Author Correction: A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines.
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ABSTRACT:
SUBMITTER: Rozen-Gagnon K
PROVIDER: S-EPMC8016948 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9-based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid-based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated U4.4 and Aag2 cell lines with reduced AGO1 expression. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.
| S-EPMC7804293 | biostudies-literature
Project description: Not available
| S-EPMC8433393 | biostudies-literature
Project description:An important advantage of delivering CRISPR reagents into cells as a ribonucleoprotein (RNP) complex is the ability to edit genes without reagents being integrated into the genome. Transient presence of RNP molecules in cells can reduce undesirable off-target effects. One method for RNP delivery into plant cells is the use of a biolistic gun. To facilitate selection of transformed cells during RNP delivery, a plasmid carrying a selectable marker gene can be co-delivered with the RNP to enrich for transformed/edited cells. In this work, we compare targeted mutagenesis in rice using three different delivery platforms: biolistic RNP/DNA co-delivery; biolistic DNA delivery; and Agrobacterium-mediated delivery. All three platforms were successful in generating desired mutations at the target sites. However, we observed a high frequency (over 14%) of random plasmid or chromosomal DNA fragment insertion at the target sites in transgenic events generated from both biolistic delivery platforms. In contrast, integration of random DNA fragments was not observed in transgenic events generated from the Agrobacterium-mediated method. These data reveal important insights that must be considered when selecting the method for genome-editing reagent delivery in plants, and emphasize the importance of employing appropriate molecular screening methods to detect unintended alterations following genome engineering.
| S-EPMC6934568 | biostudies-literature
Project description:[This corrects the article DOI: 10.1038/s41421-018-0028-z.].
| S-EPMC6411867 | biostudies-literature
Project description:CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4-20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.
| S-EPMC8179174 | biostudies-literature
Project description:A small number of HIV-1-infected individuals develop broadly neutralizing antibodies to the virus (bNAbs). These antibodies are protective against infection in animal models. However, they only emerge 1-3 yr after infection, and show a number of highly unusual features including exceedingly high levels of somatic mutations. It is therefore not surprising that elicitation of protective immunity to HIV-1 has not yet been possible. Here we show that mature, primary mouse and human B cells can be edited in vitro using CRISPR/Cas9 to express mature bNAbs from the endogenous Igh locus. Moreover, edited B cells retain the ability to participate in humoral immune responses. Immunization with cognate antigen in wild-type mouse recipients of edited B cells elicits bNAb titers that neutralize HIV-1 at levels associated with protection against infection. This approach enables humoral immune responses that may be difficult to elicit by traditional immunization.
| S-EPMC6547862 | biostudies-literature
Project description:Screening for successful CRISPR/Cas9 editing events remains a time consuming technical bottleneck in the field of Drosophila genome editing. This step can be particularly laborious for events that do not cause a visible phenotype, or those which occur at relatively low frequency. A promising strategy to enrich for desired CRISPR events is to co-select for an independent CRISPR event that produces an easily detectable phenotype. Here, we describe a simple negative co-selection strategy involving CRISPR-editing of a dominant female sterile allele, ovoD1 In this system ("ovoD co-selection"), the only functional germ cells in injected females are those that have been edited at the ovoD1 locus, and thus all offspring of these flies have undergone editing of at least one locus. We demonstrate that ovoD co-selection can be used to enrich for knock-out mutagenesis via nonhomologous end-joining (NHEJ), and for knock-in alleles via homology-directed repair (HDR). Altogether, our results demonstrate that ovoD co-selection reduces the amount of screening necessary to isolate desired CRISPR events in Drosophila.
| S-EPMC6071590 | biostudies-other
Project description:Genetically modified nonhuman primates (NHP) are useful models for biomedical research. Gene editing technologies have enabled production of target-gene knock-out (KO) NHP models. Target-gene-KO/knock-in (KI) efficiency of CRISPR/Cas9 has not been extensively investigated in marmosets. In this study, optimum conditions for target gene modification efficacies of CRISPR/mRNA and CRISPR/nuclease in marmoset embryos were examined. CRISPR/nuclease was more effective than CRISPR/mRNA in avoiding mosaic genetic alteration. Furthermore, optimal conditions to generate KI marmoset embryos were investigated using CRISPR/Cas9 and 2 different lengths (36 nt and 100 nt) each of a sense or anti-sense single-strand oligonucleotide (ssODN). KIs were observed when CRISPR/nuclease and 36 nt sense or anti-sense ssODNs were injected into embryos. All embryos exhibited mosaic mutations with KI and KO, or imprecise KI, of c-kit. Although further improvement of KI strategies is required, these results indicated that CRISPR/Cas9 may be utilized to produce KO/KI marmosets via gene editing.
| S-EPMC6722079 | biostudies-literature
Project description:Goat's milk, considered a substitute for cow's milk, has a high nutritional value. However, goat's milk contains various allergens, predominantly ?-lactoglobulin (BLG). In this study, we employed the CRISPR/Cas9 system to target the BLG locus in goat fibroblasts for sgRNA optimization and generate BLG knock-out goats through co-injection of Cas9 mRNA and small guide RNAs (sgRNAs) into goat embryos at the one-cell stage. We firstly tested sgRNA editing efficiencies in goat fibroblast cells, and approximately 8.00%-9.09% of the cells were modified in single sgRNA-guided targeting experiment. Among the kids, the genome-targeting efficiencies of single sgRNA were 12.5% (10 ng/?L sg1) and 0% (10 ng/?L sg2) and efficiencies of dual sgRNAs were 25.0% (25 ng/?L sg2+sg3 group) and 28.6% (50 ng/?L sg2+sg3 group). Relative expression of BLG in BLG knock-out goat mammary glands significantly (p < 0.01) decreased as well as other milk protein coding genes, such as CSN1S1, CSN1S2, CSN2, CSN3 and LALBA (p < 0.05). As expected, BLG protein had been abolished in the milk of the BLG knock-out goat. In addition, most of the targeted kids were chimeric (3/4), and their various body tissues were edited simultaneously. Our study thus provides a basis for optimizing the quality of goat milk, which can be applied to biomedical and agricultural research.
| S-EPMC5634636 | biostudies-literature
Project description:Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Technologies that facilitate specific and precise genome editing, such as knock-in, are critical for determining the functions of genes and for understanding fundamental biological processes. The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in mammals. Rosa26 gene can encode a non-essential nuclear RNA in almost all organizations, and become a hot point of exogenous gene insertion. Here, we describe efficient, precise CRISPR/Cas9-mediated Integration using a donor vector with tGFP sequence targeted in the sheep genomic Rosa26 locus. We succeeded in integrating with high efficiency an exogenous tGFP (turboGFP) gene into targeted genes in frame. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic sheep.
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