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Camels possess exceptional adaptability, allowing them to withstand extreme temperatures in desert environments. They conserve water by reducing their metabolic rate and regulating body temperature. The heart of the camel plays a crucial role in this adaptation, with specific genes expressed in cardiac tissue that are essential for mammalian adaptation, regulating cardiac function and responding to environmental stressors. One such gene, nebulin‐related‐anchoring protein (NRAP), is involved in the assembly of myofibrils and the transmission of force within the heart. In our study of the NRAP gene across various livestock species, including three camel species, we identified a camel‐specific exon region in the NRAP transcripts. This additional exon (exon 4) contains an open reading frame predicted in camels. To investigate its function, we generated knock‐in mice expressing camel NRAP exon 4. These ‘camelized mice’ exhibited normal phenotypic characteristics compared with wild‐type mice but showed elevated body temperatures under cold stress. Transcriptome analyses of the hearts from camelized mice under cold stress revealed differentially expressed inflammatory cytokine genes, known to influence cardiac function by modulating the contractility of cardiac muscle cells. We propose further investigations utilizing these camelized mice to explore these findings in greater depth. [ABSTRACT FROM AUTHOR]

The low knock-in efficiency, especially in primary human cells, limits the use of the genome editing technology for therapeutic purposes, rendering it important to develop approaches for increasing the knock-in levels. In this work, the efficiencies of several approaches were studied using a model of knock-in of a construct coding for the peptide HIV fusion inhibitor MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line. First, donor DNA modification was evaluated as a means to improve the efficiency of plasmid transport into the nucleus. The donor plasmid was modified to include the simian virus 40 (SV40) DNA nuclear targeting sequence (DTS) or binding sites for the transcription factor NF-κB, whose effects on the knock-in levels have not been described. The modification was ineffective in the model of MT-C34 knock-in into the CXCR4 locus. A second approach consisted in modification of Cas9 nuclease by introducing two additional nuclear localization signals (NLSs) and increased the knock-in level by 30%. Finally, blocking DNA repair via the nonhomologous end joining (NHEJ) pathway with DNA-dependent protein kinase inhibitors caused a 1.8-fold increase in knock-in. A combination of the last two approaches caused an additive effect. Thus, increasing the number of NLSs in the Cas9 protein and inhibiting DNA repair via the NHEJ pathway significantly increased the level of knock-in of the HIV-1 fusion inhibitory peptide into the clinically relevant locus CXCR4. The finding can be used to develop effective gene therapy approaches for treating HIV infection. [ABSTRACT FROM AUTHOR]

To successfully apply the genome editing technology using the CRISPR/Cas9 system in the clinic, it is necessary to achieve a high efficiency of knock-in, which is insertion of a genetic construct into a given locus of the target cell genome. One of the approaches to increase the efficiency of knock-in is to modify donor DNA with the same Cas9 targeting sites (CTS) that are used to induce double-strand breaks (DSBs) in the cell genome (the double-cut donor method). Another approach is based on introducing truncated CTS (tCTS), including a PAM site and 16 proximal nucleotides, into the donor DNA. Presumably, tCTS sites do not induce cleavage of the donor plasmid, but can support its transport into the nucleus by Cas9. However, the exact mechanisms whereby these two donor DNA modifications increase the knock-in level are unknown. In this study, the modifications were tested for effect on the knock-in efficiency of the MTC34 genetic construct encoding the HIV-1 fusion inhibitory peptide MT-C34 into the CXCR4 locus of the CEM/R5 T-cell line. When full-length CTSs were introduced into the donor plasmid DNA, the knock-in level was doubled regardless of the CTS number or position relative to the donor sequence. Modifications with tCTSs did not affect the knock-in levels. In vitro, both CTS and tCTS were efficiently cleaved by Cas9. To understand the mechanism of action of these modifications in detail, it is necessary to evaluate their cleavage both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

The CRISPR-Cas9 system has emerged as the most prevalent gene editing technology due to its simplicity, high efficiency, and low cost. However, the homology-directed repair (HDR)-mediated gene knock-in in this system suffers from low efficiency, which limits its application in animal model preparation, gene therapy, and agricultural genetic improvement. Here, we report the design and optimization of a simple and efficient reporter-based assay to visualize and quantify HDR efficiency. Through random screening of a small molecule compound library, two groups of compounds, including the topoisomerase inhibitors and PIM1 kinase inhibitors, have been identified to promote HDR. Two representative compounds, etoposide and quercetagetin, also significantly enhance the efficiency of CRISPR-Cas9 and HDR-mediated gene knock-in in mouse embryos. Our study not only provides an assay to screen compounds that may facilitate HDR but also identifies useful tool compounds to facilitate the construction of genetically modified animal models with the CRISPR-Cas9 system. [ABSTRACT FROM AUTHOR]

Although the study of many genes and their protein products is limited by the availability of high‐quality antibodies, this problem could be solved by fusing a tag/reporter to an endogenous gene using a gene‐editing approach. The type II bacterial CRISPR/Cas system has been demonstrated to be an efficient gene‐targeting technology for many insects, including the oriental fruit fly Bactrocera dorsalis. However, knocking in, an important editing method of the CRISPR/Cas9 system, has lagged in its application in insects. Here, we describe a highly efficient homology‐directed genome editing system for B. dorsalis that incorporates coinjection of embryos with Cas9 protein, guide RNA and a short single‐stranded oligodeoxynucleotide donor. This one‐step procedure generates flies carrying V5 tag (42 bp) in the BdorTRH gene. In insects, as in other invertebrates and in vertebrates, the neuronal tryptophan hydroxylase (TRH) gene encodes the rate‐limiting enzyme for serotonin biosynthesis in the central nervous system. Using V5 monoclonal antibody, the distribution of TRH in B. dorsalis at different developmental stages was uncovered. Our results will facilitate the generation of insects carrying precise DNA inserts in endogenous genes and will lay foundation for the investigation of the neural mechanisms underlying the serotonin‐mediated behaviour of B. dorsalis. [ABSTRACT FROM AUTHOR]

Abstract Background The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR–Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency. Method Here we report the development of a multicolour fluorescence assay for studying CRISPR–Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR–Cas9 strategies. Result We found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration. Conclusion Our results highlight the need for a more stringent assessment of CRISPR–Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.

Gene knock‐out (KO) mouse models for DNA polymerase beta (Polβ) revealed that loss of Polβ leads to neonatal lethality, highlighting the critical organismic role for this DNA polymerase. While biochemical analysis and gene KO cell lines have confirmed its biochemical role in base excision repair and in TET‐mediated demethylation, more long‐lived mouse models continue to be developed to further define its organismic role. The Polb‐KO mouse was the first of the Cre‐mediated tissue‐specific KO mouse models. This technology was exploited to investigate roles for Polβ in V(D)J recombination (variable‐diversity‐joining rearrangement), DNA demethylation, gene complementation, SPO11‐induced DNA double‐strand break repair, germ cell genome stability, as well as neuronal differentiation, susceptibility to genotoxin‐induced DNA damage, and cancer onset. The revolution in knock‐in (KI) mouse models was made possible by CRISPR/cas9‐mediated gene editing directly in C57BL/6 zygotes. This technology has helped identify phenotypes associated with germline or somatic mutants of Polβ. Such KI mouse models have helped uncover the importance of key Polβ active site residues or specific Polβ enzyme activities, such as the PolbY265C mouse that develops lupus symptoms. More recently, we have used this KI technology to mutate the Polb gene with two codon changes, yielding the PolbL301R/V303R mouse. In this KI mouse model, the expressed Polβ protein cannot bind to its obligate heterodimer partner, Xrcc1. Although the expressed mutant Polβ protein is proteolytically unstable and defective in recruitment to sites of DNA damage, the homozygous PolbL301R/V303R mouse is viable and fertile, yet small in stature. We expect that this and additional targeted mouse models under development are poised to reveal new biological and organismic roles for Polβ. [ABSTRACT FROM AUTHOR]

In the world's first pig-to-human cardiac cytomegalovirus (PCMV), xenotransplant and elevated levels of porcine key factors contributing to patient mortality were considered. This has renewed attention on PCMV, a virus widely prevalent in pigs. Currently, there are no effective drugs or vaccines targeting PCMV, and its high detection difficulty poses challenges for prevention and control research. In this study, antiviral small hairpin RNA (shRNA) was selected and inserted into the Rosa26 and miR-17-92 loci of pigs via a CRISPR/Cas9-mediated knock-in strategy. Further in vitro viral challenge experiments demonstrated that these genetically edited pig cells could effectively limit PCMV replication. Through this process, we constructed a PCMV-infected cell model, validated partial viral interference sites, enhanced gene knock-in efficiency, performed gene editing at two different gene loci, and ultimately demonstrated that RNA interference (RNAi) technology combined with CRISPR/Cas9 has the potential to generate pig cells with enhanced antiviral infection capabilities. This opens up possibilities for the future production of pig populations with antiviral functionalities. [ABSTRACT FROM AUTHOR]

Background: The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR–Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency. Method: Here we report the development of a multicolour fluorescence assay for studying CRISPR–Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR–Cas9 strategies. Result: We found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration. Conclusion: Our results highlight the need for a more stringent assessment of CRISPR–Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration. [ABSTRACT FROM AUTHOR]

The use of CRISPR/Cas9 system has rapidly grown in the last years. Here, the optimization of gene editing of a single-nucleotide polymorphism in a human non-malignant somatic cell line of thyrocytes (Nthy-Ori) was described highlighting strategies for overcoming the problems concerning the delivery and off-targets. We employed both lentivirus and chemical lipids as delivery agents and two strategies for creating the double-strand breaks (DSB). The former induced a DSB by a classical Cas9 nuclease (standard strategy), while the second one employed a modified Cas9 creating a single-strand break (SSB). The knock-in was carried out using a single-stranded donor oligonucleotide or the HR410-PA donor vector (HR). The desired cells could be obtained by combining the double nickase system with the HR vector transfected chemically. This result could be due to the type of DSB, likely processed mainly by non-homologous end joining when blunt (standard strategy) and by HR when overhanging (double nickase). Our results showed that the double nickase is suitable for knocking-in the immortalized Nthy-Ori cell line, while the standard CRISPR/Cas9 system is suitable for gene knock-out creating in/del mutations. [ABSTRACT FROM AUTHOR]

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

T lymphocytes represent a promising target for genome editing. They are primarily modified to recognize and kill tumor cells or to withstand HIV infection. In most studies, T cell genome editing is performed using the CRISPR/Cas technology. Although this technology is easily programmable and widely accessible, its efficiency of T cell genome editing was initially low. Several crucial improvements were made in the components of the CRISPR/Cas technology and their delivery methods, as well as in the culturing conditions of T cells, before a reasonable editing level suitable for clinical applications was achieved. In this review, we summarize and describe the aforementioned parameters that affect human T cell editing efficiency using the CRISPR/Cas technology, with a special focus on gene knock-in. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) genome editing technology, derived from the adaptive immune system of bacteria, has now been recognized as one of the most useful genetic tools in genome engineering. Short stretch of RNA guides the Cas enzyme to precise locations within the target genome of living organisms to accurately modify and control the functions of genes with high accuracy. Cas-mediated genome editing is scalable, practically feasible, and permits the research community to expound the functional organization of the hereditary material and helps to understand the relationship between genetic variations and biological phenotypes. Although CRISPR/Cas system has opened many possibilities in the field of genetic engineering, it still has some shortcomings, such as constraints of the protospacer-adjacent motif (PAM) sequence, relatively larger size of the Cas9 gene and higher chances of off-targeting. To overcome these shortcomings, many new Cas9 variants have been engineered with improved features such as specificity and editing efficacy. Anticipated traits can be developed in plants with high utility in agriculture. For the past decade, this editing system has made significant progress and has been widely used in genome editing to create gene knock-ins and knock-outs. Genome editing is mediated either by non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanism, consequently resulting in desired mutations that directly enhance plant performance. This paper summarizes the new variants of the CRISPR/Cas system and applications of the CRISPR system in plant science for the enhancement of various attributes such as yield, quality, nutritional factors, stress tolerance, and herbicide tolerance through knock-in and knock-out strategies. [ABSTRACT FROM AUTHOR]

Mitochondria play a crucial role in Alzheimer's disease (AD) onset and progression. Traditional transgenic AD mouse models which were widely used in the past decades share a common limitation: The overexpression of APP and overproduction of amyloid-beta (Aβ) are accompanied by other APP peptide fragments, which could introduce artificial and non-clinically relevant phenotypes. Here, we performed an in-depth and time-resolved behavioral and metabolic characterization of a clinically relevant AD mouse model engineered to express normal physiological levels of APP harboring humanized Swedish (K670N/M671L), Beyreuther/Iberian (I716F), and Arctic (E693G) mutations (App NL-G-F/NL-G-F ), termed APP knock-in (APPKI) mice. Our result showed that APPKI mice exhibited fear learning deficits at 6-m age and contextual memory deficit at 12-m age. Histopathological analysis revealed mild amyloidosis (6E10) accompanied by microgliosis (Iba1) as early as 3 months, which progressed significantly together with significant astrocytosis at 6 and 12 m. We further analyzed hippocampal mitochondrial dysfunction by multiple assays, while 3-m APPKI mice brain mitochondrial function remains a similar level as WT mice. Significant mitochondrial dysfunction characterized by decreased ATP production and higher membrane potential with subsequent overproduction of reactive oxygen species (ROS) was observed in mitochondria isolated from 7-m APPKI mice hippocampal tissue. Morphologically, these mitochondria were larger in volume with a decreased level of mitochondrial fusion protein mitofusin-2 (MFN2). At 12 months, APPKI mice exhibit a significantly decreased total mitochondrial oxygen consumption rate (OCR) in isolated hippocampal mitochondria detected by high-resolution respirometry. These data indicate early mitochondrial dysfunction in the brain at pre-symptomatic age in the App NL-G-F/NL-G-mice, which may play a key role in the progression of the disease. Moreover, the identified behavioral and bioenergetic alterations in this clinically relevant AD mouse model provide a valuable tool to optimize the temporal component for therapeutic interventions to treat AD.

BackgroundThe homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells.ResultsBy introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template.ConclusionThe allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.

The CRISPR-Cas9 system has emerged as the most prevalent gene editing technology due to its simplicity, high efficiency, and low cost. However, the homology-directed repair (HDR)-mediated gene knock-in in this system suffers from low efficiency, which limits its application in animal model preparation, gene therapy, and agricultural genetic improvement. Here, we report the design and optimization of a simple and efficient reporter-based assay to visualize and quantify HDR efficiency. Through random screening of a small molecule compound library, two groups of compounds, including the topoisomerase inhibitors and PIM1 kinase inhibitors, have been identified to promote HDR. Two representative compounds, etoposide and quercetagetin, also significantly enhance the efficiency of CRISPR-Cas9 and HDR-mediated gene knock-in in mouse embryos. Our study not only provides an assay to screen compounds that may facilitate HDR but also identifies useful tool compounds to facilitate the construction of genetically modified animal models with the CRISPR-Cas9 system.

Mutations of proline-rich transmembrane protein 2 (PRRT2) lead to dyskinetic disorders such as paroxysmal kinesigenic dyskinesia (PKD), which is characterized by attacks of involuntary movements precipitated by suddenly initiated motion, and some convulsive disorders. Although previous studies have shown that PKD might be caused by cerebellar dysfunction, PRRT2 has not been sufficiently analyzed in some motor-related regions, including the basal ganglia, where dopaminergic neurons are most abundant in the brain. Here, we generated several types of Prrt2 knock-in (KI) mice harboring mutations, such as c.672dupG, that mimics the human pathological mutation c.649dupC and investigated the contribution of Prrt2 to dopaminergic regulation. Regardless of differences in the frameshift sites, all truncating mutations abolished Prrt2 expression within the striatum and cerebral cortex, consistent with previous reports of similar Prrt2 mutant rodents, confirming the loss-of-function nature of these mutations. Importantly, administration of l -dopa, a precursor of dopamine, exacerbated rotarod performance, especially in Prrt2 -KI mice. These findings suggest that dopaminergic dysfunction in the brain by the PRRT2 mutation might be implicated in a part of motor symptoms of PKD and related disorders. [ABSTRACT FROM AUTHOR]

The determination of the functional impact of variants of uncertain significance (VUS) is one of the major bottlenecks in the diagnostic workflow of inherited genetic diseases. To face this problem, we set up a CRISPR/Cas9‐based strategy for knock‐in cellular model generation, focusing on inherited metabolic disorders (IMDs). We selected variants in seven IMD‐associated genes, including seven reported disease‐causing variants and four benign/likely benign variants. Overall, 11 knock‐in cell models were generated via homology‐directed repair in HAP1 haploid cells using CRISPR/Cas9. The functional impact of the variants was determined by analyzing the characteristic biochemical alterations of each disorder. Functional studies performed in knock‐in cell models showed that our approach accurately distinguished the functional effect of pathogenic from non‐pathogenic variants in a reliable manner in a wide range of IMDs. Our study provides a generic approach to assess the functional impact of genetic variants to improve IMD diagnosis and this tool could emerge as a promising alternative to invasive tests, such as muscular or skin biopsies. Although the study has been performed only in IMDs, this strategy is generic and could be applied to other genetic disorders. [ABSTRACT FROM AUTHOR]

p53 is critical for tumor suppression but also elicits detrimental effects when aberrantly overexpressed. Thus, multiple regulators, including RNA-binding protein RBM38, are found to tightly control p53 expression. Interestingly, RBM38 is unique in that it can either suppress or enhance p53 mRNA translation via altered interaction with eIF4E potentially mediated by serine-195 (S195) in RBM38. Thus, multiple RBM38/eIF4E knock-in (KI) cell lines were generated to investigate the significance of eIF4E-RBM38 interaction in controlling p53 activity. We showed that KI of RBM38-S195D or -Y192C enhances, whereas KI of RBM38-S195K/R/L weakens, the binding of eIF4E to p53 mRNA and subsequently p53 expression. We also showed that KI of eIF4E-D202K weakens the interaction of eIF4E with RBM38 and thereby enhances p53 expression, suggesting that D202 in eIF4E interacts with S195 in RBM38. Moreover, we generated an Rbm38 S193D KI mouse model in which human-equivalent serine-193 is substituted with aspartic acid. We showed that S193D KI enhances p53-dependent cellular senescence and that S193D KI mice have a shortened life span and are prone to spontaneous tumors, chronic inflammation, and liver steatosis. Together, we provide in vivo evidence that the RBM38-eIF4E loop can be explored to fine-tune p53 expression for therapeutic development.

Abstract Background Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells. Results In this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration. Conclusions These results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells.

– This paper shows a new methodology for evaluating the value and sensitivity of autocall knock-in type equity-linked securities. While the existing evaluation methods, Monte Carlo simulation and finite difference method, have limitations in underestimating the knock-in effect, which is one of the important characteristics of this type, this paper presents a precise joint probability formula for multiple autocall chances and knock-in events. Based on this, the calculation results obtained by utilizing numerical and Monte Carlo integration are presented and compared with those of existing models. The results of the proposed model show notable improvements in terms of accuracy and calculation time.

Human epidermal growth factor receptor 2 (HER-2)+ breast cancer has a high recurrence rate and a poor prognosis, with drug resistance contributing to disease progression. The present study aimed to establish a SKBR3 cell line with type II insulin-like growth factor receptor (IGR-IIR) gene site integration using the CRISPR/Cas9 system, and to provide a cell model for exploring the mechanism responsible for the effect of IGF-IIR on trastuzumab resistance in HER-2+ breast cancer cells. In the present study, six single guide (sg)RNA pairs according to the adeno-associated virus integration site 1 (AAVS1) gene sequence were designed and synthesized, and the Universal CRISPR Activity assay CRISPR/Cas9 rapid construction and activity detection kit was used to connect the annealed oligo with the pCS vector. The sgRNA with the highest efficiency was selected to construct a Cas9/sgRNA expression vector using AsiSI + Bstz17I restriction enzymes to cut IGF-IIR. The fragment was ligated into an human AAVS1-KI vector to construct the IGF-IIR targeting vector. The Cas9/sgRNA and IGF-IIR targeting vectors were electroporated into SKBR3 cells, screened using puromycin and identified via PCR, and the mixed cloned cells generated via IGF-IIR gene targeted integration were obtained. The semi-solid and limited dilution methods were used for monoclonal cell preparation, and the results revealed that a Cas9/sgRNA vector that targeted the AAVS1 was successfully constructed. sgRNA activity detection demonstrated that sgRNA2 had the highest efficiency, while enzyme digestion and sequencing confirmed that the IGF-IIR target vector was successfully constructed. The optimum conditions for electrotransfection were 1,200 V, 20 ms and 2 pulses, and the optimal screening concentration of puromycin was 0.5 µg/ml. Using these conditions, the IGF-IIR targeting vector and pCS-sgRNA2 plasmid were successfully transfected into SKBR3 cells, and PCR identification and sequencing verified the correct genotype of mixed clone fragments. The monoclonal cells proliferate slowly and gradually underwent apoptosis. Overall, the present study successfully obtained a mixed clone cell line with site-specific integration of the IGF-IIR gene at the AAVS1.

BackgroundAlzheimer's disease (AD) is the most frequent and costly neurodegenerative disorder. Although diverse lines of evidence suggest that the amyloid precursor protein (APP) is involved in its causation, the precise mechanisms remain unknown and no treatments are available to prevent or halt the disease. A favorite hypothesis has been that APP contributes to AD pathogenesis through the cerebral accumulation of the amyloid-β peptide (Aβ), which is derived from APP through sequential proteolytic cleavage by BACE1 and γ-secretase. However, inhibitors of these enzymes have failed in clinical trials despite clear evidence for target engagement.MethodsTo further elucidate the roles of APP and its metabolites in AD pathogenesis, we analyzed transgenic mice overexpressing wildtype human APP (hAPP) or hAPP carrying mutations that cause autosomal dominant familial AD (FAD), as well as App knock-in mice that do not overexpress hAPP but have two mouse App alleles with FAD mutations and a humanized Aβ sequence.ResultsAlthough these lines of mice had marked differences in cortical and hippocampal levels of APP, APP C-terminal fragments, soluble Aβ, Aβ oligomers and age-dependent amyloid deposition, they all developed cognitive deficits as well as non-convulsive epileptiform activity, a type of network dysfunction that also occurs in a substantive proportion of humans with AD. Pharmacological inhibition of BACE1 effectively reduced levels of amyloidogenic APP C-terminal fragments (C99), soluble Aβ, Aβ oligomers, and amyloid deposits in transgenic mice expressing FAD-mutant hAPP, but did not improve their network dysfunction and behavioral abnormalities, even when initiated at early stages before amyloid deposits were detectable.ConclusionshAPP transgenic and App knock-in mice develop similar pathophysiological alterations. APP and its metabolites contribute to AD-related functional alterations through complex combinatorial mechanisms that may be difficult to block with BACE inhibitors and, possibly, also with other anti-Aβ treatments.

In the world’s first pig-to-human cardiac cytomegalovirus (PCMV), xenotransplant and elevated levels of porcine key factors contributing to patient mortality were considered. This has renewed attention on PCMV, a virus widely prevalent in pigs. Currently, there are no effective drugs or vaccines targeting PCMV, and its high detection difficulty poses challenges for prevention and control research. In this study, antiviral small hairpin RNA (shRNA) was selected and inserted into the Rosa26 and miR-17-92 loci of pigs via a CRISPR/Cas9-mediated knock-in strategy. Further in vitro viral challenge experiments demonstrated that these genetically edited pig cells could effectively limit PCMV replication. Through this process, we constructed a PCMV-infected cell model, validated partial viral interference sites, enhanced gene knock-in efficiency, performed gene editing at two different gene loci, and ultimately demonstrated that RNA interference (RNAi) technology combined with CRISPR/Cas9 has the potential to generate pig cells with enhanced antiviral infection capabilities. This opens up possibilities for the future production of pig populations with antiviral functionalities.

Targeted knock-in of fluorescent reporters enables powerful gene and protein analyses in a physiological context. However, precise integration of long sequences remains challenging in vivo. Here, we demonstrate cloning-free and precise reporter knock-in into zebrafish genes, using PCR-generated templates for homology-directed repair with short homology arms (PCR tagging). Our novel knock-in reporter lines of vesicle-associated membrane protein (vamp) zebrafish homologues reveal subcellular complexity in this protein family. Our approach enables fast and efficient reporter integration in the zebrafish genome (in 10-40% of injected embryos) and rapid generation of stable germline-transmitting lines. [ABSTRACT FROM AUTHOR]

Genome editing, as exemplified by the CRISPR/Cas9 system, has recently been employed to effectively generate genetically modified animals and cells for the purpose of gene function analysis and disease model creation. There are at least four ways to induce genome editing in individuals: the first is to perform genome editing at the early preimplantation stage, such as fertilized eggs (zygotes), for the creation of whole genetically modified animals; the second is at post-implanted stages, as exemplified by the mid-gestational stages (E9 to E15), for targeting specific cell populations through in utero injection of viral vectors carrying genome-editing components or that of nonviral vectors carrying genome-editing components and subsequent in utero electroporation; the third is at the mid-gestational stages, as exemplified by tail-vein injection of genome-editing components into the pregnant females through which the genome-editing components can be transmitted to fetal cells via a placenta-blood barrier; and the last is at the newborn or adult stage, as exemplified by facial or tail-vein injection of genome-editing components. Here, we focus on the second and third approaches and will review the latest techniques for various methods concerning gene editing in developing fetuses. [ABSTRACT FROM AUTHOR]

Abstract Background Hyperphosphorylation and intraneuronal aggregation of the microtubule-associated protein tau is a major pathological hallmark of Alzheimer’s disease (AD) brain. Of special interest is the effect of cerebral amyloid beta deposition, the second main hallmark of AD, on human tau pathology. Therefore, studying the influence of cerebral amyloidosis on human tau in a novel human tau knock-in (htau-KI) mouse model could help to reveal new details on their interplay. Methods We studied the effects of a novel human htau-KI under fast-progressing amyloidosis in 5xFAD mice in terms of correlation of gene expression data with human brain regions, development of Alzheimer’s-like pathology, synaptic transmission, and behavior. Results The main findings are an interaction of human beta-amyloid and human tau in crossbred 5xFADxhtau-KI observed at transcriptional level and corroborated by electrophysiology and histopathology. The comparison of gene expression data of the 5xFADxhtau-KI mouse model to 5xFAD, control mice and to human AD patients revealed conspicuous changes in pathways related to mitochondria biology, extracellular matrix, and immune function. These changes were accompanied by plaque-associated MC1-positive pathological tau that required the htau-KI background. LTP deficits were noted in 5xFAD and htau-KI mice in contrast to signs of rescue in 5xFADxhtau-KI mice. Increased frequencies of miniature EPSCs and miniature IPSCs indicated an upregulated presynaptic function in 5xFADxhtau-KI. Conclusion In summary, the multiple interactions observed between knocked-in human tau and the 5xFAD-driven progressing amyloidosis have important implications for future model development in AD.

Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors. Safe harbor loci in the animal genome enable consistent overexpression of foreign genes, without side effects. However, relatively few safe harbor loci are available in pigs, a fact which has impeded the development of multi-transgenic pig research. We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain (COL1A1) gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. After the knock-in of a 2A peptide-green fluorescence protein (2A-GFP) transgene in the last codon of COL1A1 in multiple porcine cells, including porcine kidney epithelial (PK15), porcine embryonic fibroblast (PEF) and porcine intestinal epithelial (IPI-2I) cells, quantitative PCR (qPCR), Western blotting, RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus. The qPCR results showed that the GFP knock-in had no effect (P=0.29, P=0.66 and P=0.20 for PK15, PEF and IPI-2I cells, respectively) on the mRNA expression of COL1A1 gene. Similarly, no significant differences (P=0.64, P=0.48 and P=0.80 for PK15, PEF and IPI-2I cells, respectively) were found between the GFP knock-in and wild type cells by Western blotting. RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation (P

Abstract Background Genome editing using CRISPR/Cas9 has become a powerful tool in zebrafish to generate targeted gene knockouts models. However, its use for targeted knock-in remains challenging due to inefficient homology directed repair (HDR) pathway in zebrafish, highlighting the need for efficient and cost-effective screening methods. Results Here, we present our fluorescent PCR and capillary electrophoresis based screening approach for knock-in using a single-stranded oligodeoxynucleotide donor (ssODN) as a repair template for the targeted insertion of epitope tags, or single nucleotide changes to recapitulate pathogenic human alleles. For the insertion of epitope tags, we took advantage of the expected change in size of the PCR product. For point mutations, we combined fluorescent PCR with restriction fragment length polymorphism (RFLP) analysis to distinguish the fish with the knock-in allele. As a proof-of-principle, we present our data on the generation of fish lines with insertion of a FLAG tag at the tcnba locus, an HA tag at the gata2b locus, and a point mutation observed in Gaucher disease patients in the gba gene. Despite the low number of germline transmitting founders (1–5%), combining our screening methods with prioritization of founder fish by fin biopsies allowed us to establish stable knock-in lines by screening 12 or less fish per gene. Conclusions We have established a robust pipeline for the generation of zebrafish models with precise integration of small DNA sequences and point mutations at the desired sites in the genome. Our screening method is very efficient and easy to implement as it is PCR-based and only requires access to a capillary sequencer.

Abstract CRISPR tools can generate knockout and knock-in animal models easily, but the models can contain off-target genomic lesions or random insertions of donor DNAs. Simpler methods to identify off-target lesions and random insertions, using tail or earpiece DNA, are unavailable. We develop CRISPR-KRISPR (CRISPR-Knock-ins and Random Inserts Searching P Rotocol), a method to identify both off-target lesions and random insertions. CRISPR-KRISPR uses as little as 3.4 μg of genomic DNA; thus, it can be easily incorporated as an additional step to genotype founder animals for further breeding.

Recently, clinical trials of adeno-associated virus-mediated replacement therapy have suggested long-term therapeutic effects for several genetic diseases of the liver, including hemophilia. However, there remain concerns regarding decreased therapeutic effects when the liver is regenerated or when physiological proliferation occurs. Although genome editing using the clustered regularly interspaced short palindromic repeats/Cas9 system provides an opportunity to solve this problem, low knock-in efficiency may limit its application for therapeutically relevant expression. Here, we identified a novel gene, APOC3, in which a strong promoter facilitated the expression of knocked-in genes in hepatocytes. We also investigated the effects of APOC3 editing using a small Cas9 protein derived from Campylobacter jejuni (CjCas9) in a hemophilic model. We demonstrated that adeno-associated virus-mediated delivery of CjCas9 and donor led to moderate levels of human factor 9 expression in APOC3-humanized mice. Moreover, knock-in-driven expression induced substantial recovery of clotting function in mice with hemophilia B. There was no evidence of off-target editing in vitro or in vivo. Collectively, our findings demonstrated therapeutically relevant expression using a precise and efficient APOC3-editing platform, providing insights into the development of further long-term therapeutics for diverse monogenic liver diseases.

Background: Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells. Results: In this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration. Conclusions: These results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells. [ABSTRACT FROM AUTHOR]

Fluorescent protein (FP) tagging is a key method for observing protein distribution, dynamics and interaction with other proteins in living cells. However, the typical approach using overexpression of tagged proteins can perturb cell behavior and introduce localization artifacts. To preserve native expression, fluorescent proteins can be inserted directly into endogenous genes. This approach has been widely used in yeast for decades, and more recently in invertebrate model organisms with the advent of CRISPR/Cas9. However, endogenous FP tagging has not been widely used in mammalian cells due to inefficient homology-directed repair. Recently, the CRISPaint system used non-homologous end joining for efficient integration of FP tags into native loci, but it only allows C-terminal knock-ins. Here, we have enhanced the CRISPaint system by introducing new universal donors for N-terminal insertion and for multi-color tagging with orthogonal selection markers. We adapted the procedure for mouse embryonic stem cells, which can be differentiated into diverse cell types. Our protocol is rapid and efficient, enabling live imaging in less than 2 weeks post-transfection. These improvements increase the versatility and applicability of FP knock-in in mammalian cells. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9‐mediated targeted gene integration (TI) has been used to generate recombinant mammalian cell lines with predictable transgene expression. Identifying genomic hot spots that render high and stable transgene expression and knock‐in (KI) efficiency is critical for fully implementing TI‐mediated cell line development (CLD); however, such identification is cumbersome. In this study, we developed an artificial KI construct that can be used as a hot spot at different genomic loci. The ubiquitous chromatin opening element (UCOE) was employed because of its ability to open chromatin and enable stable and site‐independent transgene expression. UCOE KI cassettes were randomly integrated into CHO‐K1 and HEK293T cells, followed by TI of enhanced green fluorescent protein (EGFP) onto the artificial UCOE KI site. The CHO‐K1 random pool harboring 5′2.2A2UCOE‐CMV displayed a significant increase in EGFP expression level and KI efficiency compared with that of the control without UCOE. In addition, 5′2.2A2UCOE‐CMV showed improved Cas9 accessibility in the HEK293T genome, leading to an increase in indel frequency and homology‐independent KI. Overall, this assessment revealed the potential of UCOE KI constructs as artificial integration sites in streamlining the screening of high‐production targeted integrants by mitigating the selection of genomic hot spots. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been used for the insertion of large transgenes into Chinese hamster ovary cells via co-transfection of a Cas9/guide RNA expression vector and donor plasmid. The Cas9 protein includes nuclear localization sequences that are used as peptide tags for the import of Cas9 into the nucleus. However, the import of a donor plasmid into the nucleus is passive because of the absence of such localization signals; thus, the delivery of Cas9 and the donor plasmid is not synchronized, resulting in low knock-in (KI) efficiency. Here, we modified the Cas9 expression vector expressing a Cas9 protein fused to a zinc finger (ZF) domain, Cas9-ZF, to expedite the translocation of the donor plasmid into the nucleus and the co-localization of the donor plasmid with a CRISPR/Cas9-mediated DNA double-strand break site by tethering Cas9-ZF and the donor plasmid. Compared to the typical donor plasmid and wild-type Cas9, the donor plasmid harboring the ZF-binding motif showed increased homology-mediated KI efficiency, while the engineered Cas9 protein showed decreased expression and gene-editing efficiency. Moreover, the pair of Cas9-ZF and the donor plasmid with the ZF motif did not improve KI efficiency, but rather negated the positive effect of the donor plasmid with the ZF motif. This study demonstrates the importance of the transport of donor plasmids and the limitations of using the plasmid-based Cas9-ZF fusion system to improve KI efficiency. [ABSTRACT FROM AUTHOR]

In-locus editing of agronomically-important genes to optimize their spatiotemporal expression is becoming an important breeding approach. Compared to intensive studies on mRNA transcription, manipulating protein translation by genome editing has not been well exploited. Here, we found that precise knock-in of a regulating element into the 5’UTR of a target gene could efficiently increase its protein abundance in rice. We firstly screened a translational enhancer (AMVE) from alfalfa mosaic virus using protoplast-based luciferase assays with an 8.5-folds enhancement. Then the chemically modified donor of AMVE was synthesized and targeted inserted into the 5’UTRs of two genes (WRKY71 and SKC1) using CRISPR/Cas9. Following the in-locus AMVE knock-in, we observed up to a 2.8-fold increase in the amount of WRKY71 protein. Notably, editing of SKC1, a sodium transporter, significantly increased salt tolerance in T2 seedlings, indicating the expected regulation of AMVE knockin. These data demonstrated the feasibility of such in-locus editing to enhance protein expression, providing a new approach to manipulating protein translation for crop breeding. [ABSTRACT FROM AUTHOR]

Recent advances in CRISPR-Cas genome editing technology have been instrumental in improving the efficiency to produce genetically modified animal models. In this study we have combined four very promising approaches to come up with a highly effective pipeline to produce knock-in mouse and rat models. The four combined methods include: AAV-mediated DNA delivery, single-stranded DNA donor templates, 2-cell embryo modification, and CRISPR-Cas ribonucleoprotein (RNP) electroporation. Using this new combined approach, we were able to produce successfully targeted knock-in rat models containing either Cre or Flp recombinase sequences with knock-in efficiencies over 90%. Furthermore, we were able to produce a knock-in mouse model containing a Cre recombinase targeted insertion with over 50% knock-in efficiency directly comparing efficiencies to other commonly used approaches. Our modified AAV-mediated DNA delivery with 2-cell embryo CRISPR-Cas9 RNP electroporation technique has proven to be highly effective for generating both knock-in mouse and knock-in rat models.

T lymphocytes represent a promising target for genome editing. They are primarily modified to recognize and kill tumor cells or to withstand HIV infection. In most studies, T cell genome editing is performed using the CRISPR/Cas technology. Although this technology is easily programmable and widely accessible, its efficiency of T cell genome editing was initially low. Several crucial improvements were made in the components of the CRISPR/Cas technology and their delivery methods, as well as in the culturing conditions of T cells, before a reasonable editing level suitable for clinical applications was achieved. In this review, we summarize and describe the aforementioned parameters that affect human T cell editing efficiency using the CRISPR/Cas technology, with a special focus on gene knock-in.

Lysosomal network abnormalities are an increasingly recognised feature of Alzheimer’s disease (AD), which appear early and are progressive in nature. Sandhoff disease and Tay-Sachs disease (neurological lysosomal storage diseases caused by mutations in genes that code for critical subunits of β-hexosaminidase) result in accumulation of amyloid-β (Aβ) and related proteolytic fragments in the brain. However, experiments that determine whether mutations in genes that code for β-hexosaminidase are risk factors for AD are currently lacking. To determine the relationship between β-hexosaminidase and AD, we investigated whether a heterozygous deletion of Hexb, the gene that encodes the beta subunit of β-hexosaminidase, modifies the behavioural phenotype and appearance of disease lesions in AppNL-G-F/NL-G-F (AppKI/KI) mice. AppKI/KI and Hexb+/- mice were crossed and evaluated in a behavioural test battery. Neuropathological hallmarks of AD and ganglioside levels in the brain were also examined. Heterozygosity of Hexb in AppKI/KI mice reduced learning flexibility during the Reversal Phase of the Morris water maze. Contrary to expectation, heterozygosity of Hexb caused a small but significant decrease in amyloid beta deposition and an increase in the microglial marker IBA1 that was region- and age-specific. Hexb heterozygosity caused detectable changes in the brain and in the behaviour of an AD model mouse, consistent with previous reports that described a biochemical relationship between HEXB and AD. This study reveals that the lysosomal enzyme gene Hexb is not haplosufficient in the mouse AD brain.

Abstract CRISPR/Cas9‐mediated targeted gene integration (TI) has been used to generate recombinant mammalian cell lines with predictable transgene expression. Identifying genomic hot spots that render high and stable transgene expression and knock‐in (KI) efficiency is critical for fully implementing TI‐mediated cell line development (CLD); however, such identification is cumbersome. In this study, we developed an artificial KI construct that can be used as a hot spot at different genomic loci. The ubiquitous chromatin opening element (UCOE) was employed because of its ability to open chromatin and enable stable and site‐independent transgene expression. UCOE KI cassettes were randomly integrated into CHO‐K1 and HEK293T cells, followed by TI of enhanced green fluorescent protein (EGFP) onto the artificial UCOE KI site. The CHO‐K1 random pool harboring 5′2.2A2UCOE‐CMV displayed a significant increase in EGFP expression level and KI efficiency compared with that of the control without UCOE. In addition, 5′2.2A2UCOE‐CMV showed improved Cas9 accessibility in the HEK293T genome, leading to an increase in indel frequency and homology‐independent KI. Overall, this assessment revealed the potential of UCOE KI constructs as artificial integration sites in streamlining the screening of high‐production targeted integrants by mitigating the selection of genomic hot spots.

Simple Summary: The application of CRISPR/Cas9 genome editing in fish is limited to date. An easy-to-use, economical and effective genome editing system is greatly needed. In this study, we established an integrated pCas9-U6sgRNA plasmid system, the sgRNA of which was driven by U6 promoters from different fish species. Taking medaka as an example, our results suggest that pCas9-U6sgRNA driven by endogenous U6 promoter (pCas9-mU6sgRNA) can very effectively mediate gene knock-out in medaka cultured cells, but not by exogenous U6 promoter. The gene editing efficiency of pCas9-mU6sgRNA was up to 93.7% with no detectable off-target. By one-cell embryo microinjection, pCas9-mU6sgRNA can effectively mediate gene knock-out in vivo as well. Furthermore, the gene knock-in at a specific site was efficiently achieved in vitro as well as in vivo through application of pCas9-mU6sgRNA. In conclusion, we have successfully developed a simple, low-cost and effective CRISPR/Cas9 system suitable for medaka gene knock-out and knock-in at a specific site in vitro and in vivo. This study provides an insight into other fish gene manipulation and greatly promotes functional gene studies. Although CRISPR/Cas9 has been used in gene manipulation of several fish species in vivo, its application in fish cultured cells is still challenged and limited. In this study, we established an integrated CRISPR/Cas9 plasmid system and evaluated its efficiency of gene knock-out or knock-in at a specific site in medaka (Oryzias latipes) in vitro and in vivo. By using the enhanced green fluorescent protein reporter plasmid pGNtsf1, we demonstrate that pCas9-U6sgRNA driven by endogenous U6 promoter (pCas9-mU6sgRNA) mediated very high gene editing efficiency in medaka cultured cells, but not by exogenous U6 promoters. After optimizing the conditions, the gene editing efficiencies of eight sites targeting for four endogenous genes were calculated, and the highest was up to 94% with no detectable off-target. By one-cell embryo microinjection, pCas9-mU6sgRNA also mediated efficient gene knock-out in vivo. Furthermore, pCas9-mU6sgRNA efficiently mediated gene knock-in at a specific site in medaka cultured cells as well as embryos. Collectively, our study demonstrates that the genetic relationship of U6 promoter is critical to gene editing efficiency in medaka cultured cells, and a simple and efficient system for medaka genome editing in vitro and in vivo has been established. This study provides an insight into other fish genome editing and promotes gene functional analysis. [ABSTRACT FROM AUTHOR]

Recombineering is a highly efficient DNA cloning and modification technique by using the recombinase-mediated homologous recombination. Selection/counterselection cassette is often used in chromosomal DNA or large episomal DNA manipulation, in which the selection marker is used for the first step cassette selection while deleting the target gene via allelic exchange, and the counterselection marker is used for the second step replacement of the cassette by the foreign DNA fragment. A variety of selection/counterselection cassettes are reported, however, the cassettes suffer from the shortcomings of the requirement of pre-engineered strain or specific culture medium. Herein, we report a novel S-tetR- PtetA-ccdB-aacC1-S selection/counterselection cassette that sidesteps the disadvantages. As a proof-of-concept, one-step gene cloning (0.7, 1.7, and 4.2 kb) and two-step Escherichia coli chromosomal gene knock-in (0.7 and 4.2 kb) were performed. The gene cloning and gene knock-in efficiencies are high up to 90%. The novel selection/counterselection cassette adds a powerful tool to the recombineering repertoire. [ABSTRACT FROM AUTHOR]

Introduction: Alcohol is the main legal drug in the world, and excessive consumption of alcohol seriously damages the morphological structure and function of various organs. The insufficiency of an essential enzyme in ethanol metabolism, aldehyde dehydrogenase-2 (ALDH2), will aggravate the alcoholinduced brain injury. The effect of ALDH2 after chronic alcohol exposure on global lipid profiling of the brain remains unclear. Methods: In this study, ALDH2*2 knock-in mice were fed the Lieber-DeCarli liquid diet containing ethanol for 8 weeks. Blood alcohol and acetaldehyde levels were examined, and the mice were tested through novel object recognition and the Y-maze test to evaluate cognitive impairment toward the end of the study. The lipidome profiling of cerebral cortex samples was investigated using a lipidomics method based on ultra-high performance liquid tandem chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOFMS). Results and Discussion: Compared with similarly treated wild-type (WT) mice, ALDH2*2 mice exhibited poor cognitive performance, though the result did not achieve statistical significance. The lipidomics results indicated that 74 differential lipid species were selected in WT mice, of which 57 species were up-regulated, and 17 were down-regulated. Moreover, 99 differential lipids were identified in ALDH2*2 mice, of which 73 were up-regulated, and 26 were down-regulated. For ALDH2*2 mice, the number of changed significantly glycerophospholipids (GPs) subtypes was lower than that of WT mice. Interestingly, compared with WT mice, a lower proportion of polyunsaturated fatty acids (PUFAs) was found in ALDH2*2 mice. Collectively, the results provide clear evidence for a lipidomic signature of marked changes in the cerebral cortex of ALDH2*2 mice after chronic alcohol exposure. [ABSTRACT FROM AUTHOR]

Background: Hyperphosphorylation and intraneuronal aggregation of the microtubule-associated protein tau is a major pathological hallmark of Alzheimer's disease (AD) brain. Of special interest is the effect of cerebral amyloid beta deposition, the second main hallmark of AD, on human tau pathology. Therefore, studying the influence of cerebral amyloidosis on human tau in a novel human tau knock-in (htau-KI) mouse model could help to reveal new details on their interplay. Methods: We studied the effects of a novel human htau-KI under fast-progressing amyloidosis in 5xFAD mice in terms of correlation of gene expression data with human brain regions, development of Alzheimer's-like pathology, synaptic transmission, and behavior. Results: The main findings are an interaction of human beta-amyloid and human tau in crossbred 5xFADxhtau-KI observed at transcriptional level and corroborated by electrophysiology and histopathology. The comparison of gene expression data of the 5xFADxhtau-KI mouse model to 5xFAD, control mice and to human AD patients revealed conspicuous changes in pathways related to mitochondria biology, extracellular matrix, and immune function. These changes were accompanied by plaque-associated MC1-positive pathological tau that required the htau-KI background. LTP deficits were noted in 5xFAD and htau-KI mice in contrast to signs of rescue in 5xFADxhtau-KI mice. Increased frequencies of miniature EPSCs and miniature IPSCs indicated an upregulated presynaptic function in 5xFADxhtau-KI. Conclusion: In summary, the multiple interactions observed between knocked-in human tau and the 5xFAD-driven progressing amyloidosis have important implications for future model development in AD. [ABSTRACT FROM AUTHOR]