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Clustered randomly interspaced short palindromic repeats (CRISPRs) and its associated endonuclease protein, i.e., Cas9, have been discovered as an immune system in bacteria and archaea; nevertheless, they are now being adopted as mainstream biotechnological/molecular scissors that can modulate ample genetic and nongenetic diseases via insertion/deletion, epigenome editing, messenger RNA editing, CRISPR interference, etc. Many Food and Drug Administration-approved and ongoing clinical trials on CRISPR adopt ex vivo strategies, wherein the gene editing is performed ex vivo, followed by reimplantation to the patients. However, the in vivo delivery of the CRISPR components is still under preclinical surveillance. This review has summarized the nonviral nanodelivery strategies for gene editing using CRISPR/Cas9 and its recent advancements, strategic points of view, challenges, and future aspects for tissue-specific in vivo delivery of CRISPR/Cas9 components using nanomaterials.

Nanoparticle-based drug delivery systems have the potential to revolutionize medicine, but their low vascular permeability and rapid clearance by phagocytic cells have limited their medical impact. Nanoparticles delivered at the in utero stage can overcome these key limitations due to the high rate of angiogenesis and cell division in fetal tissue and the under-developed immune system. However, very little is known about nanoparticle drug delivery at the fetal stage of development. In this report, using Ai9 CRE reporter mice, we demonstrate that lipid nanoparticle (LNP) mRNA complexes can deliver mRNA in utero, and can access and transfect major organs, such as the heart, the liver, kidneys, lungs and the gastrointestinal tract with remarkable efficiency and low toxicity. In addition, at 4 weeks after birth, we demonstrate that 50.99 ± 5.05%, 36.62 ± 3.42% and 23.7 ± 3.21% of myofiber in the diaphragm, heart and skeletal muscle, respectively, were transfected. Finally, we show here that Cas9 mRNA and sgRNA complexed to LNPs were able to edit the fetal organs in utero. These experiments demonstrate the possibility of non-viral delivery of mRNA to organs outside of the liver in utero, which provides a promising strategy for treating a wide variety of devastating diseases before birth.

Immuno-oncology (IO) and targeted therapies, such as small molecule inhibitors, have changed the landscape of cancer treatment and prognosis; however, durable responses have been difficult to achieve due to tumor heterogeneity, development of drug resistance, and adverse effects that limit dosing and prolonged drug use. To improve upon the current medicinal armamentarium, there is an urgent need for new ways to understand, reverse, and treat carcinogenesis. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) 9 is a powerful and efficient tool for genome editing that has shown significant promise for developing new therapeutics. While CRISPR/Cas9 has been successfully used for pre-clinical cancer research, its use in the clinical setting is still in an early stage of development. The purpose of this review is to describe the CRISPR technology and to provide an overview of its current applications and future potential as cancer therapies.

The editorial "CRISPR: the game changer in gene and cell therapy" discusses the transformative impact of CRISPR technology on gene and cell therapy. CRISPR's simplicity, precision, and efficiency have revolutionized genome engineering, leading to innovative therapies for genetic disorders, cancer, and infectious diseases. The editorial highlights recent advancements in CRISPR technology, including applications such as diagnostics, prime editing, disease modeling, and therapeutic applications, while also addressing current challenges and future directions in the field. Researchers are encouraged to contribute to the dynamic field of CRISPR technology to improve patient outcomes and revolutionize medicine. [Extracted from the article]

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.

The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex [SKR-1/2-CUL-1-F-box (SCF)], targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin derivative/mutant AtTIR1 pair [C. elegans AID version 2 (C.e.AIDv2)] that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand-binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID-targets, the addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expand the utility of the AID system and broaden the number of proteins that can be effectively targeted with it.

Cecropins are small helical secreted peptides with antimicrobial activity that are widely distributed among insects. Genes encoding Cecropins are strongly induced upon infection, pointing to their role in host defense. In Drosophila, four cecropin genes clustered in the genome (CecA1, CecA2, CecB, and CecC) are expressed upon infection downstream of the Toll and Imd pathways. In this study, we generated a short deletion ΔCecA-C removing the whole cecropin locus. Using the ΔCecA-C deficiency alone or in combination with other antimicrobial peptide (AMP) mutations, we addressed the function of Cecropins in the systemic immune response. ΔCecA-C flies were viable and resisted challenge with various microbes as wild-type. However, removing ΔCecA-C in flies already lacking 10 other AMP genes revealed a role for Cecropins in defense against Gram-negative bacteria and fungi. Measurements of pathogen loads confirm that Cecropins contribute to the control of certain Gram-negative bacteria, notably Enterobacter cloacae and Providencia heimbachae. Collectively, our work provides the first genetic demonstration of a role for Cecropins in insect host defense and confirms their in vivo activity primarily against Gram-negative bacteria and fungi. Generation of a fly line (ΔAMP14) that lacks 14 immune inducible AMPs provides a powerful tool to address the function of these immune effectors in host-pathogen interactions and beyond.

Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

Toxoplasmosis is a worldwide disease affecting all warm-blooded animals, including humans. Vaccination strategies aimed at inducing an efficient immune response while preventing transmission have been attempted in the past. While many different approaches can partially protect immunized animals against subsequent infections, full and lasting protection is rarely attained and only with live-attenuated vaccines. In addition, vaccines based on mutant strains that are deficient in forming the chronic phase of the parasite (such as Toxovax™) cannot be extensively used due to their zoonotic potential and the possibility of reversion to virulent phenotypes. An increasing number of studies using emerging genetic-engineering tools have been conducted to design novel vaccines based on recombinant proteins, DNA or delivery systems such as nanoparticles. However, these are usually less efficient due to their antigenic simplicity. In this perspective article we discuss potential target genes and novel strategies to generate live-attenuated long-lasting vaccines based on tissue cysts and oocysts, which are the environmentally resistant chronic forms of Toxoplasma. By selectively disrupting genes important for parasite dissemination, cyst formation and/or sporozoite invasion, alone or in combination, a vaccine based on a live-attenuated strain that elicits a protective immune response while preventing the transmission of Toxoplasma could be created. Finally, further improvements of protocols to generate Toxoplasma sexual stages in vitro might lead to the production of oocysts from such a strain without the need for using mice or cats.

Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR/Cas9) has transformed our ability to edit the human genome selectively. This technology has quickly become the most standardized and reproducible gene editing tool available. Catalyzing rapid advances in biomedical research and genetic engineering, the CRISPR/Cas9 system offers great potential to provide diagnostic and therapeutic options for the prevention and treatment of currently incurable single-gene and more complex human diseases. However, significant barriers to the clinical application of CRISPR/Cas9 remain. While in vitro, ex vivo, and in vivo gene editing has been demonstrated extensively in a laboratory setting, the translation to clinical studies is currently limited by shortfalls in the precision, scalability, and efficiency of delivering CRISPR/Cas9-associated reagents to their intended therapeutic targets. To overcome these challenges, recent advancements manipulate both the delivery cargo and vehicles used to transport CRISPR/Cas9 reagents. With the choice of cargo informing the delivery vehicle, both must be optimized for precision and efficiency. This review aims to summarize current bioengineering approaches to applying CRISPR/Cas9 gene editing tools towards the development of emerging cellular therapeutics, focusing on its two main engineerable components: the delivery vehicle and the gene editing cargo it carries. The contemporary barriers to biomedical applications are discussed within the context of key considerations to be made in the optimization of CRISPR/Cas9 for widespread clinical translation.

Genome editing technologies provide a powerful tool for genetic improvement of perennial ryegrass, an important forage and turfgrass species worldwide. The sole publication for gene editing in perennial ryegrass used gene-gun for plant transformation and a dual promoter based CRISPR/Cas9 system for editing. However, their editing efficiency was low (5.9% or only one gene-edited plant produced). To test the suitability of the maize Ubiquitin 1 (ZmUbi1) promoter in gene editing of perennial ryegrass, we produced ZmUbi1 promoter:RUBY transgenic plants. We observed that ZmUbi1 promoter was active in callus tissue prior to shoot regeneration, suggesting that the promoter is suitable for Cas9 and sgRNA expression in perennial ryegrass for high-efficiency production of bi-allelic mutant plants. We then used the ZmUbi1 promoter for controlling Cas9 and sgRNA expression in perennial ryegrass. A ribozyme cleavage target site between the Cas9 and sgRNA sequences allowed production of functional Cas9 mRNA and sgRNA after transcription. Using Agrobacterium for genetic transformation, we observed a 29% efficiency for editing the PHYTOENE DESATURASE gene in perennial ryegrass. DNA sequencing analyses revealed that most pds plants contained bi-allelic mutations. These results demonstrate that the expression of a single Cas9 and sgRNA transcript unit controlled by the ZmUbi1 promoter provides a highly efficient system for production of bi-allelic mutants of perennial ryegrass and should also be applicable in other related grass species.

Recent technological advances in next-generation sequencing (NGS) technologies have dramatically reduced the cost of DNA sequencing, allowing species with large and complex genomes to be sequenced. Although bread wheat (Triticum aestivum L.) is one of the world's most important food crops, efficient exploitation of molecular marker-assisted breeding approaches has lagged behind that achieved in other crop species, due to its large polyploid genome. However, an international public-private effort spanning 9 years reported over 65% draft genome of bread wheat in 2014, and finally, after more than a decade culminated in the release of a gold-standard, fully annotated reference wheat-genome assembly in 2018. Shortly thereafter, in 2020, the genome of assemblies of additional 15 global wheat accessions was released. As a result, wheat has now entered into the pan-genomic era, where basic resources can be efficiently exploited. Wheat genotyping with a few hundred markers has been replaced by genotyping arrays, capable of characterizing hundreds of wheat lines, using thousands of markers, providing fast, relatively inexpensive, and reliable data for exploitation in wheat breeding. These advances have opened up new opportunities for marker-assisted selection (MAS) and genomic selection (GS) in wheat. Herein, we review the advances and perspectives in wheat genetics and genomics, with a focus on key traits, including grain yield, yield-related traits, end-use quality, and resistance to biotic and abiotic stresses. We also focus on reported candidate genes cloned and linked to traits of interest. Furthermore, we report on the improvement in the aforementioned quantitative traits, through the use of (i) clustered regularly interspaced short-palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene-editing and (ii) positional cloning methods, and of genomic selection. Finally, we examine the utilization of genomics for the next-generation wheat breeding, providing a practical example of using in silico bioinformatics tools that are based on the wheat reference-genome sequence.

We previously identified a homozygous G178R mutation in human ASRGL1 (hASRGL1) through whole-exome analysis responsible for early onset retinal degeneration (RD) in patients with cone-rod dystrophy. The mutant G178R ASRGL1 expressed in Cos-7 cells showed altered localization, while the mutant ASRGL1 in E. coli lacked the autocatalytic activity needed to generate the active protein. To evaluate the effect of impaired ASRGL1 function on the retina in vivo, we generated a mouse model with c.578_579insAGAAA (NM_001083926.2) mutation (Asrgl1mut/mut) through the CRISPR/Cas9 methodology. The expression of ASGRL1 and its asparaginase activity were undetectable in the retina of Asrgl1mut/mut mice. The ophthalmic evaluation of Asrgl1mut/mut mice showed a significant and progressive decrease in scotopic electroretinographic (ERG) response observed at an early age of 3 months followed by a decrease in photopic response around 5 months compared with age-matched wildtype mice. Immunostaining and RT-PCR analyses with rod and cone cell markers revealed a loss of cone outer segments and a significant decrease in the expression of Rhodopsin, Opn1sw, and Opn1mw at 3 months in Asrgl1mut/mut mice compared with age-matched wildtype mice. Importantly, the retinal phenotype of Asrgl1mut/mut mice is consistent with the phenotype observed in patients harboring the G178R mutation in ASRGL1 confirming a critical role of ASRGL1 in the retina and the contribution of ASRGL1 mutations in retinal degeneration.

BackgroundThe use of CRISPR/Cas9 technologies in generating single-base pair knock-in mutations has recently exploded in the number of methods available. However, with the growing expansion of new technologies, it can be difficult to determine the best method for genome editing.ResultsIn this study, we evaluated a number of CRISPR/Cas9 approaches for deriving cell lines with knock-in base pair edits to create a phosphorylation mutation and provide a breakdown of editing efficiencies and suggestions for improvement. Overall, our studies suggest that using pre-formed ribonucleoprotein (RNP) complexes is a reliable editing method to generate homozygous single-base pair mutations. We also show that antibiotic selection coupled homologous recombination is an efficient tool for generating highly specific heterozygous mutations.ConclusionThe methods and/or combination of methods outlined in this study can be used to help other researchers with similar goals in single-base pair genome editing.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) is an efficient and precise gene-editing technology that offers a versatile solution for establishing treatments directed at genetic diseases. Currently, CRISPR/Cas9 delivery into cells relies primarily on viral vectors, which suffer from limitations in packaging capacity and safety concerns. These issues with a nonviral delivery strategy are addressed, where Cas9•sgRNA ribonucleoprotein (RNP) complexes can be encapsulated into supramolecular nanoparticles (SMNP) to form RNP⊂SMNPs, which can then be delivered into targeted cells via supramolecular nanosubstrate-mediated delivery. Utilizing the U87 glioblastoma cell line as a model system, a variety of parameters for cellular-uptake of the RNP-laden nanoparticles are examined. Dose- and time-dependent CRISPR/Cas9-mediated gene disruption is further examined in a green fluorescent protein (GFP)-expressing U87 cell line (GFP-U87). The utility of an optimized SMNP formulation in co-delivering Cas9 protein and two sgRNAs that target deletion of exons 45-55 (708 kb) of the dystrophin gene is demonstrated. Mutations in this region lead to Duchenne muscular dystrophy, a severe genetic muscle wasting disease. Efficient delivery of these gene deletion cargoes is observed in a human cardiomyocyte cell line (AC16), induced pluripotent stem cells, and mesenchymal stem cells.

Abstract Clustered regulatory interspaced short palindromic repeats (CRISPR) has changed biomedical research and provided entirely new models to analyze every aspect of biomedical sciences during the last decade. In the study of cancer, the CRISPR/CRISPR-associated protein (Cas) system opens new avenues into issues that were once unknown in our knowledge of the noncoding genome, tumor heterogeneity, and precision medicines. CRISPR/Cas-based gene-editing technology now allows for the precise and permanent targeting of mutations and provides an opportunity to target small non-coding RNAs such as microRNAs (miRNAs). However, the development of effective and safe cancer gene editing therapy is highly dependent on proper design to be innocuous to normal cells and prevent introducing other abnormalities. This study aims to highlight the cutting-edge approaches in cancer-gene editing therapy based on the CRISPR/Cas technology to target miRNAs in cancer therapy. Furthermore, we highlight the potential challenges in CRISPR/Cas-mediated miRNA gene editing and offer advanced strategies to overcome them.

As standard in vitro fertilization is not a viable technique in horses yet, many different techniques have been used to create equine embryos for research purposes. One such method is parthenogenesis in which an oocyte is induced to mature into an embryo-like state without the introduction of a spermatozoon, and thus they are not considered true embryos. Another method is somatic cell nuclear transfer (SCNT), in which a somatic cell nucleus from an extant horse is inserted into an enucleated oocyte, creating a genetic clone of the donor horse. Due to limited availability of equine oocytes in the United States, researchers have investigated the potential for combining equine somatic cell nuclei with oocytes from other species to make embryos for research purposes, which has not been successful to date. There has also been a rising interest in producing transgenic animals using sperm exposed to exogenous DNA. The successful creation of transgenic equine blastocysts shows the promise of sperm mediated gene transfer (SMGT), but this method is not ideal for other applications, like gene therapy, because it cannot be used to induce targeted mutations. That is why technologies like CRISPR/Cas9 are vital. In this review, we argue that parthenogenesis, SCNT, and interspecies SCNT can be considered genetic manipulation strategies as they create embryos that are genetically identical to their parent cell. Here, we describe how these methods are performed and their applications and we also describe the few methods that have been used to directly modify equine embryos: SMGT and CRISPR/Cas9.

Stem cell replacement offers a great potential for cardiac regenerative therapy. However, one of the critical barriers to stem cell therapy is a significant loss of transplanted stem cells from ischemia and inflammation in the host environment. Here, we tested the hypothesis that inhibition of the soluble epoxide hydrolase (sEH) enzyme using sEH inhibitors (sEHIs) to decrease inflammation and fibrosis in the host myocardium may increase the survival of the transplanted human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) in a murine postmyocardial infarction model. A specific sEHI (1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl)urea [TPPU]) and CRISPR/Cas9 gene editing were used to test the hypothesis. TPPU results in a significant increase in the retention of transplanted cells compared with cell treatment alone. The increase in the retention of hiPSC-CMs translates into an improvement in the fractional shortening and a decrease in adverse remodeling. Mechanistically, we demonstrate a significant decrease in oxidative stress and apoptosis not only in transplanted hiPSC-CMs but also in the host environment. CRISPR/Cas9-mediated gene silencing of the sEH enzyme reduces cleaved caspase-3 in hiPSC-CMs challenged with angiotensin II, suggesting that knockdown of the sEH enzyme protects the hiPSC-CMs from undergoing apoptosis. Our findings demonstrate that suppression of inflammation and fibrosis using an sEHI represents a promising adjuvant to cardiac stem cell-based therapy. Very little is known regarding the role of this class of compounds in stem cell-based therapy. There is consequently an enormous opportunity to uncover a potentially powerful class of compounds, which may be used effectively in the clinical setting.

Gene drives based on CRISPR/Cas9 have the potential to reduce the enormous harm inflicted by crop pests and insect vectors of human disease, as well as to bolster valued species. In contrast with extensive empirical and theoretical studies in diploid organisms, little is known about CRISPR gene drive in haplodiploids, despite their immense global impacts as pollinators, pests, natural enemies of pests, and invasive species in native habitats. Here, we analyze mathematical models demonstrating that, in principle, CRISPR homing gene drive can work in haplodiploids, as well as at sex-linked loci in diploids. However, relative to diploids, conditions favoring the spread of alleles deleterious to haplodiploid pests by CRISPR gene drive are narrower, the spread is slower, and resistance to the drive evolves faster. By contrast, the spread of alleles that impose little fitness cost or boost fitness was not greatly hindered in haplodiploids relative to diploids. Therefore, altering traits to minimize damage caused by harmful haplodiploids, such as interfering with transmission of plant pathogens, may be more likely to succeed than control efforts based on introducing traits that reduce pest fitness. Enhancing fitness of beneficial haplodiploids with CRISPR gene drive is also promising.

Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today's agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized more quickly than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cell's growth rate to the target gene's function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.

The homology-independent targeted integration (HITI) strategy enables effective CRISPR/Cas9-mediated knockin of therapeutic genes in nondividing cells in vivo, promising general therapeutic solutions for treating genetic diseases like X-linked juvenile retinoschisis. Herein, supramolecular nanoparticle (SMNP) vectors are used for codelivery of two DNA plasmids-CRISPR-Cas9 genome-editing system and a therapeutic gene, Retinoschisin 1 (RS1)-enabling clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) knockin of the RS1 gene with HITI. Through small-scale combinatorial screenings, two SMNP vectors, with Cas9 and single guide RNA (sgRNA)-plasmid in one and Donor-RS1 and green fluorescent protein (GFP)-plasmid in the other, with optimal delivery performances are identified. These SMNP vectors are then employed for CRISPR/Cas9 knockin of RS1/GFP genes into the mouse Rosa26 safe-harbor site in vitro and in vivo. The in vivo study involves intravitreally injecting the two SMNP vectors into the mouse eyes, followed by repeated ocular imaging by fundus camera and optical coherence tomography, and pathological and molecular analyses of the harvested retina tissues. Mice ocular organs retain their anatomical integrity, a single-copy 3.0-kb RS1/GFP gene is precisely integrated into the Rosa26 site in the retinas, and the integrated RS1/GFP gene is expressed in the retinas, demonstrating CRISPR/Cas9 knockin of RS1/GFP gene.

Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species.

Small airway epithelial cells (SAECs) play a central role in the pathogenesis of lung diseases and are now becoming a crucial cellular model for target identification and validation in drug discovery. However, primary cell lines such as SAECs are often difficult to transfect using traditional lipofection methods; therefore, gene editing using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is often carried out through ribonucleoprotein (RNP) electroporation. Here we have established a robust, scalable, and automated arrayed CRISPR nuclease (CRISPRn) screening workflow for SAECs which can be combined with a myriad of disease-specific endpoint assays.

Clustered regulatory interspaced short palindromic repeats (CRISPR) has changed biomedical research and provided entirely new models to analyze every aspect of biomedical sciences during the last decade. In the study of cancer, the CRISPR/CRISPR-associated protein (Cas) system opens new avenues into issues that were once unknown in our knowledge of the noncoding genome, tumor heterogeneity, and precision medicines. CRISPR/Cas-based gene-editing technology now allows for the precise and permanent targeting of mutations and provides an opportunity to target small non-coding RNAs such as microRNAs (miRNAs). However, the development of effective and safe cancer gene editing therapy is highly dependent on proper design to be innocuous to normal cells and prevent introducing other abnormalities. This study aims to highlight the cutting-edge approaches in cancer-gene editing therapy based on the CRISPR/Cas technology to target miRNAs in cancer therapy. Furthermore, we highlight the potential challenges in CRISPR/Cas-mediated miRNA gene editing and offer advanced strategies to overcome them. [ABSTRACT FROM AUTHOR]

Newly assembled herpesvirus nucleocapsids traverse the intact nuclear envelope by a vesicle-mediated nucleo-cytoplasmic transport for final virion maturation in the cytoplasm. For this, they bud at the inner nuclear membrane resulting in primary enveloped particles in the perinuclear space (PNS) followed by fusion of the primary envelope with the outer nuclear membrane (ONM). While the conserved viral nuclear egress complex orchestrates the first steps, effectors of fusion of the primary virion envelope with the ONM are still mostly enigmatic but might include cellular proteins like SUN2 or ESCRT-III components. Here, we analyzed the influence of the only known AAA+ ATPases located in the endoplasmic reticulum and the PNS, the Torsins (Tor), on nuclear egress of the alphaherpesvirus pseudorabies virus. For this overexpression of wild type and mutant proteins as well as CRISPR/Cas9 genome editing was applied. Neither single overexpression nor gene knockout (KO) of TorA or TorB had a significant impact. However, TorA/B double KO cells showed decreased viral titers at early time points of infection and an accumulation of primary virions in the PNS pointing to a delay in capsid release during nuclear egress.

Interferon regulatory factor 7 (IRF7) is known as the master transcription factor of the type I interferon response in mammalian species along with IRF3. Yet birds only have IRF7, while they are missing IRF3, with a smaller repertoire of immune-related genes, which leads to a distinctive immune response in chickens compared to in mammals. In order to understand the functional role of IRF7 in the regulation of the antiviral response against avian influenza virus in chickens, we generated IRF7-/- chicken embryonic fibroblast (DF-1) cell lines and respective controls (IRF7wt) by utilizing the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system. IRF7 knockout resulted in increased viral titers of low pathogenic avian influenza viruses. Further RNA-sequencing performed on H6N2-infected IRF7-/- and IRF7wt cell lines revealed that the deletion of IRF7 resulted in the significant down-regulation of antiviral effectors and the differential expression of genes in the MAPK (mitogen-activated protein kinase) and mTOR (mechanistic target of rapamycin) signaling pathways. Dynamic gene expression profiling of the host response between the wildtype and IRF7 knockout revealed potential signaling pathways involving AP1 (activator protein 1), NF-κB (nuclear factor kappa B) and inflammatory cytokines that may complement chicken IRF7. Our findings in this study provide novel insights that have not been reported previously, and lay a solid foundation for enhancing our understanding of the host antiviral response against the avian influenza virus in chickens.

Genetically encoded fluorescent voltage indicators, such as ArcLight, have been used to report action potentials (APs) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the ArcLight expression, in all cases, relied on a high number of lentiviral vector-mediated random genome integrations (8-12 copy/cell), raising concerns such as gene disruption and alteration of global and local gene expression, as well as loss or silencing of reporter genes after differentiation. Here, we report the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease technique to develop a hiPSC line stably expressing ArcLight from the AAVS1 safe harbor locus. The hiPSC line retained proliferative ability with a growth rate similar to its parental strain. Optical recording with conventional epifluorescence microscopy allowed the detection of APs as early as 21 days postdifferentiation, and could be repeatedly monitored for at least 5 months. Moreover, quantification and analysis of the APs of ArcLight-CMs identified two distinctive subtypes: a group with high frequency of spontaneous APs of small amplitudes that were pacemaker-like CMs and a group with low frequency of automaticity and large amplitudes that resembled the working CMs. Compared with FluoVolt voltage-sensitive dye, although dimmer, the ArcLight reporter exhibited better optical performance in terms of phototoxicity and photostability with comparable sensitivities and signal-to-noise ratios. The hiPSC line with targeted ArcLight engineering design represents a useful tool for studying cardiac development or hiPSC-derived cardiac disease models and drug testing.

Recent advances in human stem cell and genome engineering have enabled the generation of genetically defined human cellular models for brain disorders. These models can be established from a patient's own cells and can be genetically engineered to generate isogenic, controlled systems for mechanistic studies. Given the challenges of obtaining and working with primary human brain tissue, these models fill a critical gap in our understanding of normal and abnormal human brain development and provide an important complement to animal models. Recently, there has been major progress in modeling the neuropathophysiology of the canonical "mTORopathy" tuberous sclerosis complex (TSC) with such approaches. Studies using two- and three-dimensional cultures of human neurons and glia have provided new insights into how mutations in the TSC1 and TSC2 genes impact human neural development and function. Here we discuss recent progress in human stem cell-based modeling of TSC and highlight challenges and opportunities for further efforts in this area.

Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. The pathogenesis of bAVM has not been fully understood. Animal models are important tools for dissecting bAVM pathogenesis and testing new therapies. We have developed several mouse bAVM models using genetically modified mice. However, due to the body size, mouse bAVM models have some limitations. Recent studies identified somatic mutations in sporadic human bAVM. To develop a feasible tool to create sporadic bAVM in rodent and animals larger than rodent, we made tests using the CRISPR/Cas9 technique to induce somatic gene mutations in mouse brain in situ. Two sequence-specific guide RNAs (sgRNAs) targeting mouse Alk1 exons 4 and 5 were cloned into pAd-Alk1e4sgRNA + e5sgRNA-Cas9 plasmid. These sgRNAs were capable to generate mutations in Alk1 gene in mouse cell lines. After packaged into adenovirus, Ad-Alk1e4sgRNA + e5sgRNA-Cas9 was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brains of wild-type C57BL/6J mice. Eight weeks after viral injection, bAVMs were detected in 10 of 12 mice. Compared to the control (Ad-GFP/AAV-VEGF-injected) brain, 13% of Alk1 alleles were mutated and Alk1 expression was reduced by 26% in the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF-injected brains. Around the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF injected site, Alk1-null endothelial cells were detected. Our data demonstrated that CRISPR/Cas9 is a feasible tool for generating bAVM model in animals.

Proteolysis associated with recombinant protein expression in Chinese Hamster Ovary (CHO) cells has hindered the development of biologics including HIV vaccines. When expressed in CHO cells, the recombinant HIV envelope protein, gp120, undergoes proteolytic clipping by a serine protease at a key epitope recognized by neutralizing antibodies. The problem is particularly acute for envelope proteins from clade B viruses that represent the major genetic subtype circulating in much of the developed world, including the US and Europe. In this paper, we have identified complement Component 1's (C1s), a serine protease from the complement cascade, as the protease responsible for the proteolysis of gp120 in CHO cells. CRISPR/Cas9 knockout of the C1s protease in a CHO cell line was shown to eliminate the proteolytic activity against the recombinantly expressed gp120. In addition, the C1s-/- MGAT1- CHO cell line, with the C1s protease and the MGAT1 glycosyltransferase knocked out, enabled the production of unclipped gp120 from a clade B isolate (BaL-rgp120) and enriched for mannose-5 glycans on gp120 that are required for the binding of multiple broadly neutralizing monoclonal antibodies (bN-mAbs). The availability of this technology will allow for the scale-up and testing of multiple vaccine concepts in regions of the world where clade B viruses are in circulation. Furthermore, the proteolysis issues caused by the C1s protease suggests a broader need for a C1s-deficient CHO cell line to express other recombinant proteins that are susceptible to serine protease activity in CHO cells. Similarly, the workflow described here to identify and knockout C1s in a CHO cell line can be applied to remedy the proteolysis of biologics by other CHO proteases.

CRISPR/Cas9, base editors and prime editors comprise the contemporary genome editing toolbox. Many studies have optimized the use of CRISPR/Cas9, as the original CRISPR genome editing system, in substituting single nucleotides by homology directed repair (HDR), although this remains challenging. Studies describing modifications that improve editing efficiency fall short of isolating clonal cell lines or have not been validated for challenging loci or cell models. We present data from 95 transfections using a colony forming and an immortalized cell line comparing the effect on editing efficiency of donor template modifications, concentration of components, HDR enhancing agents and cold shock. We found that in silico predictions of guide RNA efficiency correlated poorly withactivity in cells. Using NGS and ddPCR we detected editing efficiencies of 5–12% in the transfected populations which fell to 1% on clonal cell line isolation. Our data demonstrate the variability of CRISPR efficiency by cell model, target locus and other factors. Successful genome editing requires a comparison of systems and modifications to develop the optimal protocol for the cell model and locus. We describe the steps in this process in a flowchart for those embarking on genome editing using any system and incorporate validated HDR-boosting modifications for those using CRISPR/Cas9.

MicroRNAs (miRNAs) are post-transcriptional regulators that target specific mRNAs for repression and thus play key roles in many biological processes, including insect wing morphogenesis. miR-2 is an invertebrate-specific miRNA family that has been predicted in the fruit fly, Drosophila melanogaster, to be involved in regulating the Notch signaling pathway. We show here that miR-2 plays a critical role in wing morphogenesis in the silkworm, Bombyx mori, a lepidopteran model insect. Transgenic over-expression of a miR-2 cluster using a Gal4/UAS system results in deformed adult wings, supporting the conclusion that miR-2 regulates functions essential for normal wing morphogenesis. Two genes, abnormal wing disc (awd) and fringe (fng), which are positive regulators in Notch signaling, are identified as miR-2 targets and validated by a dual-luciferase reporter assay. The relative abundance of both awd and fng expression products was reduced significantly in transgenic animals, implicating them in the abnormal wing phenotype. Furthermore, somatic mutagenesis analysis of awd and fng using the CRISPR/Cas9 system and knock-out mutants also resulted in deformed wings similar to those observed in the miR-2 overexpression transgenic animals. The critical role of miR-2 in Bombyx wing morphogenesis may provide a potential target in future lepidopteran pest control.

Background : We previously identified the transcriptional regulator Zbtb32 as a factor that can promote T cell tolerance in the Non-Obese Diabetic (NOD) mouse, a model of Type 1 diabetes. Antigen targeted to DCIR2 + dendritic cells (DCs) in vivo inhibited both diabetes and effector T cell expansion in NOD mice. Furthermore, Zbtb32 was preferentially induced in autoreactive CD4 T cells stimulated by these tolerogenic DCIR2 + DCs, and overexpression of Zbtb32 in islet-specific T cells inhibited the diabetes development by limiting T cell proliferation and cytokine production. Methods : To further understand the role of Zbtb32 in T cell tolerance induction, we have now used CRISPR to target the Zbtb32 gene for deletion directly in NOD mice and characterized the mutant mice. We hypothesized that the systemic loss of Zbtb32 in NOD mice would lead to increased T cell activation and increased diabetes pathogenesis. Results : Although NOD.Zbtb32 -/- male NOD mice showed a trend towards increased diabetes incidence compared to littermate controls, the difference was not significant. Furthermore, no significant alteration in lymphocyte number or function was observed. Importantly, in vitro stimulation of lymphocytes from NOD.Zbtb32 -/- mice did not produce the expected hypersensitive phenotype observed in other genetic strains, potentially due to compensation by homologous genes. Conclusions : The loss of Zbtb32 in the NOD background does not result in the expected T cell activation phenotype.

Background : We previously identified the transcriptional regulator Zbtb32 as a factor that can promote T cell tolerance in the Non-Obese Diabetic (NOD) mouse, a model of Type 1 diabetes. Antigen targeted to DCIR2 + dendritic cells (DCs) in vivo inhibited both diabetes and effector T cell expansion in NOD mice. Furthermore, Zbtb32 was preferentially induced in autoreactive CD4 T cells stimulated by these tolerogenic DCIR2 + DCs, and overexpression of Zbtb32 in islet-specific T cells inhibited the diabetes development by limiting T cell proliferation and cytokine production. Methods : To further understand the role of Zbtb32 in T cell tolerance induction, we have now used CRISPR to target the Zbtb32 gene for deletion directly in NOD mice and characterized the mutant mice. We hypothesized that the systemic loss of Zbtb32 in NOD mice would lead to increased T cell activation and increased diabetes pathogenesis. Results : Although NOD.Zbtb32 -/- male NOD mice showed a trend towards increased diabetes incidence compared to littermate controls, the difference was not significant. Furthermore, no significant alteration in lymphocyte number or function was observed. Importantly, in vitro stimulation of lymphocytes from NOD.Zbtb32 -/- mice did not produce the expected hypersensitive phenotype observed in other genetic strains, potentially due to compensation by homologous genes. Conclusions : The loss of Zbtb32 in the NOD background does not result in the expected T cell activation phenotype.

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The adaptive immunity system in bacteria and archaea, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-associate (CRISPR/Cas), has been adapted as a powerful gene editing tool and got a broad application in genome research field due to its ease of use and cost-effectiveness. The performance of CRISPR/Cas relies on well-designed single-guide RNA (sgRNA), so a lot of bioinformatic tools have been developed to assist the design of highly active and specific sgRNA. These tools vary in design specifications, parameters, genomes and so on. To help researchers to choose their proper tools, we reviewed various sgRNA design tools, mainly focusing on their on-target efficiency prediction model and off-target detection algorithm. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small endogenous RNAs that regulate gene-expression posttranscriptionally. MiRNA research in allergy is expanding because miRNAs are crucial regulators of gene expression and promising candidates for biomarker development. MiRNA mimics and miRNA inhibitors currently in preclinical development have shown promise as novel therapeutic agents. Multiple technological platforms have been developed for miRNA isolation, miRNA quantitation, miRNA profiling, miRNA target detection, and modulating miRNA levels in vitro and in vivo . Here we will review the major technological platforms with consideration given for the advantages and disadvantages of each platform. [ABSTRACT FROM AUTHOR]