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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.
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Mutación Puntual , Pez Cebra , Humanos , Animales , Pez Cebra/genética , Epítopos , InvestigaciónRESUMEN
Genome editing is a powerful tool that allows study of the properties of genes or changes to be made to the genetic sequence. Programmable nucleases that can induce double-strand breaks in the genomic sequence of interest have been developed over the past few decades. After initiation of a double-strand break (DSB) in DNA, the DSB can be repaired by the NHEJ (non-homologous end joining), which leads to various errors and gene knockout. Other repair options, HDR (homology directed repair) or SSTR (single-strand template repair), allow researchers to make desired changes in the gene. HDR occurs in the presence of a donor template, in natural conditions the donor template is a sister chromatid. The efficiency of HDR and SSTR is significantly lower than the efficiency of NHEJ in genome editing. Double-stranded, single-stranded and long single-stranded DNAs are used to increase efficiency and to make desired changes in genomic DNA. In this review, we discuss donor molecules that are used for DSB repair using HDR or SSTR during genome editing, their application, and modifications to increase the efficiency of HDR and SSTR.
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Reparación del ADN por Unión de Extremidades , Edición Génica , Animales , ADN/genética , Reparación del ADN por Unión de Extremidades/genética , Endonucleasas/genética , Reparación del ADN por RecombinaciónRESUMEN
Rice is a staple food crop, which ensures the calorie requirement of half of the world's population. With the continued increase in population, rice will play a key role in achieving the food security. However, in the constantly shrinking scenario of rice fields, the necessity of these extra grains of rice must be met by reducing the yield loss due to various abiotic and biotic stresses. The adverse effects of climate impact both quality and quantity of rice production. One of the most desirable applications of CRISPR/Cas technology would be to develop climate smart rice crop to sustain and enhance its productivity in the changing environment. In this review, we analyze the desirable phenotypes and responsible genetic factors, which can be utilized to develop tolerance against major abiotic stresses imposed by climate change through genome engineering. The possibility of utilizing the information from wild resources to engineer the corresponding alleles of cultivated rice has been presented. We have also shed light on available resources for generating genome edited rice lines. The CRISPR/Cas mediated genome editing strategies for engineering of novel genes were proposed to create a plant phenotype, which can face the adversities of climate change. Further, challenges of off-targets and undesirable phenotype were discussed.
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Sistemas CRISPR-Cas/genética , Edición Génica , Genoma de Planta/genética , Oryza/genéticaRESUMEN
BACKGROUND: The generation of point mutations is a major tool for evaluating the roles of specific nucleotides or amino acids within the regulatory or functional landscape. However, examination of these mutations in vivo requires the generation of animals carrying only the relevant point mutations at the endogenous genomic loci, which is technically challenging. The CRISPR-Cas9 based genome editing greatly facilitates the generation of such genetically modified animals; however, most of the described methods use double-strand DNA (dsDNA) as the donor template. The dsDNA plasmids frequently undergo undesired integration events into the targeted genomic locus. The use of a single-strand oligodeoxynucleotide (ssODN) as the donor template prevents this complication and is therefore the preferred choice for introducing point mutations, as well as short sequences such as protein tags. RESULTS: We successfully applied the CRISPR-based white co-conversion strategy with a ssODN template, instead of the originally described dsDNA plasmid, to create genetically modified Drosophila melanogaster strains. We used the technique to easily introduce point mutations in two distinct chromosomes. Using the generated flies, we were able to demonstrate the in vivo importance of the respective mutations. For the Nucleoporin107 (Nup107) gene, the 1090G > A mutation was confirmed to affect ovarian development, while for the tinman (tin) gene, the regulatory role of the downstream core promoter element (DPE) was demonstrated within the developing Drosophila melanogaster embryo. CONCLUSIONS: The described approach has facilitated the successful generation of point mutations in two different chromosomes, by two different labs. Distinct phenotypes associated with the newly-generated genotype were identified, thus exemplifying the importance of investigating the in vivo role of specific nucleotides. In addition, detailed guidelines, recommendations and crossing schemes are provided in order to support the generation of additional genetically modified animals by the scientific community.
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The green alga Chlamydomonas reinhardtii is an invaluable reference organism to research fields including algal, plant, and ciliary biology. Accordingly, decades-long standing inefficiencies in targeted nuclear gene editing broadly hinder Chlamydomonas research. Here we report that single-step codelivery of CRISPR/Cpf1 ribonucleoproteins with single-stranded DNA repair templates results in precise and targeted DNA replacement with as much as â¼10% efficiency in C. reinhardtii We demonstrate its use in transgene- and selection-free generation of sequence-specific mutations and epitope tagging at an endogenous locus. As the direct delivery of gene-editing reagents bypasses the use of transgenes, this method is potentially applicable to a wider range of species without the need to develop methods for stable transformation.
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Proteínas Bacterianas/metabolismo , Chlamydomonas reinhardtii/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endonucleasas/metabolismo , Edición Génica/métodos , Proteínas Bacterianas/genética , Endonucleasas/genética , Oligonucleótidos/genéticaRESUMEN
In homology-directed repair, mediated knock-in single-stranded oligodeoxynucleotides (ssODNs) can be used as a homologous template and present high efficiency, but there is still a need to improve efficiency. Previous studies have mainly focused on controlling double-stranded break size, ssODN stability, and the DNA repair cycle. Nevertheless, there is a lack of research on the correlation between the cell cycle and single-strand template repair (SSTR) efficiency. Here, we investigated the relationship between cell cycle and SSTR efficiency. We found higher SSTR efficiency during mitosis, especially in the metaphase and anaphase. A Cas9 protein with a nuclear localization signal (NLS) readily migrated to the nucleus; however, the nuclear envelope inhibited the nuclear import of many nucleotide templates. This seemed to result in non-homologous end joining (NHEJ) before the arrival of the homologous template. Thus, we assessed whether NLS-tagged ssODNs and free NLS peptides could circumvent problems posed by the nuclear envelope. NLS-tagging ssODNs enhanced SSTR and indel efficiency by 4-fold compared to the control. Our results suggest the following: (1) mitosis is the optimal phase for SSTR, (2) the donor template needs to be delivered to the nucleus before nuclease delivery, and (3) NLS-tagging ssODNs improve SSTR efficiency, especially high in mitosis.
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Proteína 9 Asociada a CRISPR/genética , Edición Génica/métodos , Técnicas de Sustitución del Gen/métodos , Mitosis , Señales de Localización Nuclear , Animales , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reparación del ADN por RecombinaciónRESUMEN
Targeted insertion of large pieces of DNA is an important goal of genetic engineering. However, this goal has been elusive since classical methods for homology-directed repair are inefficient and often not feasible in many systems. Recent advances are described here that enable site-specific genomic insertion of relatively large DNA with much improved efficiency. Using the preferred repair pathway in the cell of nonhomologous end-joining, DNA of up to several kb could be introduced with remarkably good precision by the methods of HITI and ObLiGaRe with an efficiency up to 30-40%. Recent advances utilizing homology-directed repair (methods of PITCh; short homology arms including ssODN; 2H2OP) have significantly increased the efficiency for DNA insertion, often to 40-50% or even more depending on the method and length of DNA. The remaining challenges of integration precision and off-target site insertions are summarized. Overall, current advances provide major steps forward for site-specific insertion of large DNA into genomes from a broad range of cells and organisms.
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Sistemas CRISPR-Cas , Reparación del ADN , Edición Génica/métodos , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Nucleasas con Dedos de Zinc/genética , Animales , Cromosomas , ADN , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Genoma , Humanos , Plásmidos/genética , ARN Guía de Kinetoplastida , Recombinación Genética , Nucleasas con Dedos de Zinc/metabolismoRESUMEN
In the past few years, new technologies have arisen that enable higher efficiency of gene editing. With the increase ease of using gene editing technologies, it is important to consider the best method for transferring new genetic material to livestock animals. Microinjection is a technique that has proven to be effective in mice but is less efficient in large livestock animals. Over the years, a variety of methods have been used for cloning as well as gene transfer including; nuclear transfer, sperm mediated gene transfer (SMGT), and liposome-mediated DNA transfer. This review looks at the different success rate of these methods and how they have evolved to become more efficient. As well as gene editing technologies, including Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the most recent clustered regulatory interspaced short palindromic repeats (CRISPRs). Through the advancements in gene-editing technologies, generating transgenic animals is now more accessible and affordable. The goals of producing transgenic animals are to 1) increase our understanding of biology and biomedical science; 2) increase our ability to produce more efficient animals; and 3) produce disease resistant animals. ZFNs, TALENs, and CRISPRs combined with gene transfer methods increase the possibility of achieving these goals.
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Animales Modificados Genéticamente/genética , Edición Génica/métodos , Genoma/genética , Ganado/genética , Animales , Sistemas CRISPR-CasRESUMEN
Bone Morphogenetic Proteins play a significant role in ovarian physiology and contribute to the reproductive fitness of mammals. The BMPR-1B/FecB mutation, a loss of function mutation increases litter size by 1-2 with each number of mutated alleles in sheep. Considering demand-supply gap of the meat industry, and low replacement rate of indigenous caprine species, the conservative BMPR-1B locus can be explored, and FecB mutated goats can be produced. The experiment one produced CRISPR/Cas mediated KO transferable caprine embryos, and experiment two generated caprine embryos with desired FecB mutation using Easi-CRISPR strategy. In the KO experiment, Cas9 and BMPR-1B guide RNA (100:100ng/ul) were electroporated into single stage caprine zygotes at 750V, 10 ms and 1pulse using Neon transfection system. In the second experiment, phosphorothioate (PS) modified single-stranded oligodeoxynucleotide (ssODN) was used as an HDR template along with CRISPR components (100:100ng/ul, ssODN 100ng/ul). The precise time and method of electroporation, RNP format of CRISPR components and PS modified asymmetric ssODN were the factors that affected the production of mosaicism free BMPR-1B edited caprine embryos. The editing efficiency of KO and KI experiments was 68.52 and 63.16% respectively, and successful production of goats with higher mean ovulation rate can be realized with addition of embryo transfer technology to these experiments.
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Sistemas CRISPR-Cas , Cabras , Femenino , Animales , Ovinos , Cabras/genética , Mutación , Alelos , Terapia de Electroporación/veterinariaRESUMEN
Knock-in mice are useful for evaluating endogenous gene expressions and functions in vivo. Instead of the conventional gene-targeting method using embryonic stem cells, an exogenous DNA sequence can be inserted into the target locus in the zygote using genome-editing technology. In this chapter, I describe the generation of epitope-tagged mice using engineered endonuclease and single-strand oligodeoxynucleotide through the mouse zygote as an example of how to generate a knock-in mouse by genome editing.
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Edición Génica , Marcación de Gen , Ratones , Animales , Edición Génica/métodos , Marcación de Gen/métodos , Células Madre Embrionarias , Oligodesoxirribonucleótidos/genética , Sistemas CRISPR-Cas/genética , Técnicas de Sustitución del Gen , Cigoto/metabolismoRESUMEN
Just one amino acid at the carboxy-terminus of the B chain distinguishes human insulin from porcine insulin. By introducing a precise point mutation into the porcine insulin (INS) gene, we were able to generate genetically modified pigs that secreted human insulin; these pigs may be suitable donors for islet xenotransplantation. The electroporation of the CRISPR/Cas9 gene-editing system into zygotes is frequently used to establish genetically modified rodents, as it requires less time and no micromanipulation. However, electroporation has not been used to generate point-mutated pigs yet. In the present study, we introduced a point mutation into porcine zygotes via electroporation using the CRISPR/Cas9 system to generate INS point-mutated pigs as suitable islet donors. We first optimized the efficiency of introducing point mutations by evaluating the effect of Scr7 and the homology arm length of ssODN on improving homology-directed repair-mediated gene modification. Subsequently, we prepared electroporated zygotes under optimized conditions and transferred them to recipient gilts. Two recipients became pregnant and delivered five piglets. Three of the five piglets carried only the biallelic frame-shift mutation in the INS gene, whereas the other two successfully carried the desired point mutation. One of the two pigs mated with a WT boar, and this desired point mutation was successfully inherited in the next F1 generation. In conclusion, we successfully established genetically engineered pigs with the desired point mutation via electroporation-mediated introduction of the CRISPR/Cas9 system into zygotes, thereby avoiding the time-consuming and complicated micromanipulation method.
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Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene. PKD-erythroid cells suffer from an energy imbalance caused by a reduction of erythroid pyruvate kinase (RPK) enzyme activity. PKD is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. More than 300 disease-causing mutations have been identified as causing PKD. Most mutations are missense mutations, commonly present as compound heterozygous. Therefore, specific correction of these point mutations might be a promising therapy for the treatment of PKD patients. We have explored the potential of precise gene editing for the correction of different PKD-causing mutations, using a combination of single-stranded oligodeoxynucleotides (ssODN) with the CRISPR/Cas9 system. We have designed guide RNAs (gRNAs) and single-strand donor templates to target four different PKD-causing mutations in immortalized patient-derived lymphoblastic cell lines, and we have detected the precise correction in three of these mutations. The frequency of the precise gene editing is variable, while the presence of additional insertions/deletions (InDels) has also been detected. Significantly, we have identified high mutation-specificity for two of the PKD-causing mutations. Our results demonstrate the feasibility of a highly personalized gene-editing therapy to treat point mutations in cells derived from PKD patients.
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Generation of zebrafish (Danio rerio) models with targeted insertion of epitope tags and point mutations is highly desirable for functional genomics and disease modeling studies. Currently, CRISPR/Cas9-mediated knock-in is the method of choice for insertion of exogeneous sequences by providing a repair template for homology-directed repair (HDR). A major hurdle in generating knock-in models is the labor and cost involved in screening of injected fish to identify the precise knock-in events due to low efficiency of the HDR pathway in zebrafish. Thus, we developed fluorescent PCR-based high-throughput screening methods for precise knock-in of epitope tags and point mutations in zebrafish. Here, we provide a step-by-step guide that describes selection of an active sgRNA near the intended knock-in site, design of single-stranded oligonucleotide (ssODN) templates for HDR, quick validation of somatic knock-in using injected embryos, and screening for germline transmission of precise knock-in events to establish stable lines. Our screening method relies on the size-based separation of all fragments in an amplicon by fluorescent PCR and capillary electrophoresis, thus providing a robust and cost-effective strategy. Although we present the use of this protocol for insertion of epitope tags and point mutations, it can be used for insertion of any small DNA fragments (e.g., LoxP sites, in-frame codons). Furthermore, the screening strategy described here can be used to screen for precise knock-in of small DNA sequences in any model system, as PCR amplification of the target region is its only requirement. Key features This protocol expands the use of fluorescent PCR and CRISPR-STAT for screening of precise knock-in of small insertions and point mutations in zebrafish. Allows validation of selected sgRNA and HDR template within two weeks by somatic knock-in screening. Allows robust screening of point mutations by combining restriction digest with CRISPR-STAT. Graphical overview Overview of the three-phase knock-in pipeline in zebrafish (created with BioRender.com).
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Electroporation of mouse embryos with CRISPR/Cas9 endonuclease tool is a facile and efficient method to edit endogenous genome sequences for generating genetically engineered mouse models (GEMMs). Common genome engineering projects, such as knock-out (KO), conditional knock-out (cKO), point mutation, and small foreign DNA (<1 Kb) knock-in (KI) alleles, can be effectively accomplished with a simple electroporation procedure. The use of electroporation in sequential gene editing at the one-cell (0.7 days post-coitum (dpc)) and at two-cell (1.5 dpc) embryonic stages provides a fast and compelling protocol to safely introduce multiple gene modifications on the same chromosome by limiting chromosomal fractures. In addition, the co-electroporation of the ribonucleoprotein (RNP) complex and single-stranded oligodeoxynucleotide (ssODN) donor DNA with the strand exchange protein Rad51 can significantly increase the number of homozygous founders. Here we describe a comprehensive guideline for mouse embryo electroporation to generate GEMMs and the implementation of Rad51 in RNP/ssODN complex EP medium protocol.
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Proteína 9 Asociada a CRISPR , Edición Génica , Animales , Ratones , Edición Génica/métodos , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Alelos , Electroporación/métodos , ADN , Técnicas de Sustitución del GenRESUMEN
X-linked severe combined immunodeficiency (X-SCID) is a primary immunodeficiency that is caused by mutations in the interleukin-2 receptor gamma (IL2RG) gene. Some patients present atypical X-SCID with mild clinical symptoms due to somatic revertant mosaicism. CRISPR/Cas9 and prime editing are two advanced genome editing tools that paved the way for treating immune deficiency diseases. Prime editing overcomes the limitations of the CRISPR/Cas9 system, as it does not need to induce double-strand breaks (DSBs) or exogenous donor DNA templates to modify the genome. Here, we applied CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs) and prime editing methods to generate an in vitro model of the disease in K-562 cells and healthy donors' T cells for the c. 458T>C point mutation in the IL2RG gene, which also resulted in a useful way to optimize the gene correction approach for subsequent experiments in patients' cells. Both methods proved to be successful and were able to induce the mutation of up to 31% of treated K-562 cells and 26% of treated T cells. We also applied similar strategies to correct the IL2RG c. 458T>C mutation in patient T cells that carry the mutation with revertant somatic mosaicism. However, both methods failed to increase the frequency of the wild-type sequence in the mosaic T cells of patients due to limited in vitro proliferation of mutant cells and the presence of somatic reversion. To the best of our knowledge, this is the first attempt to treat mosaic cells from atypical X-SCID patients employing CRISPR/Cas9 and prime editing. We showed that prime editing can be applied to the formation of specific-point IL2RG mutations without inducing nonspecific on-target modifications. We hypothesize that the feasibility of the nucleotide substitution of the IL2RG gene using gene therapy, especially prime editing, could provide an alternative strategy to treat X-SCID patients without revertant mutations, and further technological improvements need to be developed to correct somatic mosaicism mutations.
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Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X , Humanos , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/genética , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/terapia , Sistemas CRISPR-Cas/genética , Mosaicismo , Edición Génica/métodos , Terapia Genética/métodosRESUMEN
BACKGROUND & AIMS: Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism caused by loss-of-function mutations in ATP7B, which encodes a copper-transporting protein. It is characterized by excessive copper deposition in tissues, predominantly in the liver and brain. We sought to investigate whether gene-corrected patient-specific induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps) could serve as an autologous cell source for cellular transplantation therapy in WD. METHODS: We first compared the in vitro phenotype and cellular function of ATP7B before and after gene correction using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) in iHeps (derived from patients with WD) which were homozygous for the ATP7B R778L mutation (ATP7BR778L/R778L). Next, we evaluated the in vivo therapeutic potential of cellular transplantation of WD gene-corrected iHeps in an immunodeficient WD mouse model (Atp7b -/- / Rag2 -/- / Il2rg -/- ; ARG). RESULTS: We successfully created iPSCs with heterozygous gene correction carrying 1 allele of the wild-type ATP7B gene (ATP7BWT/-) using CRISPR/Cas9 and ssODNs. Compared with ATP7BR778L/R778L iHeps, gene-corrected ATP7BWT/- iHeps restored i n vitro ATP7B subcellular localization, its subcellular trafficking in response to copper overload and its copper exportation function. Moreover, in vivo cellular transplantation of ATP7BWT/- iHeps into ARG mice via intra-splenic injection significantly attenuated the hepatic manifestations of WD. Liver function improved and liver fibrosis decreased due to reductions in hepatic copper accumulation and consequently copper-induced hepatocyte toxicity. CONCLUSIONS: Our findings demonstrate that gene-corrected patient-specific iPSC-derived iHeps can rescue the in vitro and in vivo disease phenotypes of WD. These proof-of-principle data suggest that iHeps derived from gene-corrected WD iPSCs have potential use as an autologous ex vivo cell source for in vivo therapy of WD as well as other inherited liver disorders. LAY SUMMARY: Gene correction restored ATP7B function in hepatocytes derived from induced pluripotent stem cells that originated from a patient with Wilson's disease. These gene-corrected hepatocytes are potential cell sources for autologous cell therapy in patients with Wilson's disease.
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Background & Aims: The truncating mutations in tight junction protein 2 (TJP2) cause progressive cholestasis, liver failure, and hepatocyte carcinogenesis. Due to the lack of effective model systems, there are no targeted medications for the liver pathology with TJP2 deficiency. We leveraged the technologies of patient-specific induced pluripotent stem cells (iPSC) and CRISPR genome-editing, and we aim to establish a disease model which recapitulates phenotypes of patients with TJP2 deficiency. Methods: We differentiated iPSC to hepatocyte-like cells (iHep) on the Transwell membrane in a polarized monolayer. Immunofluorescent staining of polarity markers was detected by a confocal microscope. The epithelial barrier function and bile acid transport of bile canaliculi were quantified between the two chambers of Transwell. The morphology of bile canaliculi was measured in iHep cultured in the Matrigel sandwich system using a fluorescent probe and live-confocal imaging. Results: The iHep differentiated from iPSC with TJP2 mutations exhibited intracellular inclusions of disrupted apical membrane structures, distorted canalicular networks, altered distribution of apical and basolateral markers/transporters. The directional bile acid transport of bile canaliculi was compromised in the mutant hepatocytes, resembling the disease phenotypes observed in the liver of patients. Conclusions: Our iPSC-derived in vitro hepatocyte system revealed canalicular membrane disruption in TJP2 deficient hepatocytes and demonstrated the ability to model cholestatic disease with TJP2 deficiency to serve as a platform for further pathophysiologic study and drug discovery. Lay summary: We investigated a genetic liver disease, progressive familial intrahepatic cholestasis (PFIC), which causes severe liver disease in newborns and infants due to a lack of gene called TJP2. By using cutting-edge stem cell technology and genome editing methods, we established a novel disease modeling system in cell culture experiments. Our experiments demonstrated that the lack of TJP2 induced abnormal cell polarity and disrupted bile acid transport. These findings will lead to the subsequent investigation to further understand disease mechanisms and develop an effective treatment.
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This study aimed to investigate the efficiency of KRAS gene editing via CRISPR/Cas9 delivery by electroporation and analyzed the effects of the non-homologous end-joining pathway inhibitor Scr7 and single-stranded oligodeoxynucleotide (ssODN) homology arm length on introducing a point mutation in KRAS. Various concentrations (0-2 µM) of Scr7 were evaluated; all concentrations of Scr7 including 0 µM resulted in the generation of blastocysts with a point mutation and the wild-type sequence or indels. No significant differences in the blastocyst formation rates of electroporated zygotes were observed among ssODN homology arm lengths, irrespective of the gRNA (gRNA1 and gRNA2). The proportion of blastocysts carrying a point mutation with or without the wild-type sequence and indels was significantly higher in the ssODN20 group (i.e., the group with a ssODN homology arm of 20 bp) than in the ssODN60 group (gRNA1: 25.7% vs. 5.4% and gRNA2: 45.5% vs. 5.9%, p < .05). In conclusion, the CRISPR/Cas9 delivery with ssODN via electroporation is feasible for the generation of point mutations in porcine embryos. Further studies are required to improve the efficiency and accuracy of the homology-directed repair.
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Sistemas CRISPR-Cas , Electroporación/métodos , Fertilización In Vitro/veterinaria , Edición Génica/métodos , Edición Génica/veterinaria , Oligodesoxirribonucleótidos , Mutación Puntual , Proteínas Proto-Oncogénicas p21(ras)/genética , Porcinos/embriología , Porcinos/genética , Cigoto , Animales , BlastocistoRESUMEN
Clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR/Cas9), derived from bacterial and archean immune systems, has received much attention from the scientific community as a powerful, targeted gene editing tool. The CRISPR/Cas9 system enables a simple, relatively effortless and highly specific gene targeting strategy through temporary or permanent genome regulation or editing. This endonuclease has enabled gene correction by taking advantage of the endogenous homology directed repair (HDR) pathway to successfully target and correct disease-causing gene mutations. Numerous studies using CRISPR support the promise of efficient and simple genome manipulation, and the technique has been validated in in vivo and in vitro experiments, indicating its potential for efficient gene correction at any genomic loci. In this chapter, we detailed various strategies related to gene editing using the CRISPR/Cas9 system. We also outlined strategies to improve the efficiency of gene correction via the HDR pathway and to improve viral and non-viral mediated gene delivery methods, with an emphasis on their therapeutic potential for correcting genetic disorder in humans and other mammals.
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Sistemas CRISPR-Cas , Edición Génica , Animales , Sistemas CRISPR-Cas/genética , Endonucleasas/genética , Endonucleasas/metabolismo , Marcación de Gen , Humanos , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
The ultimate goal of technology development in genome editing is to enable precisely targeted genomic changes in any cells or organisms. Here we describe protoplast systems for precise and efficient DNA sequence changes with preassembled Cas9 ribonucleoprotein (RNP) complexes in Arabidopsis thaliana, Nicotiana benthamiana, Brassica rapa, and Camelina sativa. Cas9 RNP-mediated gene disruption with dual gRNAs could reach â¼90% indels in Arabidopsis protoplasts. To facilitate facile testing of any Cas9 RNP designs, we developed two GFP reporter genes, which led to sensitive detection of nonhomologous end joining (NHEJ) and homology-directed repair (HDR), with editing efficiency up to 85 and 50%, respectively. When co-transfected with an optimal single-stranded oligodeoxynucleotide (ssODN) donor, precise editing of the AtALS gene via HDR reached 7% by RNPs. Significantly, precise mutagenesis mediated by preassembled primer editor (PE) RNPs led to 50% GFP reporter gene recovery in protoplasts and up to 4.6% editing frequency for the specific AtPDS mutation in the genome. The rapid, versatile and efficient gene editing by CRISPR RNP variants in protoplasts provides a valuable platform for development, evaluation and optimization of new designs and tools in gene and genomic manipulation and is applicable in diverse plant species.