Progenitor Hyperpolarization Regulates the Sequential Generation of Neuronal Subtypes in the Developing Neocortex.

During corticogenesis, ventricular zone progenitors sequentially generate distinct subtypes of neurons, accounting for the diversity of neocortical cells and the circuits they form. While activity-dependent processes are critical for the differentiation and circuit assembly of postmitotic neurons, how bioelectrical processes affect nonexcitable cells, such as progenitors, remains largely unknown. Here, we reveal that, in the developing mouse neocortex, ventricular zone progenitors become more hyperpolarized as they generate successive subtypes of neurons. Experimental in vivo hyperpolarization shifted the transcriptional programs and division modes of these progenitors to a later developmental status, with precocious generation of intermediate progenitors and a forward shift in the laminar, molecular, morphological, and circuit features of their neuronal progeny. These effects occurred through inhibition of the Wnt-beta-catenin signaling pathway by hyperpolarization. Thus, during corticogenesis, bioelectric membrane properties are permissive for specific molecular pathways to coordinate the temporal progression of progenitor developmental programs and thus neocortical neuron diversity.

Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia.

The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR T cells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.

Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL.

Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in treating haematological malignancies1-7. However, CAR-T cell therapy currently has several limitations8-12. Here we successfully developed a two-in-one approach to generate non-viral, gene-specific targeted CAR-T cells through CRISPR-Cas9. Using the optimized protocol, we demonstrated feasibility in a preclinical study by inserting an anti-CD19 CAR cassette into the AAVS1 safe-harbour locus. Furthermore, an innovative type of anti-CD19 CAR-T cell with PD1 integration was developed and showed superior ability to eradicate tumour cells in xenograft models. In adoptive therapy for relapsed/refractory aggressive B cell non-Hodgkin lymphoma (ClinicalTrials.gov, NCT04213469 ), we observed a high rate (87.5%) of complete remission and durable responses without serious adverse events in eight patients. Notably, these enhanced CAR-T cells were effective even at a low infusion dose and with a low percentage of CAR+ cells. Single-cell analysis showed that the electroporation method resulted in a high percentage of memory T cells in infusion products, and PD1 interference enhanced anti-tumour immune functions, further validating the advantages of non-viral, PD1-integrated CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, gene-specific integrated CAR-T cells, thus providing an innovative technology for CAR-T cell therapy.

Local circuit amplification of spatial selectivity in the hippocampus.

Local circuit architecture facilitates the emergence of feature selectivity in the cerebral cortex1. In the hippocampus, it remains unknown whether local computations supported by specific connectivity motifs2 regulate the spatial receptive fields of pyramidal cells3. Here we developed an in vivo electroporation method for monosynaptic retrograde tracing4 and optogenetics manipulation at single-cell resolution to interrogate the dynamic interaction of place cells with their microcircuitry during navigation. We found a local circuit mechanism in CA1 whereby the spatial tuning of an individual place cell can propagate to a functionally recurrent subnetwork5 to which it belongs. The emergence of place fields in individual neurons led to the development of inverse selectivity in a subset of their presynaptic interneurons, and recruited functionally coupled place cells at that location. Thus, the spatial selectivity of single CA1 neurons is amplified through local circuit plasticity to enable effective multi-neuronal representations that can flexibly scale environmental features locally without degrading the feedforward input structure.

Transdermal microarrayed electroporation for enhanced cancer immunotherapy based on DNA vaccination.

Despite the tremendous clinical potential of nucleic acid-based vaccines, their efficacy to induce therapeutic immune response has been limited by the lack of efficient local gene delivery techniques in the human body. In this study, we develop a hydrogel-based organic electronic device (µEPO) for both transdermal delivery of nucleic acids and in vivo microarrayed cell electroporation, which is specifically oriented toward one-step transfection of DNAs in subcutaneous antigen-presenting cells (APCs) for cancer immunotherapy. The µEPO device contains an array of microneedle-shaped electrodes with pre-encapsulated dry DNAs. Upon a pressurized contact with skin tissue, the electrodes are rehydrated, electrically triggered to release DNAs, and then electroporate nearby cells, which can achieve in vivo transfection of more than 50% of the cells in the epidermal and upper dermal layer. As a proof-of-concept, the µEPO technique is employed to facilitate transdermal delivery of neoantigen genes to activate antigen-specific immune response for enhanced cancer immunotherapy based on a DNA vaccination strategy. In an ovalbumin (OVA) cancer vaccine model, we show that high-efficiency transdermal transfection of APCs with OVA-DNAs induces robust cellular and humoral immune responses, including antigen presentation and generation of IFN-γ+ cytotoxic T lymphocytes with a more than 10-fold dose sparing over existing intramuscular injection (IM) approach, and effectively inhibits tumor growth in rodent animals.

Optimizing CRISPR/Cas9 approaches in the polymorphic tunicate Ciona intestinalis.

CRISPR/Cas9 became a powerful tool for genetic engineering and in vivo knockout also in the invertebrate chordate Ciona intestinalis. Ciona (ascidians, tunicates) is an important model organism because it shares developmental features with the vertebrates, considered the sister group of tunicates, and offers outstanding experimental advantages: a compact genome and an invariant developmental cell lineage that, combined with electroporation mediated transgenesis allows for precise and cell type specific targeting in vivo. A high polymorphism and the mosaic expression of electroporated constructs, however, often hamper the efficient CRISPR knockout, and an optimization in Ciona is desirable. Furthermore, seasonality and artificial maintenance settings can profit from in vitro approaches that would save on animals. Here we present improvements for the CRISPR/Cas9 protocol in silico, in vitro and in vivo. Firstly, in designing sgRNAs, prior sequencing of target genomic regions from experimental animals and alignment with reference genomes of C. robusta and C. intestinalis render a correction possible of subspecies polymorphisms. Ideally, the screening for efficient and non-polymorphic sgRNAs will generate a database compatible for worldwide Ciona populations. Secondly, we challenged in vitro assays for sgRNA validation towards reduced in vivo experimentation and report their suitability but also overefficiency concerning mismatch tolerance. Thirdly, when comparing Cas9 with Cas9:Geminin, thought to synchronize editing and homology-direct repair, we could indeed increase the in vivo efficiency and notably the access to an early expressed gene. Finally, for in vivo CRISPR, genotyping by next generation sequencing (NGS) ex vivo streamlined the definition of efficient single guides. Double CRISPR then generates large deletions and reliable phenotypic excision effects. Overall, while these improvements render CRISPR more efficient in Ciona, they are useful when newly establishing the technique and very transferable to CRISPR in other organisms.

A simple method for gene expression in endo- and ectodermal cells in mouse embryos before neural tube closure.

The lack of a widely accessible method for expressing genes of interest in wild-type embryos is a fundamental obstacle to understanding genetic regulation during embryonic development. In particular, only a few methods are available for introducing gene expression vectors into cells prior to neural tube closure, which is a period of drastic development for many tissues. In this study, we present a simple technique for injecting vectors into the amniotic cavity and allowing them to reach the ectodermal cells and the epithelia of endodermal organs of mouse embryos at E8.0 via in utero injection, using only a widely used optical fiber with an illuminator. Using this technique, retroviruses can be introduced to facilitate the labeling of cells in various tissues, including the brain, spinal cord, epidermis, and digestive and respiratory organs. We also demonstrated in utero electroporation of plasmid DNA into E7.0 and E8.0 embryos. Taking advantage of this method, we reveal the association between Ldb1 and the activity of the Neurog2 transcription factor in the mouse neocortex. This technique can aid in analyzing the roles of genes of interest during endo- and ectodermal development prior to neural tube closure.

Breasi-CRISPR: an efficient genome-editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex.

In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. Here, we describe the Breasi-CRISPR approach (Brain Easi-CRISPR), combining Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. Breasi-CRISPR enables knock-in of both short and long epitope tag sequences with high efficiency. We visualized epitope-tagged proteins with varied expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1 and RPL22. Detection was possible by immunohistochemistry as soon as 1 day after electroporation and we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores, allowing visualization of endogenous proteins by live imaging in organotypic brain slices. Finally, we used Breasi-CRISPR to perform co-immunoprecipitation mass-spectrometry analyses of the autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data demonstrate that Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

Nanoinjection: A Platform for Innovation in Ex Vivo Cell Engineering.

ConspectusIn human cells, intracellular access and therapeutic cargo transport, including gene-editing tools (e.g., CRISPR-Cas9 and transposons), nucleic acids (e.g., DNA, mRNA, and siRNA), peptides, and proteins (e.g., enzymes and antibodies), are tightly constrained to ensure healthy cell function and behavior. This principle is exemplified in the delivery mechanisms of chimeric antigen receptor (CAR)-T cells for ex-vivo immunotherapy. In particular, the clinical success of CAR-T cells has established a new standard of care by curing previously incurable blood cancers. The approach involves the delivery, typically via the use of electroporation (EP) and lentivirus, of therapeutic CAR genes into a patient's own T cells, which are then engineered to express CARs that target and combat their blood cancer. But the key difficulty lies in genetically manipulating these cells without causing irreversible damage or loss of function─all the while minimizing complexities of manufacturing, safety concerns, and costs, and ensuring the efficacy of the final CAR-T cell product.Nanoinjection─the process of intracellular delivery using nanoneedles (NNs)─is an emerging physical delivery route that efficiently negotiates the plasma membrane of many cell types, including primary human T cells. It occurs with minimal perturbation, invasiveness, and toxicity, with high efficiency and throughput at high spatial and temporal resolutions. Nanoinjection promises greatly improved delivery of a broad range of therapeutic cargos with little or no damage to those cargos. A nanoinjection platform allows these cargos to function in the intracellular space as desired. The adaptability of nanoinjection platforms is now bringing major advantages in immunomodulation, mechanotransduction, sampling of cell states (nanobiopsy), controlled intracellular interrogation, and the primary focus of this account─intracellular delivery and its applications in ex vivo cell engineering.Mechanical nanoinjection typically exerts direct mechanical force on the cell membrane, offering a straightforward route to improve membrane perturbation by the NNs and subsequent transport of genetic cargo into targeted cell type (adherent or suspension cells). By contrast, electroactive nanoinjection is controlled by coupling NNs with an electric field─a new route for activating electroporation (EP) at the nanoscale─allowing a dramatic reduction of the applied voltage to a cell and so minimizing post-EP damage to cells and cargo, and overcoming many of the limitations of conventional bulk EP. Nanoinjection transcends mere technique; it is an approach to cell engineering ex vivo, offering the potential to endow cells with new, powerful features such as generating chimeric antigen receptor (CAR)-T cells for future CAR-T cell technologies.We first discuss the manufacturing of NN devices (Section 2), then delve into nanoinjection-mediated cell engineering (Section 3), nanoinjection mechanisms and interfacing methodologies (Section 4), and emerging applications in using nanoinjection to create functional CAR-T cells (Section 5).

M-TUBE enables large-volume bacterial gene delivery using a high-throughput microfluidic electroporation platform.

Conventional cuvette-based and microfluidics-based electroporation approaches for bacterial gene delivery have distinct advantages, but they are typically limited to relatively small sample volumes, reducing their utility for applications requiring high throughput such as the generation of mutant libraries. Here, we present a scalable, large-scale bacterial gene delivery approach enabled by a disposable, user-friendly microfluidic electroporation device requiring minimal device fabrication and straightforward operation. We demonstrate that the proposed device can outperform conventional cuvettes in a range of situations, including across Escherichia coli strains with a range of electroporation efficiencies, and we use its large-volume bacterial electroporation capability to generate a library of transposon mutants in the anaerobic gut commensal Bifidobacterium longum.

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage.

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

2D electrical admittance lattice model of biological cellular system for modeling electroporation.

In this work, a new modeling approach is presented to obtain a two-dimensional transport lattice of a biological cellular system for the calculation of the potential distribution throughout the system and investigation of the corresponding membrane electroporation. The presented model has been obtained by a modified bilayer model of the cell membrane. This modified membrane model allows for an effective inclusion of the shape of the cell membrane in the potential calculation. The results of the model have shown good agreement with the results of the well-known Schwan equation and COMSOL Multiphysics for the circular cell. The simulation results show that both membranes of a mitochondrion can be simultaneously electroporated by an alternating voltage source with frequencies between 1 MHz and 1 GHz.

Real-time imaging of individual electropores proves their longevity in cells.

With over 50 years of electroporation research, the nature of cell membrane permeabilization remains elusive. The lifetime of electropores in molecular models is limited to nano- or microseconds, whereas the permeabilization of electroporated cells can last minutes. This study aimed at resolving a longstanding debate on whether the prolonged permeabilization is due to the formation of long-lived pores in cells. We developed a method for dynamic monitoring and conductance measurements of individual electropores. This was accomplished by time-lapse total internal reflection fluorescence (TIRF) imaging in HEK cells loaded with CAL-520 dye and placed on an indium tin oxide (ITO) surface. Applying a 1-ms, 0 to -400 mV pulse between the patch pipette and ITO evoked focal Ca2+ transients that identified individual electropores. Some transients disappeared in milliseconds but others persisted for over a minute. Persistent transients ("Ca2+ plumes") faded over time to a stable or a randomly fluctuating level that could include periods of full quiescence. Single pore conductance, measured by 0 to -50 mV, 50 ms steps at 30 and 60 s after the electroporation, ranged from 80 to 200 pS. These experiments proved electropore longevity in cells, in stark contrast to molecular simulations and many findings in lipid bilayers.

A solution for highly efficient electroporation of primary cytotoxic T lymphocytes.

BACKGROUND: Cytotoxic T lymphocytes (CTLs) are central players in the adaptive immune response. Their functional characterization and clinical research depend on efficient and reliable transfection. Although various methods have been utilized, electroporation remains the preferred technique for transient gene over-expression. However, the efficiency of electroporation is reduced for human and mouse primary CTLs. Lonza offers kits that effectively improve plasmid DNA transfection quality. Unfortunately, the removal of key components of the cell recovery medium considerably reduced the efficiency of their kit for CTLs. Our aim was to develop a new recovery medium to be used with Lonza's Nucleofector system that would significantly enhance transfection rates. RESULTS: We assessed the impact of different media in which the primary CTLs were placed to recover after electroporation on cell survival, transfection rate and their ability to form an immunological synapse and to perform exocytosis. We transfected the cells with pmax-GFP and large constructs encoding for either CD81-super ecliptic pHluorin or granzyme B-pHuji. The comparison of five different media for mouse and two for human CTLs demonstrated that our new recovery medium composed of Opti-MEM-GlutaMAX supplemented with HEPES, DMSO and sodium pyruvate gave the best result in cell survival (> 50%) and transfection rate (> 30 and 20% for mouse and human cells, respectively). More importantly, the functionality of CTLs was at least twice as high as with the original Lonza recovery medium. In addition, our RM significantly improved transfection efficacy of natural killer cells that are notoriously hard to electroporate. CONCLUSION: Our results show that successful transfection depends not only on the electroporation medium and pulse sequence but also on the medium applied for cell recovery. In addition, we have reduced our reliance on proprietary products by designing an effective recovery medium for both mouse and human primary CTLs and other lymphocytes that can be easily implemented by any laboratory. We expect that this recovery medium will have a significant impact on both fundamental and applied research in immunology.

Hes1 overexpression leads to expansion of embryonic neural stem cell pool and stem cell reservoir in the postnatal brain.

Neural stem cells (NSCs) gradually alter their characteristics during mammalian neocortical development, resulting in the production of various neurons and glial cells, and remain in the postnatal brain as a source of adult neurogenesis. Notch-Hes signaling is a key regulator of stem cell properties in the developing and postnatal brain, and Hes1 is a major effector that strongly inhibits neuronal differentiation and maintains NSCs. To manipulate Hes1 expression levels in NSCs, we generated transgenic (Tg) mice using the Tet-On system. In Hes1-overexpressing Tg mice, NSCs were maintained in both embryonic and postnatal brains, and generation of later-born neurons was prolonged until later stages in the Tg neocortex. Hes1 overexpression inhibited the production of Tbr2+ intermediate progenitor cells but instead promoted the generation of basal radial glia-like cells in the subventricular zone (SVZ) at late embryonic stages. Furthermore, Hes1-overexpressing Tg mice exhibited the expansion of NSCs and enhanced neurogenesis in the SVZ of adult brain. These results indicate that Hes1 overexpression expanded the embryonic NSC pool and led to the expansion of the NSC reservoir in the postnatal and adult brain.

Transepithelial Electrical Impedance Increase Following Porous Substrate Electroporation Enables Label-Free Delivery.

Porous substrate electroporation (PSEP) is a promising new method for intracellular delivery, yet fundamentals of PSEP are not well understood, especially the intermediate processes leading to delivery. PSEP is an electrical method, yet the relationship between PSEP and electrical impedance remains underexplored. In this study, a device capable of measuring impedance and performing PSEP is developed and the changes in transepithelial electrical impedance (TEEI) are monitored. These measurements show TEEI increases following PSEP, unlike other electroporation methods. The authors then demonstrate how cell culture conditions and electrical waveforms influence this response. More importantly, TEEI response features are correlated with viability and delivery efficiency, allowing prediction of outcomes without fluorescent cargo, imaging, or image processing. This label-free delivery also allows improved temporal resolution of transient processes following PSEP, which the authors expect will aid PSEP optimization for new cell types and cargos.

Novel electroporation-based genome editing of carnation plant tissues using RNPs targeting the anthocyanidin synthase gene.

MAIN CONCLUSION: A novel electroporation method for genome editing was performed using plant tissue samples by direct RNPs-introduction in carnation. Genome editing is becoming a very useful tool in plant breeding. In this study, a novel electroporation method was performed for genome editing using plant tissue samples. The objective was to create a flower color mutant using the pink-flowered carnation 'Kane Ainou 1-go'. For this purpose, a ribonucleoprotein consisting of guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) was introduced into the stem tissue to induce mutations in the anthocyanidin synthase (ANS) gene, which is involved in anthocyanin biosynthesis. As the ANS of 'Kane Ainou 1-go' has not been previously isolated, we initially isolated the ANS gene from 'Kane Ainou 1-go' for characterization. Southern hybridization analysis confirmed that the ANS gene was present in the genome as a two-allele gene with a pair of homologous sequences (ANS-1 and 2); these sequences were used as the target for genome editing. Genome editing was performed by introducing #2_single-guide RNA into the stem tissue using the ribonucleoprotein. This molecule was used because it exhibited the highest efficiency in an analysis of cleavage activity against the target sequence in vitro. Cleaved amplified polymorphic sequence analysis of genomic DNA extracted from 85 regenerated individuals after genome editing was performed. The results indicated that mutations in the ANS gene may have been introduced into two lines. Cloning of the ANS gene in these two lines confirmed the introduction of a single nucleotide substitution mutation for ANS-1 in both lines, and a single amino acid substitution in one line. We discussed the possibility of color change by the amino acid substitution, and also the future applications of this technology.

Recent Advancements in Electroporation Technologies: From Bench to Clinic.

Over the past decade, the increased adoption of electroporation-based technologies has led to an expansion of clinical research initiatives. Electroporation has been utilized in molecular biology for mammalian and bacterial transfection; for food sanitation; and in therapeutic settings to increase drug uptake, for gene therapy, and to eliminate cancerous tissues. We begin this article by discussing the biophysics required for understanding the concepts behind the cell permeation phenomenon that is electroporation. We then review nano- and microscale single-cell electroporation technologies before scaling up to emerging in vivo applications.

The elimination of two restriction enzyme genes allows for electroporation-based transformation and CRISPR-Cas9-based base editing in the non-competent Gram-negative bacterium Acinetobacter sp. Tol 5.

Environmental isolates are promising candidates for new chassis of synthetic biology because of their inherent capabilities, which include efficiently converting a wide range of substrates into valuable products and resilience to environmental stresses; however, many remain genetically intractable and unamenable to established genetic tools tailored for model bacteria. Acinetobacter sp. Tol 5, an environmentally isolated Gram-negative bacterium, possesses intriguing properties for use in synthetic biology applications. Despite the previous development of genetic tools for the engineering of strain Tol 5, its genetic manipulation has been hindered by low transformation efficiency via electroporation, rendering the process laborious and time-consuming. This study demonstrated the genetic refinement of the Tol 5 strain, achieving efficient transformation via electroporation. We deleted two genes encoding type I and type III restriction enzymes. The resulting mutant strain not only exhibited marked efficiency of electrotransformation but also proved receptive to both in vitro and in vivo DNA assembly technologies, thereby facilitating the construction of recombinant DNA without reliance on intermediate Escherichia coli constructs. In addition, we successfully adapted a CRISPR-Cas9-based base-editing platform developed for other Acinetobacter species. Our findings provide genetic modification strategies that allow for the domestication of environmentally isolated bacteria, streamlining their utilization in synthetic biology applications.IMPORTANCERecent synthetic biology has sought diverse bacterial chassis from environmental sources to circumvent the limitations of laboratory Escherichia coli strains for industrial and environmental applications. One of the critical barriers in cell engineering of bacterial chassis is their inherent resistance to recombinant DNA, propagated either in vitro or within E. coli cells. Environmental bacteria have evolved defense mechanisms against foreign DNA as a response to the constant threat of phage infection. The ubiquity of phages in natural settings accounts for the genetic intractability of environmental isolates. The significance of our research is in demonstrating genetic modification strategies for the cell engineering of such genetically intractable bacteria. This research marks a pivotal step in the domestication of environmentally isolated bacteria, promising candidates for emerging synthetic biology chassis. Our work thus significantly contributes to advancing their applications across industrial, environmental, and biomedical fields.

Electroporation-mediated genome editing in vitrified/warmed porcine zygotes obtained in vitro.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) system is the most efficient and widely used technology for genome editing in all sorts of organisms, including livestock animals. Here, we examined the feasibility of CRISPR/Cas9-derived genome editing (GE) in vitrified porcine zygotes, where the flexible planning of experiments in time and space is expected. OCT4 and CD46 genes were targeted, and the Cas9/sgRNA ribonucleoprotein complexes (RNP) were electroporated into zygotes at 2 h after warming. Vitrification or GE alone did not significantly reduce the developmental rates to the blastocyst stage. However, vitrification followed by GE significantly reduced blastocyst development. Sequencing analysis of the resultant blastocysts revealed efficient GE for both OCT4 (nonvitrified: 91.0%, vitrified: 95.1%) and CD46 (nonvitrified: 94.5%, vitrified: 93.2%), with no significant difference among them. Immunocytochemical analysis showed that GE-blastocysts lacked detectable proteins. They were smaller in size, and the cell numbers were significantly reduced compared with the control (p < 0.01). Finally, we demonstrated that double GE efficiently occurs (100%) when the OCT4-RNP and CD46-RNP are simultaneously introduced into zygotes after vitrification/warming. This is the first demonstration that vitrified porcine zygotes can be used in GE as efficiently as nonvitrified ones.