Knock-in Approach Research Articles

Mitochondrial Iron Metabolism as a Potential Key Mediator of PD-L1 Thermal Regulation.

Glioblastoma (GBM) is the most common primary brain malignancy in the U.S. with a 5-year overall survival < 5% despite an aggressive standard of care. Laser interstitial thermal therapy (LITT) is a surgical approach to treating GBM that has gained traction, providing a safe option for reducing intracranial tumor burden. LITT is believed to potentially modulate GBM immune responses; however, the biochemical mechanisms underlying the modulation of immune checkpoints in GBM cells have been poorly characterized. The present study aimed to preliminarily evaluate the effects of thermal therapy and radiation on PD-L1 modulation in vitro, as a function of IDH mutational status. U87 cells and their IDH-mutant counterpart (U87R132H), which was generated using a crispr-cas9 knock-in approach, were utilized for this preliminary evaluation. Cell heating was achieved by harvesting with trypsin centrifugation where the cell pellets were treated on a heat block for the associated time and temperature. Following thermal therapy, cells were resuspended and irradiated using a 37-Cesium irradiator at 0.6 Gy min-1. Immediately following treatment, cells were either plated as single cells to allow colonies to form, and stained with Coomassie blue to be counted approximately 10-14 days later or harvested for Western blot analysis. Cell lysates were analyzed for PD-L1 expression with respect to various iron metabolic parameters (mortalin (HSPA9), transferrin receptor, and ferritin heavy chain) using a Western blotting approach. In both U87 and U87R132H cell lines, thermal therapy showed a temperature-dependent cell-killing effect, but U87R132H cells appeared more sensitive to thermal treatment when treated at 43 °C for 10 min. Moreover, thermal therapy had minimal effects on cell responses to 2 Gy irradiation. Treatment with thermal therapy downregulated PD-L1 expression in U87R132H cells, which was associated with increased expression of the mitochondrial iron metabolic enzyme, HSPA9. Thermal therapy reversed the radiation-induced overexpression of PD-L1, transferrin receptor, and ferritin heavy chain in U87R132H cells. No effects were observed in wild-type U87 cells. Moreover, Ga(NO3)3 depleted mitochondrial iron content which, in turn, significantly enhanced the sensitivity of U87R132H cells to thermal therapy and 2 Gy irradiation and caused a significant increase in PD-L1 expression. These results suggest that thermal therapy alone can modulate the immune checkpoint PD-L1. This effect was more pronounced when thermal therapy was combined with radiation. Mechanistically, mitochondrial iron trafficking through HSPA9 may coordinate the regulation of PD-L1 in the context of thermal therapy and ionizing radiation, which can be targeted with gallium-based therapy. These novel, preliminary findings warrant further mechanistic investigations in pre-clinical models of LITT.

Efficient CRISPR/Cas9 Knock-in Approaches for Manipulation of Endogenous Genes in Human B Lymphoma Cells.

Precise understanding of temporally controlled protein-protein interactions, localization, and expression is often difficult to achieve using traditional overexpression techniques. Recent advances have made CRISPR-based knock-in approaches efficient, which enables rapid derivation of cells with tagged endogenous proteins. However, the high degree of variability in knock-in efficiency across cell types and gene loci poses challenges, in particular with B lymphocytes, which are refractory to lipid transfection. Here, we present detailed protocols for efficient B lymphoma cell CRISPR/Cas9-mediated knock-in. We address knock-in efficiency in two ways. First, we provide a detailed approach for assessing cutting efficiency to select the most efficient single guide RNA for the gene region of interest. Second, we provide detailed approaches for tagging endogenous proteins with a fluorescent marker or instead for co-expressing them with an unlinked fluorescent marker. Either approach facilitates downstream selection of single-cell or bulk populations with the desired knock-in, particularly when knock-in efficiency is low. The utility of this approach is demonstrated via examples of engineering tags onto endogenous protein N- or C-termini, together with downstream analyses. We anticipate that this workflow can be applied more broadly to other cell types for efficient knock-in into endogenous loci. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Choosing an optimal knock-in target site and single guide RNA (sgRNA) design Basic Protocol 2: Assessment of Cas9 editing efficiency at the desired B cell genomic knock-in site Basic Protocol 3: Cloning the sgRNA dual guide construct Basic Protocol 4: Repair template design and cloning Basic Protocol 5: Electroporation and selection of engineered B cells Basic Protocol 6: Single-cell cloning of engineered B cells.

Microhomology-mediated end-joining Knock-In approaches to delete the allergenic domain of trout parvalbumin beta-1. Preliminary results in F0 animals and feedback

Background Gene editing techniques offer new opportunities to improve important traits in aquaculture. The allergenicity of fish flesh is a major problem in aquaculture. Parvalbumin (Parv) is the most prevalent fish allergen. For instance, in salmonids, a single parvalbumin beta-1 protein (parvb1) has been identified as an allergen in specific patients. Therefore, generating trout carrying two parvb1 alleles deleted from the allergenic peptide-encoding region could prevent allergies in these sensitive individuals. Methods Here, we describe the application of the Crispr/cas9 system in an attempt to delete parvb1 exon 2 encoding the allergenic peptide and, alternatively, to replace exon 2 of parvb1 with exon2 of parvalbumin beta-2 protein (parvb2,) which does not encode the allergenic peptide. Exon skipping and swapping were pursued through microhomology-mediated end-joining (MMEJ) knock-In using specifically designed double-stranded donor DNA. Results Genotyping of approximately 200 F0 fingerlings originating from eggs injected with donor DNA designed for exon 2 skipping led to the identification of only one animal carrying an allele lacking exon 2. Genotyping of approximately 150 fingerlings originating from eggs injected with donor DNA for exon 2 swapping did not result in any trout carrying the expected modified allele. Conclusions These preliminary results indicate the potential difficulties associated with the MMEJ KI experiments performed in farmed fish. Finally, new genomic techniques in aquaculture are further discussed in the context of lively debates taking place in the European parliament regarding a possible revision of the current law that determines the legal status of farm animals modified by genome editing. Gene editing, microhomology-mediated end-joining knock-in, parvalbumin, allergenicity, trout, and genetically modified organisms (GMOs).

When less is more - a fast TurboID knock-in approach for high-sensitivity endogenous interactome mapping.

In recent years, proximity labeling has established itself as an unbiased and powerful approach to map the interactome of specific proteins. Although physiological expression of labeling enzymes is beneficial for the mapping of interactors, generation of the desired cell lines remains time-consuming and challenging. Using our established pipeline for rapid generation of C- and N-terminal CRISPR-Cas9 knock-ins (KIs) based on antibiotic selection, we were able to compare the performance of commonly used labeling enzymes when endogenously expressed. Endogenous tagging of the µ subunit of the adaptor protein (AP)-1 complex with TurboID allowed identification of known interactors and cargo proteins that simple overexpression of a labeling enzyme fusion protein could not reveal. We used the KI strategy to compare the interactome of the different AP complexes and clathrin and were able to assemble lists of potential interactors and cargo proteins that are specific for each sorting pathway. Our approach greatly simplifies the execution of proximity labeling experiments for proteins in their native cellular environment and allows going from CRISPR transfection to mass spectrometry analysis and interactome data in just over a month.

Modeling Choroideremia Disease with Isogenic Induced Pluripotent Stem Cells.

Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy causing progressive vision loss due to mutations in the CHM gene, leading to Rab escort protein 1 loss of function. CHM disease is characterized by a progressive degeneration of the choroid, the retinal pigment epithelium (RPE), and the retina. The RPE is a monolayer of polarized cells that supports photoreceptors, providing nutrients, growth factors, and ions, and removes retinal metabolism waste products, having a central role in CHM pathogenesis. Commonly used models such as ARPE-19 cells do not reproduce accurately the nature of RPE cells. Human induced pluripotent stem cells (hiPSCs) can be differentiated into RPE cells (hiPSC-RPE), which mimic key features of native RPE, being more suited to study retinal diseases. Therefore, we took advantage of hiPSCs to generate new human-based CHM models. Two isogenic hiPSC lines were generated through CRISPR/Cas9: a CHM knock-out line from a healthy donor and a corrected CHM patient line using a knock-in approach. The differentiated hiPSC-RPE lines exhibited critical morphological and physiological characteristics of native RPE, including the presence of the tight junction markers Claudin-19 and Zonula Occludens-1, phagocytosis of photoreceptor outer segments, pigmentation, a postmitotic state, and the characteristic polygonal shape. In addition, all the studied cells were able to form retinal organoids. This work resulted in the establishment of isogenic hiPSC lines, representing a new and important CHM cellular model. To our knowledge, this is the first time that isogenic cell lines have been developed to model CHM disease, providing a valuable tool for studying the mechanisms at the onset of RPE degeneration.

Enzyme Is the Name-Adapter Is the Game.

Signaling proteins in eukaryotes usually comprise a catalytic domain coupled to one or several interaction domains, such as SH2 and SH3 domains. An additional class of proteins critically involved in cellular communication are adapter or scaffold proteins, which fulfill their purely non-enzymatic functions by organizing protein-protein interactions. Intriguingly, certain signaling enzymes, e.g., kinases and phosphatases, have been demonstrated to promote particular cellular functions by means of their interaction domains only. In this review, we will refer to such a function as "the adapter function of an enzyme". Though many stories can be told, we will concentrate on several proteins executing critical adapter functions in cells of the immune system, such as Bruton´s tyrosine kinase (BTK), phosphatidylinositol 3-kinase (PI3K), and SH2-containing inositol phosphatase 1 (SHIP1), as well as in cancer cells, such as proteins of the rat sarcoma/extracellular signal-regulated kinase (RAS/ERK) mitogen-activated protein kinase (MAPK) pathway. We will also discuss how these adaptor functions of enzymes determine or even undermine the efficacy of targeted therapy compounds, such as ATP-competitive kinase inhibitors. Thereby, we are highlighting the need to develop pharmacological approaches, such as proteolysis-targeting chimeras (PROTACs), that eliminate the entire protein, and thus both enzymatic and adapter functions of the signaling protein. We also review how genetic knock-out and knock-in approaches can be leveraged to identify adaptor functions of signaling proteins.

Building a novel TRUCK by harnessing the endogenous IFN-gamma promoter for cytokine expression

Despite the remarkable success of CAR T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous IFN-γ promoter for transgenic IL-15 expression. We demonstrate that IFN-γ expression is tightly regulated by TCR signaling. By introducing IRES-IL15 into the 3’-UTR of the IFN-γ gene via HDR-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen-stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Lastly, as part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.

A Novel CRISPR-Cas9 Strategy to Target DYSTROPHIN Mutations Downstream of Exon 44 in Patient-Specific DMD iPSCs.

Mutations in the DMD gene cause fatal Duchenne Muscular Dystrophy (DMD). An attractive therapeutic approach is autologous cell transplantation utilizing myogenic progenitors derived from induced pluripotent stem cells (iPSCs). Given that a significant number of DMD mutations occur between exons 45 and 55, we developed a gene knock-in approach to correct any mutations downstream of exon 44. We applied this approach to two DMD patient-specific iPSC lines carrying mutations in exons 45 and 51 and confirmed mini-DYSTROPHIN (mini-DYS) protein expression in corrected myotubes by western blot and immunofluorescence staining. Transplantation of gene-edited DMD iPSC-derived myogenic progenitors into NSG/mdx4Cv mice produced donor-derived myofibers, as shown by the dual expression of human DYSTROPHIN and LAMIN A/C. These findings further provide proof-of-concept for the use of programmable nucleases for the development of autologous iPSC-based therapy for muscular dystrophies.

The G932C mutation of chitin synthase 1 gene (CHS1) mediates buprofezin resistance as confirmed by CRISPR/Cas9-mediated knock-in approach in the brown planthopper, Nilaparvata lugens

The brown planthopper (Nilaparvata lugens) is a major destructive rice pest in Asia. High levels of insecticide resistance have been frequently reported, and the G932C mutation in the chitin synthase 1 (CHS1) gene has been found to mediate buprofezin resistance. However, there has been no direct evidence to confirm the functional significance of the single G932C substitution mutation leading to buprofezin resistance in N. lugens. Here, we successfully constructed a knock-in homozygous strain (Nl-G932C) of N. lugens using CRISPR/Cas9 coupled with homology-directed repair (HDR). Compared with the background strain susceptible to buprofezin (Nl-SS), the knock-in strain (Nl-G932C) showed a 94.9-fold resistance to buprofezin. Furthermore, resistant strains (Nl-932C) isolated from the field exhibited a 2078.8-fold resistance to buprofezin, indicating that there are other mechanisms contributing to buprofezin resistance in the field. Inheritance analysis showed that the resistance trait is incomplete dominance. In addition, the Nl-G932C strain had a relative fitness of 0.33 with a substantially decreased survival rate, emergence rate, and fecundity. This study provided in vivo functional evidence for the causality of G932C substitution mutation of CHS1 with buprofezin resistance and valuable information for facilitating the development of resistance management strategies in N. lugens. This is the first example of using CRISPR/Cas9 gene-editing technology in a hemipteran insect to directly confirm the role of a candidate target site mutation in insecticide resistance.

Expression of endogenous epitope-tagged GPR4 in the mouse brain.

GPR4 is a proton-sensing G protein-coupled receptor implicated in many peripheral and central physiological processes. GPR4 expression has previously been assessed only via detection of the cognate transcript or indirectly, by use of fluorescent reporters. In this work, CRISPR/Cas9 knock-in technology was used to encode a hemagglutinin (HA) epitope tag within the endogenous locus of Gpr4 and visualize GPR4-HA in the mouse central nervous system using a specific, well characterized HA antibody; GPR4 expression was further verified by complementary Gpr4 mRNA detection. HA immunoreactivity was found in a limited set of brain regions, including in the retrotrapezoid nucleus (RTN), serotonergic raphe nuclei, medial habenula, lateral septum, and several thalamic nuclei. GPR4 expression was not restricted to cells of a specific neurochemical identity as it was observed in excitatory, inhibitory, and aminergic neuronal cell groups. HA immunoreactivity was not detected in brain vascular endothelium, despite clear expression of Gpr4 mRNA in endothelial cells. In the RTN, GPR4 expression was detected at the soma and in proximal dendrites along blood vessels and the ventral surface of the brainstem; HA immunoreactivity was not detected in RTN projections to two known target regions. This localization of GPR4 protein in mouse brain neurons corroborates putative sites of expression where its function has been previously implicated (e.g., CO2-regulated breathing by RTN), and provides a guide for where GPR4 could contribute to other CO2/H+ modulated brain functions. Finally, GPR4-HA animals provide a useful reagent for further study of GPR4 in other physiological processes outside of the brain.Significance Statement GPR4 is a proton-sensing G-protein coupled receptor whose expression is necessary for a number of diverse physiological processes including acid-base sensing in the kidney, immune function, and cancer progression. In the brain, GPR4 has been implicated in the hypercapnic ventilatory response mediated by brainstem neurons. While knockout studies in animals have clearly demonstrated its necessity for normal physiology, descriptions of GPR4 expression have been limited due to a lack of specific antibodies for use in mouse models. In this paper, we implemented a CRISPR/Cas9 knock-in approach to incorporate the coding sequence for a small epitope tag into the locus of GPR4. Using these mice, we were able to describe GPR4 protein expression directly for the first time.

In vivo liver targeted genome editing as therapeutic approach: progresses and challenges.

The liver is an essential organ of the body that performs several vital functions, including the metabolism of biomolecules, foreign substances, and toxins, and the production of plasma proteins, such as coagulation factors. There are hundreds of genetic disorders affecting liver functions and, for many of them, the only curative option is orthotopic liver transplantation, which nevertheless entails many risks and long-term complications. Some peculiar features of the liver, such as its large blood flow supply and the tolerogenic immune environment, make it an attractive target for in vivo gene therapy approaches. In recent years, several genome-editing tools mainly based on the clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) system have been successfully exploited in the context of liver-directed preclinical or clinical therapeutic applications. These include gene knock-out, knock-in, activation, interference, or base and prime editing approaches. Despite many achievements, important challenges still need to be addressed to broaden clinical applications, such as the optimization of the delivery methods, the improvement of the editing efficiency, and the risk of on-target or off-target unwanted effects and chromosomal rearrangements. In this review, we highlight the latest progress in the development of in vivo liver-targeted genome editing approaches for the treatment of genetic disorders. We describe the technological advancements that are currently under investigation, the challenges to overcome for clinical applicability, and the future perspectives of this technology.

Genetic Knockin Approaches to Reconstructing DLBCL-Subtypes from Human Germinal Center B-Cells

Background and Significance: The clinical and genetic heterogeneity of DLBCL has led to the recent classification of the disease into five genetically distinct subtypes. The MCD/C5 subtype of DLBCL is defined by genetic alterations of CD79B at the first tyrosine residue of its ITAM motif (CD79B Y196H), crucial for signaling downstream of the BCR, and MYD88 (MYD88 L265P), a signaling adaptor protein essential for Toll Like receptor signaling. CD79B Y196H and MYD88 L265P mutations together define the MCD/C5 subtype and were found to be co-dependent for proliferation and oncogenic BCR-signaling in vitro. However, the mechanism of epistatic interaction between CD79B Y196H and MYD88 L265P is unknown. The MCD/C5 DLBCL subtype is associated with particularly poor clinical outcomes, however only a few human cell lines and a genetic mouse model are available for preclinical testing. Here, we developed a novel genetic approach to engineer the MCD/C5 subtype of DLBCL in primary human tonsillar germinal center B cells. To this end, we introduced the CD79B Y196H and MYD88 L265P mutations as genetic knockin alleles utilizing CRISPR-Cas9 homology directed repair templates (HDRT). In addition, we developed humanized NBSGW mice carrying a mouse Tslp-transgene to enable development of lymph nodes and secondary lymphoid organs to propagate genetically engineered GC B-cells carrying CD79B Y196H and MYD88 L265P knockin mutations for lymphoma development ( Figure 1). Results: Primary human germinal center B-cells were isolated from tonsils and grown on follicular dendritic feeder cells expressing CD40L and IL21. Single strand DNA templates were designed for CD79B, with and without Y196H mutation. Constructs were linked to fluorescent reporters by T2A sites and flanked by homology arms of ~280 bp targeting the guide RNA cut site. The target site was selected to disrupt the endogenous gene, while placing the construct under transcriptional control of endogenous promoter/enhancer elements. We achieved a knock-in efficiency of 5-8%, prior to single-cell cloning and genotyping to confirm the presence of heterozygous CD79B Y196H mutant CD79B Y196 wild-type mutations ( Figure 1). Preliminary biochemical studies show a delayed response and reduced intensity of signaling downstream of the BCR upon stimulation in CD79B Y196H mutant, compared to CD79B Y196 wild-type knockin germinal center B-cells. A similar approach was taken to design temples encoding Exon 5 of MYD88, with and without the L265P mutation, utilizing two guide RNAs to excise and replace Exon 5, linked to a mCherry reporter by a T2A site. Upon successfully introducing the MYD88 HDR templates into our CD79B wild-type and mutant clones to generate four distinct genotypes based on wildtype and CD79B Y196H and MYD88 L265P mutations alone or in combination. In addition, traditional NSG mice lack lymph nodes and secondary lymphoid organs. Lack of lymphoid follicles in NSG mice, owing to defective IL7Rα-signaling, contributed to lack of lymphoma formation observed in the previous model. In our model, we substituted the missing IL7Rα-signal with transgenic expression of murine Tslp, which enables development of embryonic lymphoid tissue inducer (LTi) cells that are required for the development of secondary lymphoid organs and germinal center follicles supporting initiation of human DLBCL development. Conclusion: Previous approaches to model DLBCL starting from germinal center B-cells were based on lentiviral overexpression of mutant oncogenes. Thereby, tonsillar germinal center B-cells with lentiviral BCL2 and BCL6 overexpression did not form lymphomas in NSG mice, however, were permissive to malignant transformation by MYC. Building on this previous work, we propose an improved model for preclinical testing for genetically defined DLBCL subtypes. Our two main improvements involve (i) genetic modeling based on knockin-strategies targeting endogenous loci and transcriptional control elements to phenocopy physiological gene expression levels and transcriptional regulation, and (ii) substitution of defective IL7Rα-signaling by transgenic expression of murine Tslp to rescue development of LTi cells. Transgenic expression of mTslp enables LTi-dependent formation of lymph nodes and secondary lymphoid organs supporting lymphoma development.

Abstract 17551: Mapping the Interactome of FLNC in Restrictive Cardiomyopathy

Introduction: Filamin C (FLNC) is an actin crosslinking protein that organizes structural components of the sarcomere and signal transduction complexes. Human mutations in FLNC have been linked to dilated, hypertrophic and restrictive cardiomyopathies. The mechanism connecting mutations in FLNC to sarcomeric impairment remains undefined. We recently reported the ability to model FLNC cardiomyopathy using induced pluripotent stem cell (iPSC) derived engineered heart tissue. Here, we employ a knock-in approach to epitope tag endogenous alleles in iPSC derived cardiomyocytes to explore FLNC genotype-interactome relationships using affinity purification mass spectrometry. Methods: An iPSC line was generated from a patient with restrictive cardiomyopathy caused by an insertion deletion mutation in the ROD2 domain FLNC (c.7416_7418delGAA, p.Glu2472_Asn2473delinAsp). CRISPR-Cas9 was used to establish a corrected cell line as a wild type control. 3X-Flag motif was introduced to the N terminus of FLNC using CRISPR-Cas9. iPSCs were then differentiated into cardiomyocytes using standard techniques. Affinity purification mass spectrometry (AP-MS) was performed using the Flag epitope. Interaction data was analyzed using Perseus software. Gene ontology (GO) enrichment analysis was performed using ToppFun. Results: 3X-Flag insertion was confirmed using genomic sequencing. AP-MS identified 281 high confidence interactors of mutant FLNC and 208 interactors of wild type FLNC (FDR 0.01, log2FC &gt;1.5) when compared to IgG controls. Interestingly, 161 proteins were shared between the two genotypes. These were enriched in mitochondrial ribosomal proteins and phosphorylase kinase subunits, potentially revealing an unknown role of FLNC in mitochondria translation and metabolism. Thirty-three proteins were significantly differentially bound to mutant FLNC protein, including tropomyosin 4, caldesmon 1, and filamin A (FDR &lt;0.05, log2FC&gt;1). GO analysis of these proteins revealed enrichment of actin cytoskeleton elements, muscle contraction, and muscle system processes. Conclusions: Human mutations in FLNC lead to disruption of key protein interactions, revealing a potential mechanism for disease pathogenesis in FLNC cardiomyopathy.

A robust knock-in approach using a minimal promoter and a minicircle

Knock-in reporter (KI) animals are essential tools in biomedical research to study gene expression impacting diverse biological events. While CRISPR/Cas9-mediated genome editing allows for the successful generation of KI animals, several factors should be considered, such as low expression of the target gene, prevention of bacterial DNA integration, and in-frame editing. To circumvent these challenges, we developed a new strategy that utilizes minicircle technology and introduces a minimal promoter. We demonstrated that minicircles serve as an efficient donor DNA in zebrafish, significantly enhancing KI events compared to plasmids containing bacterial backbones. In an attempt to generate a KI reporter for scn8ab, we precisely integrated a fluorescence gene at the start codon. However, the seamlessly integrated reporter was unable to direct expression that recapitulates endogenous scn8ab expression. To overcome this obstacle, we introduced the hsp70 minimal promoter to provide an ectopic transcription initiation site and succeeded in establishing stable KI transgenic reporters for scn8ab. This strategy also created a fgf20b KI reporter line with a high success rate. Furthermore, our data revealed that an unexpectedly edited genome can inappropriately influence the integrated reporter gene expression, highlighting the importance of selecting a proper KI line. Overall, our approach utilizing a minicircle and an ectopic promoter establishes a robust and efficient strategy for KI generation, expanding our capacity to create KI animals.

Establishment of transgenic epithelium-specific Cre-recombinase driving medaka (Oryzias latipes) by homology repair mediated knock-in.

Deletion of gene expression in the target tissues and cells is an effective strategy for elucidating the physiological functions of the protein of interest. For tissue-specific and/or inducible gene deletion, the Cre-loxP system has been widely used in various model organisms including medaka (Oryzias latipes). The epithelium is the key tissue, locating at the outermost area and playing a role in barrier to external stimuli. Despite a large genetic toolbox developed in medaka, there is no available Cre-driver line that works in an epithelium-specific manner. Here, we established epithelium-specific Cre-driver lines in medaka using a homology-directed repair mediated knock-in approach with CRISPR/Cas9, targeting each of periplakin and keratin genes. We show that Cre-recombinase is expressed exclusively in the epithelium in the knock-in lines and that it efficiently and specifically induces recombination in the tissues. These Cre-driver lines are useful for studying the functions of proteins expressed in the epithelium.

A robust genetic toolbox for fine-tuning gene expression in the CO2-Fixing methanogenic archaeon Methanococcus maripaludis

Libraries of well-characterized genetic elements for fine-tuning gene expression are essential for biological and biotechnological research and applications. The fast-growing and genetically tractable methanogen, Methanococcus maripaludis, is a promising host organism for biotechnological conversion of carbon dioxide and renewable hydrogen into fuels and value-added products, as well as fundamental biological studies of archaea. However, the lack of molecular tools for gene expression has hindered its application as a workhorse to fine-tune gene and metabolic pathway expressions. In this study, we developed a genetic toolbox, including libraries of promoters, ribosome binding sites (RBS), and neutral sites for chromosomal integration, to facilitate precise gene expression in M. maripaludis. We generated a promoter library consisting of 81 constitutive promoters with expression strengths spanning a ∼104-fold dynamic range. Importantly, we identified a base composition rule for strong archaeal promoters and successfully remodeled weak promoters, enhancing their activities by up to 120-fold. We also established an RBS library containing 42 diverse RBS sequences with translation strengths covering a ∼100-fold dynamic range. Additionally, we identified eight neutral sites and developed a one-step, Cas9-based marker-less knock-in approach for chromosomal integration. We successfully applied the characterized promoter and RBS elements to significantly improve recombinant protein expression by 41-fold and modulate essential gene expression to generate corresponding physiological changes in M. maripaludis. Therefore, this work establishes a solid foundation for utilizing this autotrophic methanogen as an ideal workhorse for archaeal biology and biotechnological studies and applications.

Optogenetic control of medaka behavior with channelrhodopsin.

Optogenetics enables the manipulation of neural activity with high spatiotemporal resolution in genetically defined neurons. The method is widely used in various model animals in the neuroscience and physiology fields. Channelrhodopsins are robust tools for optogenetic manipulation, but they have not yet been used for studies in medaka. In the present study, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated knock-in approach to establish a transgenic medaka strain expressing the Chloromonas oogama channelrhodopsin (CoChR) in the ISL LIM homeobox 1 (isl1) locus. We demonstrated that light stimuli elicited specific behavioral responses, such as bending or turning locomotion in the embryos and pectoral fin movements in the larvae and adults. The response probabilities and intensities of these movements could be controlled by adjusting the intensity, duration, or wavelength of each light stimulus. Furthermore, we demonstrated that the pectoral fin movements in the adult stage could be elicited using a laser pointer to irradiate region including the caudal hind brain and the rostral spinal cord. Our results indicate that CoChR allows for manipulation of medaka behaviors by activating targeted neurons, which will further our understanding of the detailed neural mechanisms of motor control or social behaviors in medaka.

Investigator-initiated phase 1 clinical trial of synthetic TIL: Autologous peripheral T cells engineered with polyclonal TIL-derived TCRs.

e14525 Background: The efficacy of tumor-infiltrating lymphocyte (TIL) therapy is limited by the rarity of anti-tumor TCRs and exhausted status of the product. Its improvement should focus on a high frequency of diverse anti-tumor TCRs with stem-like phenotype, which can be achieved synthetically by identifying multiple anti-tumor TCRs from the patient's intra-tumor TCR repertoire and subsequently engineering them into the patient’s peripheral T cells. Such a ‘synthetic TIL’ is cost-prohibitive using existing technologies. To enable a synthetic TIL product, we developed ultra-high throughput TCR gene synthesis and screening methods to synthesize and screen &gt; 50,000 TCRs from scRNA-Seq data from &gt; 30 patients and identified &gt; 100 tumor/NeoAg-reactive TCRs. The gene expression patterns of the T cells carrying such experimentally validated tumor/NeoAg-reactive TCRs gave rise to an algorithm called Interpreter to predict potentially anti-tumor TCRs from scRNA-Seq data. Methods: To translate synthetic TIL to the clinic, we developed a method to generate up to 100 sequence-verified TCR-encoding plasmids (selected by Interpreter) from fresh tumor biopsy in just 3 weeks. Our GMP-grade production uses a collective knock-in approach, where a mixture of up to 100 plasmids (each encoding a distinct TCR) at equal proportion are packaged into AAV which serve as template to precisely insert the TCR genes into a primarily monoallelic locus and simultaneously inhibit the endogenous TCR. This gene editing is characterized by a modified scRNA-Seq method called EntrySeq. In first-in-human, N-of-1 trials, 109 to 6x109 synthetic TIL cells were administered to solid tumor patients with or without lymphodepletion and IL2. An investigator-initiated Phase 1 trial was then opened to evaluate safety, preliminary efficacy, and cellular activity of synthetic TIL and to determine RP2D and MTD. Subjects with relapsed, unresectable locally advanced solid tumors who received at least 1 standard systemic therapy are allowed to enroll in this study. Those patients with no standard treatment regimens available, or those who are unable to obtain effective treatment due to economic conditions are also permitted. Results: EntrySeq confirmed that &gt; 80% of the T cells express only 1 TCR, avoiding mispairing of different TCRs. The frequencies of different TCRs were largely uniform and were stable through plasmid, AAV and T-cell mixtures. Up to 1.5x1010 synthetic TIL cells were produced from a patient’s leukopak. 40 to 60% of the T cells in the product for infusion express an exogenous TCR. In first-in-human trials, the administered T cells showed favorable persistence and safety profile. The Phase 1 trial is ongoing. Conclusions: We established a fast, affordable, and GMP-compliant process to manufacture synthetic TIL products, enabling a Phase 1 trial.

Rapid and precise genome engineering in a naturally short-lived vertebrate.

The African turquoise killifish is a powerful vertebrate system to study complex phenotypes at scale, including aging and age-related disease. Here, we develop a rapid and precise CRISPR/Cas9-mediated knock-in approach in the killifish. We show its efficient application to precisely insert fluorescent reporters of different sizes at various genomic loci in order to drive cell-type- and tissue-specific expression. This knock-in method should allow the establishment of humanized disease models and the development of cell-type-specific molecular probes for studying complex vertebrate biology.

Neurogenetic identification of mosquito sensory neurons

Anopheles mosquitoes, as vectors for the malaria parasite, are a global threat to human health. To find and bite a human, they utilize neurons within their sensory appendages. However, the identity and quantification of sensory appendage neurons are lacking. Here we use a neurogenetic approach to label all neurons in Anopheles coluzzii mosquitoes. We utilize the homology assisted CRISPR knock-in (HACK) approach to generate a T2A-QF2w knock-in of the synaptic gene bruchpilot. We use a membrane-targeted GFP reporter to visualize the neurons in the brain and to quantify neurons in all major chemosensory appendages (antenna, maxillary palp, labella, tarsi, and ovipositor). By comparing labeling of brp>GFP and Orco>GFP mosquitoes, we predict the extent of neurons expressing ionotropic receptors (IRs) or other chemosensory receptors. This work introduces a valuable genetic tool for the functional analysis of Anopheles mosquito neurobiology and initiates characterization of the sensory neurons that guide mosquito behavior.