Efficient Genetic Manipulation Research Articles

Methylation of foreign DNA overcomes the restriction barrier of Flavobacterium psychrophilum and allows efficient genetic manipulation.

Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum is a problem for salmonid aquaculture worldwide, and current control measures are inadequate. An obstacle in understanding and controlling BCWD is that most F. psychrophilum strains resist DNA transfer, thus limiting genetic studies of their virulence mechanisms. F. psychrophilum restriction enzymes that destroy foreign DNA were suspected to contribute to this problem. Here, we used F. psychrophilum DNA methyltransferases to modify and protect foreign DNA from digestion. This allowed efficient conjugative DNA transfer into nine diverse F. psychrophilum strains that had previously resisted DNA transfer. Using this approach, we constructed a gene deletion mutant that failed to cause disease in rainbow trout. Further genetic studies could help determine the molecular factors involved in pathogenesis and may aid development of innovative BCWD control strategies.

Developing a RecT-assisted endogenous CRISPR/SzCas9 system for precise genome editing in Streptococcus zooepidemicus.

Streptococcus zooepidemicus is the most commonly utilized strain for industrial-scale hyaluronic acid (HA) production due to its capacity to produce high-quality HA. However, the lack of efficient genetic manipulation approaches has significantly hindered the industrial application of this species. In this study, we developed a RecT-assisted endogenous CRISPR/SzCas9 system to enable markerless gene deletion, gene substitution, stop codon insertion, and iterative editing in the industrially significant strain S. zooepidemicus for hyaluronic acid production. A variety of editing outcomes were achieved, including a 3186-bp deletion of the hylb gene, replacement of VHb, and insertion of stop codons into the sagA gene. Ultimately, we successfully developed an engineered strain, designated SD-3, which exhibited reduced toxicity and non-hemolytic properties. The hyaluronic acid production of SD-3 increased by 39.7 %, reaching 10.8 g/L in a 5-L fermenter. This achievement represents the highest titer of high molecular weight hyaluronic acid to date, characterized by a uniform and high molecular weight of up to 2.8 × 106 Da.

Gene expression screening and cell factory engineering for enhancing echinocandin B production in Aspergillus nidulans NRRL8112

BackgroundEchinocandin B (ECB) is a key precursor of the antifungal drug anidulafungin and its biosynthesis occurs via ani gene cluster in Aspergillus nidulans NRRL8112. Strain improvement for industrial ECB production has mainly relied on mutation breeding due to the lack of genetic tools.ResultsHere, a CRISPR-base-editing tool was developed in A. nidulans NRRL8112 for simultaneous inactivation of the nkuA gene and two marker genes, pryoA and riboB, which enabled efficient genetic manipulation. Then, in-vivo plasmid assembly was harnessed for ani gene expression screening, identifying the rate-limiting enzyme AniA and a pathway-specific transcription factor AniJ. Stepwise titer enhancement was achieved by overexpressing aniA and/or aniJ, and ECB production reached 1.5 g/L during 5-L fed-batch fermentation, an increase of ~ 30-fold compared with the parent strain.ConclusionThis study, for the first time, revealed the regulatory mechanism of ECB biosynthesis and harnessed genetic engineering for the development of an efficient ECB-producing strain.

New vectors and optimal conditions for allelic exchange in hypervirulent Klebsiella pneumoniae

The emergence of antibiotic-resistant Klebsiella pneumoniae is a significant global health threat that has led to increased morbidity and mortality. This resistance also hinders basic research, as many strains are no longer susceptible to antibiotics commonly used in microbial genetics. Addressing this requires the development of new genetic tools with alternative selective markers. In this report, we introduce new allelic exchange vectors for use in drug-resistant strains. These vectors feature a conditional R6K origin of replication, an origin of transfer, SacB counter-selection, and alternative selectable markers. We validated the vectors by generating unmarked deletions in the K. pneumoniae KPPR1S bla (β-lactamase) and lacZ (β-galactosidase) genes. During this process, we defined optimized conditions for SacB-mediated allelic exchange in KPPR1S, significantly enhancing the efficiency of mutant generation. Furthermore, we demonstrated that lacZ is dispensable for virulence and that the lacZ mutant can serve as a surrogate for wild-type strains in competition assays using the Galleria mellonella infection model. Our findings provide new tools for the efficient genetic manipulation of K. pneumoniae and other drug-resistant bacteria.

An Enhanced Retroviral Vector for Efficient Genetic Manipulation and Selection in Mammalian Cells.

Introducing genetic material into hard-to-transfect mammalian cell lines and primary cells is often best achieved through retroviral infection. An ideal retroviral vector should offer a compact, selectable, and screenable marker while maximizing transgene delivery capacity. However, a previously published retroviral vector featuring an EGFP/Puromycin fusion protein failed to meet these criteria in our experiments. We encountered issues such as low infection efficiency, weak EGFP fluorescence, and selection against infected cells. To address these shortcomings, we developed a novel retroviral vector based on the Moloney murine leukemia virus. This vector includes a compact bifunctional EGFP and Puromycin resistance cassette connected by a 2A peptide. Our extensively tested vector demonstrated superior EGFP expression, efficient Puromycin selection, and no growth penalty in infected cells compared with the earlier design. These benefits were consistent across multiple mammalian cell types, underscoring the versatility of our vector. In summary, our enhanced retroviral vector offers a robust solution for efficient infection, reliable detection, and effective selection in mammalian cells. Its improved performance and compact design make it an ideal choice for a wide range of applications involving precise genetic manipulation and characterization in cell-based studies.

Establishment and comprehensive characterization of a novel dark-reared zebrafish model for myopia studies

Myopia is predicted to impact approximately 5 billion people by 2050, necessitating mechanistic understanding of its development. Myopia results from dysregulated genetic mechanisms of emmetropization, caused by over-exposure to aberrant visual environments; however, these genetic mechanisms remain unclear. Recent human genome-wide association studies have identified a range of novel myopia-risk genes. To facilitate large-scale in vivo mechanistic examination of gene-environment interactions, this study aims to establish a myopia model platform that allows efficient environmental and genetic manipulations. We established an environmental zebrafish myopia model by dark-rearing. Ocular biometrics including relative ocular refraction were quantified using optical coherence tomography images. Spatial vision was assessed using optomotor response (OMR). Retinal function was analyzed via electroretinography (ERG). Myopia-associated molecular contents or distributions were examined using RT-qPCR or immunohistochemistry. Our model produces robust phenotypic changes, showing myopia after 2 weeks of dark-rearing, which were recoverable within 2 weeks after returning animals to normal lighting. 2-week dark-reared zebrafish have reduced spatial-frequency tuning function. ERG showed reduced photoreceptor and bipolar cell function (a- and b-waves) after only 2 days of dark-rearing, which worsened after 2 weeks of dark-rearing. We also found dark-rearing-induced changes to expression of myopia-risk genes, including egr1, vegfaa, vegfab, rbp3, gjd2a and gjd2b, inner retinal distribution of EFEMP1, TIMP2 and MMP2, as well as transiently reduced PSD95 density in the inner plexiform layer. Coupled with the gene editing tools available for zebrafish, our environmental myopia model provides an excellent platform for large-scale investigation of gene-environment interactions in myopia development.

CRISPR/Cas9-Based Genome Editing for Protein Expression and Secretion in Kluyveromyces lactis.

The budding yeast Kluyveromyces lactis has emerged as a promising microbial chassis in industrial biotechnology. However, a lack of efficient molecular genetic manipulation tools and strategies has hindered the development of K. lactis as a biomanufacturing platform. In this study, we developed and applied a CRISPR/Cas9-based genome editing method to K. lactis. Single-gene editing efficiency was increased to 80% by disrupting the nonhomologous end-joining-related gene KU80 and performing a series of process optimizations. Subsequently, the CRISPR/Cas9 system was explored based on different sgRNA delivery modes for simultaneous multigene editing. With the aid of the color indicator, the editing efficiencies of two and three genes reached 73.3 and 36%, respectively, in the KlΔKU80 strain. Furthermore, the CRISPR/Cas9 system was used for multisite integration to enhance lactase production and combinatorial knockout of TMED10 and HSP90 to characterize the extracellular secretion of lactase in K. lactis. Generally, genome editing is a powerful tool for constructing K. lactis cell factories for protein and chemical production.

An upgraded Myxococcus xanthus chassis with enhanced growth characteristics for efficient genetic manipulation

Myxobacteria are well known for multicellular social behaviors and valued for biosynthesis of natural products. Myxobacteria social behaviors such as clumping growth severely hamper strain cultivation and genetic manipulation. Using Myxococcus xanthus DK1622, we engineered Hu04, which is deficient in multicellular behavior and pigmentation. Hu04, while maintaining nutritional growth and a similar metabolic background, exhibits improved dispersed growth, streamlining operational procedures. It achieves high cell densities in culture and is promising for synthetic biology applications.

Efficient markerless genetic manipulation of Pasteurella multocida using lacZ and pheSm as selection markers.

Pasteurella multocida is a zoonotic conditional pathogen that infects multiple livestock species, causing substantial economic losses in the animal husbandry industry. An efficient markerless method for gene manipulation may facilitate the investigations of P. multocida gene function and pathogenesis of P. multocida. Herein, a temperature-sensitive shuttle vector was constructed using lacZ as a selection marker, and markerless glgB, opa, and hyaE mutants of P. multocida were subsequently constructed through blue-white colony screening. The screening efficiency of markerless deletion strains was improved by the lacZ system, and the method could be used for multiple gene deletions. However, the fur mutant was unavailable via this method. Therefore, we constructed a pheSm screening system based on mutated phenylalanine tRNA synthetase as a counterselection marker to achieve fur deletion mutant. The transformed strain was sensitive to 20 mM p-chloro-phenylalanine, demonstrating the feasibility of pheSm as a counter-selective marker. The pheSm system was used for markerless deletions of glgB, opa, and hyaE as well as fur that could not be screened by the lacZ system. A comparison of screening efficiencies of the system showed that the pheSm counterselection system was more efficient than the lacZ system and broadly applicable for mutant screening. The methods developed herein may provide valuable tools for genetic manipulation of P. multocida.IMPORTANCEPasteurella multocida is a highly contagious zoonotic pathogen. An understanding of its underlying pathogenic mechanisms is of considerable importance and requires efficient species-specific genetic tools. Herein, we propose a screening system for P. multocida mutants using lacZ or pheSm screening markers. We evaluated the efficiencies of both systems, which were used to achieve markerless deletion of multiple genes. The results of this study support the use of lacZ or pheSm as counterselection markers to improve counterselection efficiency in P. multocida. This study provides an effective genetic tool for investigations of the virulence gene functions and pathogenic mechanisms of P. multocida.

CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus

Monascus pilosus has been used to produce lipid-lowering drugs rich in monacolin K (MK) for a long period. Genome mining reveals there are still many potential genes worth to be explored in this fungus. Thereby, efficient genetic manipulation tools will greatly accelerate this progress. In this study, we firstly developed the protocol to prepare protoplasts for recipient of CRISPR/Cas9 system. Subsequently, the vector and donor DNA were co-transformed into recipients (106 protoplasts/mL) to produce 60–80 transformants for one test. Three genes (mpclr4, mpdot1, and mplig4) related to DNA damage response (DDR) were selected to compare the gene replacement frequencies (GRFs) of Agrobacterium tumefaciens-mediated transformation (ATMT) and CRISPR/Cas9 gene editing system (CGES) in M. pilosus MS-1. The results revealed that GRF of CGES was approximately five times greater than that of ATMT, suggesting that CGES was superior to ATMT as a targeting gene editing tool in M. pilosus MS-1. The inactivation of mpclr4 promoted DDR via the non-homologous end-joining (NHEJ) and increased the tolerances to DNA damaging agents. The inactivation of mpdot1 blocked DDR and led to the reduced tolerances to DNA damaging agents. The inactivation of mplig4 mainly blocked the NHEJ pathway and led to obviously reduced tolerances to DNA damaging agents. The submerged fermentation showed that the ability to produce MK in strain Δmpclr4 was improved by 52.6% compared to the wild type. This study provides an idea for more effective exploration of gene functions in Monascus strains.Key points• A protocol of high-quality protoplasts for CGES has been developed in M. pilosus.• The GRF of CGES was about five times that of ATMT in M. pilosus.• The yield of MK for Δmpclr4 was enhanced by 52.6% compared with the wild type.

Development of an Efficient and Seamless Genetic Manipulation Method for Xenorhabdus and Its Application for Enhancing the Production of Fabclavines.

Xenorhabdus can produce numerous natural products, but their development has been hampered by the lack of a seamless genetic manipulation method. In this study, we compared several lethal genes and determined the sacB gene as the most effective counter-selection marker and then established a dual selection/counter-selection system by integrating neo and sacB genes into one cassette. This provides an efficient and seamless genetic manipulation method for Xenorhabdus. Using this method, DNA fragments ranging from 205 to 47,788 bp in length were seamlessly knocked out or replaced with impressively high positive rates of 80 to 100% in Xenorhabdus budapestensis XBD8. In addition, the method was successfully applied with good efficiency (45-100%) in Xenorhabdus nematophila CB6. To further validate the method, different constitutive promoters were used to replace the native fclC promoter in a batch experiment. The positivity rate remained consistently high, at 46.3%. In comparison to WT XBD8, the recombinant strain MX14 demonstrated a significant increase in the production of fabclavine 7 and fabclavine 8 by 4.97-fold and 3.22-fold, respectively, while the overall production of fabclavines was enhanced by 3.52-fold.

A toolkit to study microglia states using stem cell‐derived models

AbstractBackgroundEmerging genetic studies of late‐onset Alzheimer’s Disease (LOAD) implicate microglia, the brain’s resident macrophages. More than half the risk genes associated with LOAD are expressed in microglia, yet we know little about the underlying biology or how myeloid cells contribute to LOAD pathogenesis. Single‐cell transcriptomic studies reveal diverse microglial states in patients and mouse models; however, we lack tools to track these states and understand the impact of environmental challenges or genetic susceptibility.MethodWe used human embryonic and induced pluripotent stem cell models to gain insights into microglia. These cells can be differentiated into microglia, enabling the broad characterization of cells in vitro and understanding their impact on the other cell types in xenograft models, allowing us to gain biological insight into disease pathogenesis.ResultOur lab has developed a novel human stem cell‐based platform1 which induces stem cell‐derived microglia (iMGLs) to take on diverse transcriptional signatures similar to those found in the human brain in response to exposure to brain‐relevant substrates. Moreover, we conceived a lentiviral protocol that allows for efficient genetic manipulation of microglia (>90% transduction). Building on these systems, we generated reporters of microglia state to track the expression of key microglial genes and adapted iPSC “villages”2, or pooled cultures, that combine iPSC cell lines in the same dish. Using these tools in vitro and in xenograft models3,4, we identified genetic regulators and functional changes of microglia states in LOAD.ConclusionTogether, these tools represent a broad toolset that will allow us to answer some of the most pressing questions, such as when and how in disease these states are formed, what is their impact on disease progression, and how plastic they are. These tools will be invaluable to understanding the role of microglia in AD and the brain in general.1. Dolan, M.‐J. et al. Biorxiv 2022.05.02.490100 (2022)2. Wells, M. F. et al. Biorxiv 2021.11.08.467815 (2021)3. Hasselmann, J. et al. Neuron 103, 1016‐1033.e10 (2019)4. Mancuso, R. et al. Nature Neuroscience 22, 2111‐2116 (2019)

Removal of innate immune barriers allows efficient transduction of quiescent human hematopoietic stem cells

Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities; however, they have not been exploited extensively because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in long-term repopulating quiescent HSC, allowing their efficient genetic manipulation. Mechanistically, lentiviral vector transduction of quiescent HSC was found to be restricted at the level of vector entry and by limited pyrimidine pools. These restrictions were overcome by the combined addition of cyclosporin H (CsH) and deoxynucleosides (dNs) during lentiviral vector transduction. Clinically relevant transduction levels were paired with higher polyclonal engraftment of long-term repopulating HSC as compared with standard exvivo cultured controls. These findings identify the cell-intrinsic barriers that restrict the transduction of quiescent HSC and provide a means to overcome them, paving the way for the genetic engineering of unstimulated HSC.

GENETIC REDESIGN: CRISPR-CAS AND MICROBIAL GENOME EDITING

Microbial biotechnology and genome editing have undergone a revolutionary transformation with the discovery and advancement of CRISPR-Cas systems. Initially recognized as an adaptive immune system in bacteria and archaea, these systems have been repurposed into highly effective tools for accurately modifying genetic material. In this comprehensive review, we delve into the remarkable influence of CRISPR-Cas systems on microbial biotechnology and genome editing. We explore their fundamental mechanisms, recent breakthroughs, existing challenges, and future prospects in this rapidly evolving field. By unraveling the intricate workings of CRISPR-Cas systems, we shed light on their ability to facilitate precise and efficient genetic manipulation. With a focus on microbial biotechnology and genome editing, we uncover how CRISPR-Cas systems have revolutionized these areas, presenting new possibilities for genetic engineering. Moreover, we discuss the challenges CRISPR-Cas systems face, such as off-target effects and delivery methods, while highlighting the ongoing research endeavors that aim to overcome these obstacles. By examining the transformative impact of CRISPR-Cas systems, this review reveals this groundbreaking technology's immense potential and promising future in microbial biotechnology and genome editing. Keywords: CRISPR-Cas systems, microbial biotechnology, genome editing, adaptive immune system, genetic material, precise manipulation, transformative impact, off-target effects, delivery methods, genetic engineering.

Screening of antibiotics to obtain axenic cell cultures of a marine microalga Chrysotila roscoffensis.

Due to high growth rate, outstanding abiotic stress tolerance, and rich value-added substances, Chrysotila roscoffensis, belonging to the phylum of Haptophyta, can be considered as a versatile resource for industrial exploitation of bioactive compounds. However, the application potential of C. roscoffensis has drawn attention until just recently, and the understanding related to the biological properties of this species is still scarce. For example, the sensitivities of C. roscoffensis to antibiotics, which is essential for the verification of heterotrophic capacity and the establishment of efficient genetic manipulation system is still unavailable. Aiming to provide fundamental information for future exploitation, the sensitivities of C. roscoffensis to nine types of antibiotics were tested in this study. The results demonstrated that C. roscoffensis exhibited relatively high resistances to ampicillin, kanamycin, streptomycin, gentamicin, and geneticin, while was sensitive to bleomycin, hygromycin B, paromomycin, and chloramphenicol. Using the former five types of antibiotics, a bacteria removal strategy was established tentatively. Finally, the axenicity of treated C. roscoffensis was confirmed based on a multi-strategy method including solid plate, 16S rDNA amplification, and nuclear acid staining. This report can provide valuable information for the development of optimal selection markers, which are meaningful for more extensive transgenic studies in C. roscoffensis. Moreover, our study also paves the way for the establishment of heterotrophic/mixotrophic cultivation modes of C. roscoffensis.

Ex vivo factor VIII-modified proliferating human hepatocytes therapy for haemophilia A.

Ex vivo gene manipulation in human hepatocytes is a promising therapeutic strategy in the treatment of inherited liver diseases. However, a major limitation is the lack of a highly efficient and safe genetic manipulation system for transplantable primary human hepatocytes (PHHs). Here, we reported that proliferating human hepatocytes (ProliHHs) cultured in vitro showed high susceptibility to lentivirus-mediated genetic modification and maintained cellular phenotypes after lentiviral infection. Human factor VIII expression was introduced through F8-Lentivirus-mediated transduction of ProliHHs followed by xenotransplantation into immunocompromised haemophilia A mice. We demonstrated that these F8-modified ProliHHs could effectively repopulate the mouse liver, resulting in therapeutic benefits in mouse models. Furthermore, no genotoxicity was detected in F8-modified ProliHHs using lentiviral integration site analysis. Thus, this study demonstrated, for the first time, the feasibility and safety of lentiviral modification in ProliHHs to induce the expression of coagulation factor VIII in the treatment of haemophilia A.

A modified Agrobacterium-mediated transformation for two oomycete pathogens.

Oomycetes are a group of filamentous microorganisms that include some of the biggest threats to food security and natural ecosystems. However, much of the molecular basis of the pathogenesis and the development in these organisms remains to be learned, largely due to shortage of efficient genetic manipulation methods. In this study, we developed modified transformation methods for two important oomycete species, Phytophthora infestans and Plasmopara viticola, that bring destructive damage in agricultural production. As part of the study, we established an improved Agrobacterium-mediated transformation (AMT) method by prokaryotic expression in Agrobacterium tumefaciens of AtVIP1 (VirE2-interacting protein 1), an Arabidopsis bZIP gene required for AMT but absent in oomycetes genomes. Using the new method, we achieved an increment in transformation efficiency in two P. infestans strains. We further obtained a positive GFP transformant of P. viticola using the modified AMT method. By combining this method with the CRISPR/Cas12a genome editing system, we successfully performed targeted mutagenesis and generated loss-of-function mutations in two P. infestans genes. We edited a MADS-box transcription factor-encoding gene and found that a homozygous mutation in MADS-box results in poor sporulation and significantly reduced virulence. Meanwhile, a single-copy avirulence effector-encoding gene Avr8 in P. infestans was targeted and the edited transformants were virulent on potato carrying the cognate resistance gene R8, suggesting that loss of Avr8 led to successful evasion of the host immune response by the pathogen. In summary, this study reports on a modified genetic transformation and genome editing system, providing a potential tool for accelerating molecular genetic studies not only in oomycetes, but also other microorganisms.

Silencing of heat shock protein 90 (hsp90): Effect on development and infectivity of Ichthyophthirius multifiliis

BackgroundRecently, an increasing number of ichthyophthiriasis outbreaks has been reported, leading to high economic losses in fisheries and aquaculture. Although several strategies, including chemotherapeutics and immunoprophylaxis, have been implemented to control the parasite, no effective method is available. Hence, it is crucial to discover novel drug targets and vaccine candidates against Ichthyophthirius multifiliis. For this reason, understanding the parasite stage biology, host–pathogen interactions, molecular factors, regulation of major aspects during the invasion, and signaling pathways of the parasite can promote further prospects for disease management. Unfortunately, functional studies have been hampered in this ciliate due to the lack of robust methods for efficient nucleic acid delivery and genetic manipulation. In the current study, we used antisense technology to investigate the effects of targeted gene knockdown on the development and infectivity of I. multifiliis. Antisense oligonucleotides (ASOs) and their gold nanoconjugates were used to silence the heat shock protein 90 (hsp90) of I. multifiliis. Parasite stages were monitored for motility and development. In addition, the ability of the treated parasites to infect fish and cause disease was evaluated.ResultsWe demonstrated that ASOs were rapidly internalized by I. multifiliis and distributed diffusely throughout the cytosol. Knocking down of I. multifiliis hsp90 dramatically limited the growth and development of the parasite. In vivo exposure of common carp (Cyprinus carpio) showed reduced infectivity of ASO-treated theronts compared with the control group. No mortalities were recorded in the fish groups exposed to theronts pre-treated with ASOs compared with the 100% mortality observed in the non-treated control fish.ConclusionThis study presents a gene regulation approach for investigating gene function in I. multifiliis in vitro. In addition, we provide genetic evidence for the crucial role of hsp90 in the growth and development of the parasite, suggesting hsp90 as a novel therapeutic target for successful disease management. Further, this study introduces a useful tool and provides a significant contribution to the assessing and understanding of gene function in I. multifiliis.

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells.

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.