Targeted Gene Disruption Research Articles

Targeted gene disruption in a marsupial, Monodelphis domestica, by CRISPR/Cas9 genome editing

Marsupials represent one of three extant mammalian subclasses with very unique characteristics not shared by other mammals. Most notably, much of the development of neonates immaturely born after a relatively short gestation takes place in the external environment. Among marsupials, the gray short-tailed opossum (Monodelphis domestica; hereafter "the opossum") is one of very few established laboratory models. Due to many biologically unique characteristics and experimentally advantageous features, the opossum is used as a prototype species for basic research on marsupial biology.1,2 However, invivo studies of gene function in the opossum, and thus marsupials in general, lag far behind those of eutherian mammals due to the lack of reliable means to manipulate their genomes. In this study, we describe the successful generation of genome edited opossums by a combination of refined methodologies in reproductive biology and embryo manipulation. We took advantage of the opossum's resemblance to popular rodent models, such as the mouse and rat, in body size and breeding characteristics. First, we established a tractable pipeline of reproductive technologies, from induction of ovulation, timed copulation, and zygote collection to embryo transfer to pseudopregnant females, that warrant an essential platform to manipulate opossum zygotes. Further, we successfully demonstrated the generation of gene knockout opossums at the Tyr locus by microinjection of pronuclear stage zygotes using CRISPR/Cas9 genome editing, along with germline transmission of the edited alleles to the F1 generation. This study provides a critical foundation for venues to expand mammalian reverse genetics into the metatherian subclass.

Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1.

Background: Advancement in genome engineering enables rapid and targeted disruption of any coding sequences to study gene functions or establish human disease models. We explored whether this approach can be used to study Gaucher disease, one of the most common types of lysosomal storage diseases (LSDs) in a near-haploid human cell line (HAP1). Results: CRISPR-Cas9 targeting to coding sequences of β-glucocerebrosidase (GBA), the causative gene of Gaucher disease, resulted in an insertional mutation and premature termination of GBA. We confirmed the GBA knockout at both the gene and enzyme levels by genotyping and GBA enzymatic assay. Characterization of the knockout line showed no significant changes in cell morphology and growth. Lysosomal staining revealed more granular lysosomes in the cytosol of the GBA-knockout line compared to its parental control. Flow cytometry analysis further confirmed that more lysosomes accumulated in the cytosol of the knockout line, recapturing the disease phenotype. Finally, we showed that this knockout cell line could be used to evaluate a replacement therapy by recombinant human GBA. Conclusions: Targeted gene disruption in human HAP1 cells enables rapid establishment of the Gaucher model to capture the key pathology and to test replacement therapy. We expect that this streamlined method can be used to generate human disease models of other LSDs, most of which are still lacking both appropriate human disease models and specific treatments to date.

Development of a Targeted Gene Disruption System in the Poly(Ethylene Terephthalate)-Degrading Bacterium Ideonella sakaiensis and Its Applications to PETase and MHETase Genes

Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however, its nonbiodegradability results in a large amount of waste accumulation that has a negative impact on the environment. Recently, a PET-degrading bacterium, Ideonella sakaiensis 201-F6 strain, was isolated, and the enzymes involved in PET digestion, PET hydrolase (PETase), and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase) were identified. Despite the great potentials of I. sakaiensis in bioremediation and biorecycling, approaches to studying this bacterium remain limited. In this study, to enable the functional analysis of PETase and MHETase genes in vivo, we have developed a gene disruption system in I. sakaiensis. The pT18mobsacB-based disruption vector harboring directly connected 5'- and 3'-flanking regions of the target gene for homologous recombination was introduced into I. sakaiensis cells via conjugation. First, we deleted the orotidine 5'-phosphate decarboxylase gene (pyrF) from the genome of the wild-type strain, producing the ΔpyrF strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the ΔpyrF strain as a parent strain and pyrF as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both Δpetase and Δmhetase strains on terephthalic acid (TPA; one of the PET hydrolytic products) was comparable to that of the parent strain. However, these mutant strains dramatically decreased the growth level on PET to that on a no-carbon source. Moreover, the Δpetase strain completely abolished PET degradation capacity. These results demonstrate that PETase and MHETase are essential for I. sakaiensis metabolism of PET. IMPORTANCE The poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis possesses two unique enzymes able to serve in PET hydrolysis. PET hydrolase (PETase) hydrolyzes PET into mono(2-hydroxyethyl) terephthalic acid (MHET), and MHET hydrolase (MHETase) hydrolyzes MHET into terephthalic acid (TPA) and ethylene glycol (EG). These enzymes have attracted global attention, as they have potential to be used for bioconversion of PET. Compared to many in vitro studies, including biochemical and crystal structure analyses, few in vivo studies have been reported. Here, we developed a targeted gene disruption system in I. sakaiensis, which was then applied for constructing Δpetase and Δmhetase strains. Growth of these disruptants revealed that PETase is the sole enzyme responsible for PET degradation in I. sakaiensis, while PETase and MHETase play essential roles in its PET assimilation.

CRISPR/Cas9: Principle, Applications, and Delivery through Extracellular Vesicles.

The establishment of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) technology for eukaryotic gene editing opened up new avenues not only for the analysis of gene function but also for therapeutic interventions. While the original methodology allowed for targeted gene disruption, recent technological advancements yielded a rich assortment of tools to modify genes and gene expression in various ways. Currently, clinical applications of this technology fell short of expectations mainly due to problems with the efficient and safe delivery of CRISPR/Cas9 components to living organisms. The targeted in vivo delivery of therapeutic nucleic acids and proteins remain technically challenging and further limitations emerge, for instance, by unwanted off-target effects, immune reactions, toxicity, or rapid degradation of the transfer vehicles. One approach that might overcome many of these limitations employs extracellular vesicles as intercellular delivery devices. In this review, we first introduce the CRISPR/Cas9 system and its latest advancements, outline major applications, and summarize the current state of the art technology using exosomes or microvesicles for transporting CRISPR/Cas9 constituents into eukaryotic cells.

Significance of two intracellular triacylglycerol lipases of Aspergillus oryzae in lipid mobilization: A perspective in industrial implication for microbial lipid production.

Microbial lipid production of oleaginous strains involves in a complex cellular metabolism controlling lipid biosynthesis, accumulation and degradation. Particular storage lipid, triacylglycerol (TAG), contributes to dynamic traits of intracellular lipids and cell growth. To explore a basis of TAG degradation in the oleaginous strain of Aspergillus oryzae, the functional role of two intracellular triacylglycerol lipases, AoTgla and AoTglb, were investigated by targeted gene disruption using CRISPR/Cas9 system. Comparative lipid profiling of different cultivation stages between the control, single and double disruptant strains (ΔAotgla, ΔAotglb and ΔAotglaΔAotglb strains) showed that the inactivation of either AoTgla or AoTglb led to the increase of total lipid contents, particularly in the TAG fraction. Moreover, the prolonged lipid-accumulating stage of all disruptant strains was obtained as indicated by a reduction in specific rate of lipid turnover, in which a holding capacity in maximal lipid and TAG levels was achieved. The involvement of AoTgls in spore production of A. oryzae was also discovered. In addition to the significance in lipid physiology of the oleaginous fungi, this study provides an impact on industrial practice by overcoming the limitation in short lipid-accumulating stage of the fungal strain, which facilitate the cell harvesting step at the maximum lipid production yield.

A novel therapeutic modality using CRISPR-engineered dendritic cells to treat allergies

Despite the important roles of dendritic cells (DCs) in airway allergies, current therapeutic strategies such as drugs, allergen immunotherapy and biologics haven't been targeted at them. In this study, we established a promising DC-based therapeutic approach for the alleviation of allergic rhinitis (AR)-associated allergic reactions, using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated targeted gene disruption. RNA sequencing analysis revealed upregulation of vacuolar protein sorting 37 B (VPS37B) in AR-derived DCs, indicating a novel molecular target. Following antigen presentation, VPS37A and VPS37B enabled endocytosis of the mannose receptor, which recognizes the house dust mite (HDM) allergen Der p 1. DCs with targeted disruption of VPS37A/B alleviated Th2 cytokine production when co-cultured in vitro with allogeneic naïve CD4+ T cell from patients with AR. Furthermore, nasal administration of Vps37a/b-disrupted bone marrow DCs to a mouse model of AR resulted in strongly reduced AR-related symptoms. Thus, this novel modality using genetically engineered DCs can provide an effective therapeutic and preventative strategy for allergic diseases.

Cas9-expressing chickens and pigs as resources for genome editing in livestock

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.

Development of a CRISPR/Cpf1 system for targeted gene disruption in Aspergillus aculeatus TBRC 277

BackgroundCRISPR-Cas genome editing technologies have revolutionized biotechnological research particularly in functional genomics and synthetic biology. As an alternative to the most studied and well-developed CRISPR/Cas9, a new class 2 (type V) CRISPR-Cas system called Cpf1 has emerged as another versatile platform for precision genome modification in a wide range of organisms including filamentous fungi.ResultsIn this study, we developed AMA1-based single CRISPR/Cpf1 expression vector that targets pyrG gene in Aspergillus aculeatus TBRC 277, a wild type filamentous fungus and potential enzyme-producing cell factory. The results showed that the Cpf1 codon optimized from Francisella tularensis subsp. novicida U112, FnCpf1, works efficiently to facilitate RNA-guided site-specific DNA cleavage. Specifically, we set up three different guide crRNAs targeting pyrG gene and demonstrated that FnCpf1 was able to induce site-specific double-strand breaks (DSBs) followed by an endogenous non-homologous end-joining (NHEJ) DNA repair pathway which caused insertions or deletions (indels) at these site-specific loci.ConclusionsThe use of FnCpf1 as an alternative class II (type V) nuclease was reported for the first time in A. aculeatus TBRC 277 species. The CRISPR/Cpf1 system developed in this study highlights the feasibility of CRISPR/Cpf1 technology and could be envisioned to further increase the utility of the CRISPR/Cpf1 in facilitating strain improvements as well as functional genomics of filamentous fungi.

Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice

CRISPR-Cas systems have emerged as a powerful tool to generate genetic models for studying normal and diseased central nervous system (CNS). Targeted gene disruption at specific loci has been demonstrated successfully in non-dividing neurons. Despite its simplicity, high specificity and low cost, the efficiency of CRISPR-mediated knockout in vivo can be substantially impacted by many parameters. Here, we used CRISPR-Cas9 to disrupt the neuronal-specific gene, NeuN, and optimized key parameters to achieve effective gene knockout broadly in the CNS in postnatal mice. Three cell lines and two primary neuron cultures were used to validate the disruption of NeuN by single-guide RNAs (sgRNA) harboring distinct spacers and scaffold sequences. This triage identified an optimal sgRNA design with the highest NeuN disruption in in vitro and in vivo systems. To enhance CRISPR efficiency, AAV-PHP.B, a vector with superior neuronal transduction, was used to deliver this sgRNA in Cas9 mice via neonatal intracerebroventricular (ICV) injection. This approach resulted in 99.4% biallelic indels rate in the transduced cells, leading to greater than 70% reduction of total NeuN proteins in the cortex, hippocampus and spinal cord. This work contributes to the optimization of CRISPR-mediated knockout and will be beneficial for fundamental and preclinical research.

Differential intolerance to loss of function and missense mutations in genes that encode human matricellular proteins.

Targeted gene disruption in mice has provided valuable insights into the functions of matricellular proteins. Apart from missense and loss of function mutations that have been associated with inherited diseases, however, their functions in humans remain unclear. The availability of deep exome sequencing data from over 140,000 individuals in the Genome Aggregation Database provided an opportunity to examine intolerance to loss of function and missense mutations in human matricellular genes. The probability of loss-of-function intolerance (pLI) differed widely within members of the thrombospondin, CYR61/CTGF/NOV (CCN), tenascin, small integrin-binding ligand N-linked glycoproteins (SIBLING), and secreted protein, acidic and rich in cysteine (SPARC) gene families. Notably, pLI values in humans had limited correlation with viability of the corresponding homozygous null mice. Among the thrombospondins, only THBS1 was highly loss-intolerant (pLI = 1). In contrast, Thbs1 is not essential for viability in mice. Several known thrombospondin-1 receptors were similarly loss-intolerant, although thrombospondin-1 is not the exclusive ligand for some of these receptors. The frequencies of missense mutations in THBS1 and the gene encoding its signaling receptor CD47 indicated conservation of some residues implicated in specific receptor binding. Deficits in missense mutations were also observed for other thrombospondin genes and for SPARC, SPOCK1, SPOCK2, TNR, and DSPP. The intolerance of THBS1 to loss of function in humans and elevated pLI values for THBS2, SPARC, SPOCK1, TNR, and CCN1 support important functions for these matricellular protein genes in humans, some of which may relate to functions in reproduction or responding to environmental stresses.

CaNRT2.1 Is Required for Nitrate but Not Nitrite Uptake in Chili Pepper Pathogen Colletotrichum acutatum

Chili peppers are an important food additive used in spicy cuisines worldwide. However, the yield and quality of chilis are threatened by anthracnose disease caused by Colletotrichum acutatum. Despite the impact of C. acutatum on chili production, the genes involved in fungal development and pathogenicity in this species have not been well characterized. In this study, through T-DNA insertional mutagenesis, we identified a mutant strain termed B7, which is defective for the growth of C. acutatum on a minimal nutrient medium. Our bioinformatics analysis revealed that a large fragment DNA (19.8 kb) is deleted from the B7 genome, thus resulting in the deletion of three genes, including CaGpiP1 encoding a glycosylphosphatidyl-inisotol (GPI)-anchored protein, CaNRT2.1 encoding a membrane-bound nitrate/nitrite transporter, and CaRQH1 encoding a RecQ helicase protein. In addition, T-DNA is inserted upstream of the CaHP1 gene encoding a hypothetical protein. Functional characterization of CaGpiP1, CaNRT2.1, and CaHP1 by targeted gene disruption and bioassays indicated that CaNRT2.1 is responsible for the growth-defective phenotype of B7. Both B7 and CaNRT2.1 mutant strains cannot utilize nitrate as nitrogen sources, thus restraining the fungal growth on a minimal nutrient medium. In addition to CaNRT2.1, our results showed that CaGpiP1 is a cell wall-associated GPI-anchored protein. However, after investigating the functions of CaGpiP1 and CaHP1 in fungal pathogenicity, growth, development and stress tolerance, we were unable to uncover the roles of these two genes in C. acutatum. Collectively, in this study, our results identify the growth-defective strain B7 via T-DNA insertion and reveal the critical role of CaNRT2.1 in nitrate transportation for the fungal growth of C. acutatum.

Genome Editing Using CRISPR/Cas9 System in the Rice Blast Fungus.

Fast and flexible genome manipulation is a powerful strategy for an in-depth understanding of molecular mechanisms in biological research. In recent years, CRISPR/Cas9-mediated genome editing has been used as a reliable genome manipulation method in a broad range of biological research including studies of filamentous fungi. The CRISPR/Cas9 system comprises a single-guide RNA (sgRNA) and a Cas9 protein, and the Cas9/sgRNA complex catalyzes a DNA double-strand break at the desired genomic locus. This protocol describes a fundamental CRISPR/Cas9 methodology that includes the design of the target sequence, construction of the CRISPR/Cas9 expression vector, and transformation for genome editing in Pyricularia (Magnaporthe) oryzae. This allows efficient targeted gene disruption, base editing, and reporter gene knock-in without any additional modifications of the host components. This protocol would be suitable for applying other CRISPR/Cas technologies and various functional genomics in P. oryzae.

Molecular breeding of sporeless strains of Pleurotus ostreatus using a non-homologous DNA end-joining defective strain

Gene targeting is useful to isolate strains with mutations in a gene of interest for efficient breeding. In this study, we generated msh4 or mer3 single-gene disruptant monokaryons using a Pleurotus ostreatus Δku80 strain for efficient gene targeting. Dikaryons of P. ostreatus Δmsh4×Δmsh4 or Δmer3×Δmer3 were isolated via backcrosses, and the number of basidiospores produced was measured. The number of basidiospores fell by an average 1/13.7 in the P. ostreatus Δmsh4×Δmsh4 dikaryons versus the P. ostreatus msh4+×Δmsh4 dikaryons, and 1/82.6 in the P. ostreatus Δmer3×Δmer3 dikaryons versus the P. ostreatus mer3+×Δmer3 dikaryons. To demonstrate the effects of ku80 disruption, P. ostreatus Δku80×Δku80 dikaryon strains were isolated and no significant effects on basidiospore production were observed. Fluorescence microscopy showed meiotic progression was arrested during prophase I in the msh4 or mer3 disruptants. To our knowledge, this is the first report on molecular breeding of sporeless strains in cultivated mushrooms using an efficient method for targeted gene disruption.

The receptor tyrosine kinase torso regulates ecdysone homeostasis to control developmental timing in Bombyx mori.

Insect growth and development are precisely controlled by hormone homeostasis. The prothoracicotropic hormone (PTTH) receptor, Torso, is a member of the receptor tyrosine kinase family in insects. Activation of Torso by PTTH triggers biosynthesis and release of the steroid hormone in the prothoracic gland (PG). Although numbers of genes functioning in steroid hormone synthesis and metabolism have been identified in insects, the PTTH transduction pathway via its receptor Torso is poorly understood. In the current study, we describe a loss‐of‐function analysis of Torso in the silkworm, Bombyx mori, by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA‐guided Cas9 nucleases) system. Depletion of B. mori Torso (BmTorso) did not eventually affect larval ecdysis and metamorphosis processes. Instead, BmTorso deficiency resulted in significant extension of developing time during larval and pupal stages with increased pupa and cocoon sizes. The ecdysteriod titers in the hemolymph of BmTorso mutants sharpy declined. Transcriptional levels of genes involved in ecdysone biosynthesis and ecdysteroid signaling pathways were significantly reduced in BmTorso‐deficient animals. Additionally, RNA‐Seq analysis revealed that genes involved in the longevity pathway and protein processing in the endoplasmic reticulum pathway were affected after BmTorso deletion. These results indicate that Torso is critical for maintaining steroid hormone homeostasis in insects.

Targeted gene disruption of ATP synthases 6-1 and 6-2 in the mitochondrial genome of Arabidopsis thaliana by mitoTALENs.

We recently achieved targeted disruptions of cytoplasmic male sterility (CMS)-associated genes in the mitochondrial genomes of rice and rapeseed by using mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs). It was the first report of stable and heritable targeted gene modification of plant mitochondrial genomes. Here, we attempted to use mitoTALENs to disrupt two mitochondrial genes in the model plant Arabidopsis thaliana(Arabidopsis) using three different promoters and two types of TALENs. The targets were the two isoforms of the ATP synthase subunit6 gene, atp6-1 and atp6-2. Each of these genes was successfully deleted and the mitochondrial genomes were recovered in a homoplasmic state. The nuclear genome also has a copy of atp6-1, and we were able to confirm that it was the mitochondrial gene and not the nuclear pseudogene that was knocked out. Among the three mitoTALEN promoters tried, the RPS5A promoter was the most effective. Conventional mitoTALENs were more effective than single-molecule mito-compactTALENs. Targeted mitochondrial gene deletion was achieved by crossing as well as by floral-dip transformation to introduce the mitoTALEN constructs into the nucleus. The gene disruptions were caused by large (kb-size) deletions. The ends of the remaining sequences were connected to distant loci, mostly by illegitimate homologous recombinations between repeats.

Identification of 8-Azaguanine Biosynthesis-Related Genes Provides Insight Into the Enzymatic and Non-enzymatic Biosynthetic Pathway for 1,2,3-Triazole.

8-Azaguanine (1) is a special 1,2,3-triazole containing natural product that possesses potent antibacterial and antitumor activities. In the present study, the entire 8-azaguanine biosynthetic gene cluster was located from Streptomyces CGMCC4.1633. Targeted gene disruption, heterologous expression analysis, and feeding experiments identified crucial genes for 8-azaguanine production. Moreover, we characterized the structure of two novel metabolites, analyzed NO (or reactive nitrogen species) related genes 8-azgA/B and radical SAM enzyme homologous 8-AzgG, and verified the non-enzymatic ring formation reaction of 8-azaguanine 1,2,3-triazole. All of the data and presumptions provide insight into the timing and mechanism of the enzymatic and non-enzymatic pathway that produce 8-azaguanine-type 1,2,3-triazole.

MaPmt1, a protein O-mannosyltransferase, contributes to virulence through governing the appressorium turgor pressure in Metarhizium acridum

O-glycosylation is a very important post-translational modification of protein and involved in many cell processes in fungi. There exist three protein O-manosyltransferanse genes (MaPmt1, MaPmt2, MaPmt4) in Metarhizium acridum based on sequence homology. Here, MaPmt1, a gene for Pmt1 O-manosyltransferanse in M. acridum, was characterized and functionally analyzed through targeted gene disruption and complementation methods. Deletion of MaPmt1 had no effect on conidial germination, but slightly increased the conidial yield and significantly impaired fungal tolerances to UV-B radiation and wet-heat. Deletion of MaPmt1 made the fungus become more sensitive to cell wall disturbing agents and exhibit a thinner cell wall with changed components. Insect bioassays showed that disruption of MaPmt1 attenuated the fungal virulence significantly by topical inoculation but not by injection, indicating that MaPmt1 is required for penetration during the infection of M. acridum. Interestingly, deletion of MaPmt1 did not affect appressorium formation but significantly decreased appressorium turgor pressure. Moreover, the decreased virulence of MaPmt1 disruptant is mainly due to the reduced appressorium turgor pressure, which may be resulted from the declined glycerol concentration, combined with the weakened cell wall that could not hold the normal appressorium turgor pressure to penetrate the host cuticle.

P53 Hinders CRISPR/Cas9-Mediated Targeted Gene Disruption in Memory CD8 T Cells In Vivo.

CRISPR/Cas9 technology has revolutionized rapid and reliable gene editing in cells. Although many cell types have been subjected to CRISPR/Cas9-mediated gene editing, there is no evidence of success in genetic alteration of Ag-experienced memory CD8 T cells. In this study, we show that CRISPR/Cas9-mediated gene editing in memory CD8 T cells precludes their proliferation after Ag re-encounter in vivo. This defect is mediated by the proapoptotic transcription factor p53, a sensor of DNA damage. Temporarily inhibiting p53 function offers a window of opportunity for the memory CD8 T cells to repair the DNA damage, facilitating robust recall responses on Ag re-encounter. We demonstrate this by functionally altering memory CD8 T cells using CRISPR/Cas9-mediated targeted gene disruption under the aegis of p53siRNA in the mouse model. Our approach thus adapts the CRISPR/Cas9 technology for memory CD8 T cells to undertake gene editing in vivo, for the first time, to our knowledge.

Non-viral Gene Disruption by CRISPR/Cas9 Delivery Using Cell-permeable and Protein-stabilizing 30Kc19 Protein

CRISPR/Cas9 system has served a new insight in genome editing of eukaryotes, including human cells. In this system, delivery of Cas9 nuclease with guide RNA has been central challenge in developing safe and efficient techniques. The viral delivery of genes encoding these two components i.e. Cas9 and guide RNA may cause unexpected integration of the DNA sequence into the host cell genome, and lead to potential safety problems such as tumorigenesis. Herein, we report that the Cas9 protein can be directly delivered into the human cells through fusion with 30Kc19, a cell-penetrating and protein-stabilizing protein originating from silkworm. The 30Kc19-conjugated Cas9 (30Kc19-Cas9) showed higher stability than native Cas9 for thermal and chemical-induced inactivation in DNA-cleavable activity. In addition, it was demonstrated that 30Kc19-Cas9 was efficiently delivered into human cells and resulted in targeted gene disruption with single guide RNA by showing gene expression and site-specific mutations in the genome. With the advantages of efficient delivery in addition to the enhancement of Cas9 stability, this method is expected to provide a versatile strategy to advance non-viral and clinically-feasible genome editing for in vivo applications.

Multiple Ehrlichia chaffeensis Genes Critical for Its Persistent Infection in a Vertebrate Host Are Identified by Random Mutagenesis Coupled with In Vivo Infection Assessment.

Ehrlichia chaffeensis, a tick-transmitted obligate intracellular rickettsial agent, causes human monocytic ehrlichiosis. In recent reports, we described substantial advances in developing random and targeted gene disruption methods to investigate the functions of E. chaffeensis genes. We reported earlier that the Himar1 transposon-based random mutagenesis is a valuable tool in defining E. chaffeensis genes critical for its persistent growth in vivo in reservoir and incidental hosts. The method also aided in extending studies focused on vaccine development and immunity. Here, we describe the generation and mapping of 55 new mutations. To define the critical nature of the bacterial genes, infection experiments were carried out in the canine host with pools of mutant organisms. Infection evaluation in the physiologically relevant host by molecular assays and by xenodiagnoses allowed the identification of many proteins critical for the pathogen's persistent in vivo growth. Genes encoding proteins involved in biotin biosynthesis, protein synthesis and fatty acid biosynthesis, DNA repair, electron transfer, and a component of a multidrug resistance (MDR) efflux pump were concluded to be essential for the pathogen's in vivo growth. Three known immunodominant membrane proteins, i.e., two 28-kDa outer membrane proteins (P28/OMP) and a 120-kDa surface protein, were also recognized as necessary for the pathogen's obligate intracellular life cycle. The discovery of many E. chaffeensis proteins crucial for its continuous in vivo growth will serve as a major resource for investigations aimed at defining pathogenesis and developing novel therapeutics for this and related pathogens of the rickettsial family Anaplasmataceae.