges us to develop more rigorous and faithful animal models in neurodegenerative diseases. As genetic manipulation of rats becomes much more accessible due to availability of CRISPR-Cas9 and other genomic editing toolboxes, rats have been emerging as a new model system for neurodegenerative diseases. Even though mouse models have been dominant over the last decades, rats may provide advantages over mice. Rats are more genetically and physiologically closer to humans than to mice. Also, certain rat models can represent deposition of tau, which is one of the key pathological features of Alzheimer's diseases and tauopathies. However, there is an unmet need for standardized, rigorous testing in rat models. We adopted two commonly used biochemical and immunofluorescence methods from mice and human postmortem brains to measure tau aggregation. Due to the intrinsic differences between mice and rats, e.g., size of rat brains, certain equipment is required for rat models to study tau pathologies. Along with specific tools, here we describe the detailed methods for rat models of neurodegenerative diseases.

CRISPR Activation in Mouse Trophoblast Stem Cells.

The placenta is a vital organ that regulates nutrient supply to the developing embryo during gestation. In mice, the placenta is composed of trophoblast lineage and mesodermal derivatives, which merge through the chorioallantoic fusion process in a critical event for the progression of placenta development. The trophoblast lineage is derived from self-renewing, multipotent cells known as mouse trophoblast stem cells (mTSCs). These cells are a valuable tool that allows scientists to comprehend the signals regulating major placental cell types' self-renewal and differentiation capacity. Recent advances in CRISPR-Cas9 genome editing applied in mTSCs have provided novel insights into the molecular networks involved in placentation. Here, we present a comprehensive CRISPR activation (CRISPRa) protocol based on the CRISPR/gRNA-directed synergistic activation mediator (SAM) method to overexpress specific target genes in mTSCs.

Recent Advancements in Reducing the Off-Target Effect of CRISPR-Cas9 Genome Editing.

The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)) and the associated protein (Cas9) system, a young but well-studied genome-editing tool, holds plausible solutions to a wide range of genetic disorders. The single-guide RNA (sgRNA) with a 20-base user-defined spacer sequence and the Cas9 endonuclease form the core of the CRISPR-Cas9 system. This sgRNA can direct the Cas9 nuclease to any genomic region that includes a protospacer adjacent motif (PAM) just downstream and matches the spacer sequence. The current challenge in the clinical applications of CRISPR-Cas9 genome-editing technology is the potential off-target effects that can cause DNA cleavage at the incorrect sites. Off-target genome editing confuses and diminishes the therapeutic potential of CRISPR-Cas9 in addition to potentially casting doubt on scientific findings regarding the activities of genes. In this review, we summarize the recent technological advancements in reducing the off-target effect of CRISPR-Cas9 genome editing.

Chromosome 8p engineering reveals increased metastatic potential targetable by patient-specific synthetic lethality in liver cancer.

Large-scale chromosomal aberrations are prevalent in human cancer, but their function remains poorly understood. We established chromosome-engineered hepatocellular carcinoma cell lines using CRISPR-Cas9 genome editing. A 33-mega-base pair region on chromosome 8p (chr8p) was heterozygously deleted, mimicking a frequently observed chromosomal deletion. Using this isogenic model system, we delineated the functional consequences of chr8p loss and its impact on metastatic behavior and patient survival. We found that metastasis-associated genes on chr8p act in concert to induce an aggressive and invasive phenotype characteristic for chr8p-deleted tumors. Genome-wide CRISPR-Cas9 viability screening in isogenic chr8p-deleted cells served as a powerful tool to find previously unidentified synthetic lethal targets and vulnerabilities accompanying patient-specific chromosomal alterations. Using this target identification strategy, we showed that chr8p deletion sensitizes tumor cells to targeting of the reactive oxygen sanitizing enzyme Nudix hydrolase 17. Thus, chromosomal engineering allowed for the identification of novel synthetic lethalities specific to chr8p loss of heterozygosity.

Manipulation of Proteostasis Networks in Transgenic ZAAT Zebrafish via CRISPR-Cas9 Gene Editing.

The CRISPR-Cas9 genome editing system is used to induce mutations in genes of interest resulting in the loss of functional protein. A transgenic zebrafish α1-antitrypsin deficiency (AATD) model displays an unusual phenotype, in that it lacks the hepatic accumulation of the misfolding Z α1-antitrypsin (ZAAT) evident in human and mouse models. Here we describe the application of the CRISPR-Cas9 system to generate mutant zebrafish with defects in key proteostasis networks likely to be involved in the hepatic processing of ZAAT in this model. We describe the targeting of the atf6a and man1b1 genes as examples.

Clustered Regularly Interspaced Short Palindromic Repeats- a new era in Genomic technology and its applications in Theranostics

Clustered Regularly Interspaced Short Palindromic Repeats is termed as CRISPR. Bacteria having "spacer" sequences of deoxyribonucleic acid between the repeats of genomic sequences that resemble genomic sequences in viruses were found to have repetitive DNA sequences known as CRISPR.The CRISPR technology in genetic engineering has revolutionalized the field of medicine in treating various genetically linked diseases that are difficult to treat such as Lebers Congenital amaurosis, in which there is bi-allelic deletion caused by mutation in mitochondrial DNA MT-ND4(NADH ubiquinone oxidoreductase chain4) 1178G are treated by intravitreous injection of AAV2-ND4 produced by CRISPR-Cas9 genomic editing technology.CRISPR-Cas9 genomic editing is used to remove the expression of receptor Enhancer Protein-6 (Reep6 p.Leu135Pro ) gene that causes retinitis pigmentosa. Open angle Glaucoma caused by mutations in myocilin (MYOR) gene was effectively removed out by CRISPR-Cas9 editing technology. Mutation in KRT-12 gene that caused Meesman epithelial corneal dystrophy ( MECD) was alleviated by Cas9/sgRNA injection into the stroma of cornea. CRISPR-Cas9 genomic sequencing used in the treatment of Haemophilia -B , an inherited disease caused by mutation of factor IX gene Y371D was successfully modified by CRISPR-

Merozoite surface protein 1 paralog is involved in the human erythrocyte invasion of a zoonotic malaria, Plasmodium knowlesi.

The zoonotic malaria parasite Plasmodium knowlesi is an important public health concern in Southeast Asia. Invasion of host erythrocytes is essential for parasite growth, and thus, understanding the repertoire of parasite proteins that enable this process is vital for identifying vaccine candidates and how some species are able to cause zoonotic infection. Merozoite surface protein 1 (MSP1) is found in all malaria parasite species and is perhaps the most well-studied as a potential vaccine candidate. While MSP1 is encoded by a single gene in P. falciparum, all other human infective species (P. vivax, P. knowlesi, P. ovale, and P. malariae) additionally encode a divergent paralogue known as MSP1P, and little is known about its role or potential functional redundancy with MSP1. We, therefore, studied the function of P. knowlesi merozoite surface protein 1 paralog (PkMSP1P), using both recombinant protein and CRISPR-Cas9 genome editing. The recombinant 19-kDa C-terminus of PkMSP1P (PkMSP1P-19) was shown to bind specifically to human reticulocytes. However, immunoblotting data suggested that PkMSP1P-19-induced antibodies can recognize PkMSP1-19 and vice versa, confounding our ability to separate the properties of these two proteins. Targeted disruption of the pkmsp1p gene profoundly impacts parasite growth, demonstrating for the first time that PkMSP1P is important in in vitro growth of P. knowlesi and likely plays a distinct role from PkMSP1. Importantly, the MSP1P KO also enabled functional characterization of the PkMSP1P-19 antibodies, revealing clear immune cross-reactivity between the two paralogues, highlighting the vital importance of genetic studies in contextualizing recombinant protein studies.

Transplastomic plants — new approaches to solving “old” problems

Transplastomic plants are capable to accumulate the significant amounts (up to 70% of TSP) of target recombinant proteins in tissues. However, the production of such forms is severely limited by the low yield of initial transformants. This problem requires the development and optimization of new approaches to the delivery of transgenes into chloroplasts and an increase in the frequency of their integration into the plastome. Transplastomic tobacco plants expressing thegfpreporter gene and theaadAselectable marker under the control of thePrrnG10Lpromoter and theTpsbAterminator were obtained in the laboratory of plant bioengineering. It is known that the selected promoter and insertion region (between the tRNA genes of isoleucine and alanine) are capable to provide a high yield of recombinant proteins in the leaves of transplastomic plants [1]. However, the content of recombinant GFP in the leaves of the obtained transplastomic plants was determined at the level of 0.12%, and the variability for this trait was minimal and ranged from 0.09 to 0.16% of TSP. Insufficient accumulation of the target protein in transformants is not associated with transcription disorders or the presence of non-transgenic copies of the plastome. Probably, the low frequency of transformation and the lack of variability between the transformants are the reasons that make it difficult to select highly productive forms. It is proposed to increase the efficiency of targeted delivery of genetic constructs to plastids using single-walled carbon nanotubes loaded with recombinant DNA. This process can also be facilitated by our proposed approach to increase the frequency of DNA double-strand breaks in target regions of the plastome through the use of the CRISPR-Cas9 genome editing system.
 This work was supported by the Russian Science Foundation grant No. 23-24-00545.

The strong base for using base editing in plants

The most common application of CRISPR-Cas9 genome editing system is a gene knockout via indel mutations introducing. It is obvious, because this approach has minimum critical conditions in guide RNA design: available PAM sequence and conservative 19–25 nucleotides within all alleles of a target gene. Precise nucleotide changing with base editing systems has more limitations: target nucleotide should locate in editing window of adenine- or cytidine-deaminase, besides this, all undesired adenines or cytosines in editing window will be likely changed. However, there is a more fundamental issue — it is very difficult to find a single aminoacid substitution, which changes protein features in a desirable side. One of the good examples of base editing target will be considered in this work.
 Nicotiana tabacumL. is a plant fromSolanaceaefamily, the same as potato, tomato and pepper. All these plants are strongly affected by potato virus Y (PVY). It is known, that PVY recruits host translation initiation factor eIF4E by the viral protein VPg in order to start synthesis its proteins. If eIF4E can’t interact with VPg, plant will be resistant.
 In our work, we established an aminoacid substitution in tobacco eIF4E factor, which disrupted interaction with PVY VPg in yeast two-hybrid conditions, but didn’t influence the factor functionality. Then we designed two genetic constructions with different sgRNAs for introducing this mutation in tobacco plants using cytidine-deaminase system. These constructions were used to plant transformation and development of edited tobacco plants.
 This work is supported by State Task No. 0431-2022-0004.

Abstract A166: CRISPR-Cas9 genome editing in iPSCs for functional genetic screening

Abstract Induced pluripotent stem cells (iPSCs) are widely used for disease modeling, drug discovery, and cell therapy development. However, iPSCs are difficult to engineer with an efficient CRISPR/Cas9 system for functional genetic screens. We used WTC11 human iPS cells to generate functionally validated, lentiviral-transduced Cas9 lines, characterized by high gene-editing activity and sustained potential to differentiate. Flow cytometry was used to check for the expression of pluripotency markers Oct3/4, TRA1-60, and SSEA-4 in the iPS-Cas9 cells, and their actual pluripotency was then confirmed by testing the ability to differentiate into the three germ layers: ectoderm, mesoderm, and endoderm. Furthermore, genome-wide transcriptome analysis was performed to confirm the expression of germ layer-specific markers in the differentiated cells. Sustained Cas9 activity was confirmed in the differentiated cells. This study provides proof-of-principle that patient-derived iPSCs can be used to enable CRISPR/Cas9 functional genetic screening technology in reconstituted patient-specific tissues or disease models. Citation Format: Nadja Isachenko, Dongfang Hu, Alex Chenchik, Paul Diehl, Donato Tedesco. CRISPR-Cas9 genome editing in iPSCs for functional genetic screening [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A166.

Self-delivering, chemically modified CRISPR RNAs for AAV co-delivery and genome editing in vivo.

Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a 'protecting oligo'), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.

Generation of a C57BL/6J mouse strain expressing the CD45.1 epitope to improve hematopoietic stem cell engraftment and adoptive cell transfer experiments.

Adoptive cell transfer between genetically identical hosts relies on the use of a congenic marker to distinguish the donor cells from the host cells. CD45, a glycoprotein expressed by all hematopoietic cells, is one of the main congenic markers used because its two isoforms, CD45.1 and CD45.2, can be discriminated by flow cytometry. As a consequence, C57BL/6J (B6; CD45.2) and B6.SJL-Ptprca Pepcb/BoyJ (B6.SJL; CD45.1) mice are widely used in adoptive cell transfer experiments, under the presumption that they differ only at the CD45 (Ptprc) locus. However, recent studies have identified genetic variations between these congenic strains and have notably highlighted a differential expression of cathepsin E (CTSE). The B6.SJL mouse presents a number of functional differences in hematopoietic stem cell engraftment potential and immune cell numbers compared with the B6 mouse. In this study, we showed that B6 and B6.SJL mice also differ in their CD8+ T cell compartment and CD8+ T cell responses to viral infection. We identified Ctse as the most differentially expressed gene between CD8+ T cells of B6 and B6.SJL and demonstrated that the differences reported between these two mouse strains are not due to CTSE. Finally, using CRISPR-Cas9 genome editing, we generated a CD45.1-expressing B6 mouse by inserting one nucleotide mutation (A904G) leading to an amino acid change (K302E) in the Ptprc gene of the B6 mouse. We showed that this new B6-Ptprcem(K302E)Jmar/J mouse resolves the experimental biases reported between the B6 and B6.SJL mouse lines and should thus represent the new gold standard for adoptive cell transfer experiments in B6.

Loss-of-function of an α-SNAP gene confers resistance to soybean cyst nematode

Plant-parasitic nematodes are one of the most economically impactful pests in agriculture resulting in billions of dollars in realized annual losses worldwide. Soybean cyst nematode (SCN) is the number one biotic constraint on soybean production making it a priority for the discovery, validation and functional characterization of native plant resistance genes and genetic modes of action that can be deployed to improve soybean yield across the globe. Here, we present the discovery and functional characterization of a soybean resistance gene, GmSNAP02. We use unique bi-parental populations to fine-map the precise genomic location, and a combination of whole genome resequencing and gene fragment PCR amplifications to identify and confirm causal haplotypes. Lastly, we validate our candidate gene using CRISPR-Cas9 genome editing and observe a gain of resistance in edited plants. This demonstrates that the GmSNAP02 gene confers a unique mode of resistance to SCN through loss-of-function mutations that implicate GmSNAP02 as a nematode virulence target. We highlight the immediate impact of utilizing GmSNAP02 as a genome-editing-amenable target to diversify nematode resistance in commercially available cultivars.

Generation of an NCS1 gene knockout human induced pluripotent stem cell line using CRISPR/Cas9

NCS1 (Neuronal calcium sensor protein 1) encodes a highly conserved calcium binding protein abundantly expressed in neurons. It modulates intracellular calcium homeostasis, calcium-dependent signaling pathways as well as neuronal transmission and plasticity. Here, we generated a NCS1 knockout human induced pluripotent stem cell (hiPSC) line using CRISPR-Cas9 genome editing. It shows regular expression of pluripotent markers, normal iPSC morphology and karyotype as well as no detectable off-target effects on top 6 potentially affected genes. This newly generated cell line constitutes a valuable tool for studying the role of NCS1 in the pathophysiology of various neuropsychiatric disorders and non-neurological disease.

Abstract 18550: Impaired Microtubule Mediated Protein Trafficking as a Mechanism of PLN R14del Dilated Cardiomyopathy

Introduction: Phospholamban (PLN) regulates calcium handling and contractility in the heart by altering its phosphorylation status, thereby activating or inhibiting the calcium ATPase SERCA2a. A pathogenic mutation in PLN at the Arginine 14 codon (R14del) results in dilated cardiomyopathy characterized by ventricular arrythmias and sudden cardiac death. Cytoplasmic aggregation of PLN is a hallmark of the disease. However, exactly how the R14del mutation causes DCM remains largely unknown. As PLN is trafficked to the cell membrane before it localizes to the t-tubules, we hypothesize that PLN protein trafficking may be impaired, contributing to the PLN aggregation and DCM. Methods: Induced pluripotent stem cell derived cardiomyocytes (iPSC- iPSC-CMs) from patients carrying the PLN R14del mutation and isogenic controls generated by CRISPR-Cas9 genome editing were tagged with biotin to quantify membrane-bound protein expression. Western blotting and mass spectrometry was used to identify differences membrane protein expression, and immunocytochemistry was used to image microtubule layout, PLN and junctional protein expression at the cell borderintercalated disc (do you mean the intercalated disk?) in micropatterned iPSC-CMs. Results: Quantification of membrane-bound proteins showed a significant decrease of PLN at the cell membrane and connexin 43 expression in the intercalated disk of R14del iPSC-CMs compared to isogenic controls. Additionally, we observed significantly reduced levels of EB1, the microtubule tip protein, at the cell border of PLN R14del iPSC-CMs by immunofluorescence imaging and western blot analysis. Finally, disruption of microtube polymerization by Nocodazole treatment, induced PLN aggregate formation in iPSC-CMs. Conclusion: Our data suggest that the transportation of PLN and Cx43 proteins by microtubules is impaired in PLN R14del iPSC-CMs, which results in PLN accumulation in the cytoplasm and a decrease of Cx43 expression in the intercalated disc. These findings provide a novel mechanism for the pathogenesis of PLN R14del cardiomyopathy and a possible therapeutic target.

Targeting Nucleotide Biosynthesis Ameliorates Neutropenia in a Zebrafish Model of Dnajc21-Mutant Shwachman-Diamond Syndrome

Germline mutations in the ribosome maturation factors, SBDS, DNAJC21 and EFL1, lead to Shwachman-Diamond syndrome (SDS), a type of inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency and growth restriction. Further, SDS patients have a 36% increased cumulative risk of developing myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML) by 30 years of age. Given the rarity of SDS, large numbers of primary human samples are not readily available for mechanistic studies, warranting the use of animal models. Mouse and zebrafish models of SBDS mutations exhibit early lethality. In contrast, no in vivo models of DNAJC21 mutations have been published to date. We generated the first in vivo model of dnajc21 loss using zebrafish ( Danio rerio), given their highly conserved hematopoietic program and ease of genetic manipulation. Introduction of a homozygous deletion mutation in the zebrafish dnajc21 gene using CRISPR-Cas9 genomic editing resulted in stunted body growth and neutropenia, recapitulating hallmark SDS patient phenotypes. Whole-mount in situ hybridization for key hematopoietic lineage markers revealed reduced l-plastin +mature leukocytes and hbbe3 + mature erythrocytes in dnajc21 mutant embryos at 48 hours post-fertilization (hpf). A concomitant accumulation of myb + hematopoietic stem and progenitor cells (HSPCs) both in the dorsal aorta (equivalent to aorta-gonad-mesonephros) and the caudal hematopoietic tissue (fetal liver equivalent) was observed. Treatment of embryos with zebrafish granulocyte colony stimulating factor (gcsf) mRNA resulted in a modest expansion of neutrophils in dnajc21 mutant zebrafish compared to wildtype. Further, we observed reduced cell proliferation marked by phosphohistone-H3 (pH3) as well as low ATP:AMP levels measured by liquid chromatography/mass-spectrometry (LC/MS) in the dnajc21 mutants, suggesting a poor bioenergetic profile. At 4 months of age, flow cytometry and histopathological analysis identified reduced mature myelomonocytes and mild erythroid dysplasia in the whole kidney marrow (human bone marrow equivalent) of mutant fish. The link between TP53 somatic mutations and MDS/AML has been established in SBDS-mutant SDS, but has not been explored for DNAJC21-mutant SDS. We hence crossed our dnajc21 mutant zebrafish with a zebrafish line carrying a gain-of-function mutation (R217H) in the tp53 gene. Compound mutant fish had improved neutrophil counts but importantly, this was accompanied by an expansion of myeloid progenitors and significant erythroid dysplasia. To understand the disease mechanisms underlying the neutropenia and hypoproliferation in SDS, we performed bulk RNA sequencing of whole zebrafish larvae at 48 hpf. Biological processes such as oxidative stress response, drug catabolism and pre-mRNA spliceosome assembly were downregulated; whereas, acyl-CoA and nucleotide metabolism were upregulated in the mutants. In parallel, we performed untargeted metabolomics by LC/MS to determine the metabolic changes induced by dnajc21 loss. Upregulated pathways included urea cycle, amino acid and phenylacetate metabolism. Pyrimidine biosynthesis, including both the de novo and salvage pathways were downregulated in the mutants. Remarkably, exogenous supplementation with uridine or thymidine significantly improved neutrophil counts in the mutants. Our findings suggest that in dnajc21-mutant SDS1) neutropenia is caused by a combination of impaired granulocyte differentiation and cell proliferation; 2) the kidney marrow features myelodysplasia-like changes and 3) nucleotide biosynthesis may be a previously unrecognized therapeutic strategy in SDS.

Design and Validation of Guide RNAs for CRISPR-Cas9 Genome Editing in Mosquitoes.

CRISPR-Cas9 has revolutionized gene editing for traditional and nontraditional model organisms alike. This tool has opened the door to new mechanistic studies of basic mosquito biology as well as the development of novel vector control strategies based on CRISPR-Cas9, including gene drives that spread genetic elements in the population. Although the promise of the specificity, flexibility, and ease of deployment CRISPR is real, its implementation still requires empirical optimization for each new species of interest, as well as to each genomic target within a given species. Here, we provide an overview of designing and testing single-guide RNAs for the use of CRISPR-based gene editing tools.