One-step homozygosity in precise gene editing by an improved CRISPR/Cas9 system.
Ontology highlight
ABSTRACT:
SUBMITTER: Zhao P
PROVIDER: S-EPMC4856768 | biostudies-literature | 2016 May
REPOSITORIES: biostudies-literature
Publications
One-step homozygosity in precise gene editing by an improved CRISPR/Cas9 system.
Zhao Pei P Zhang Zhe Z Lv Xiaoying X Zhao Xiang X Suehiro Yuji Y Jiang Yinan Y Wang Xinquan X Mitani Shohei S Gong Haipeng H Xue Ding D
Cell research 20160408 5
Similar Datasets
Project description:Pig shows multiple superior characteristics in anatomy, physiology, and genome that have made this species to be more suitable models for human diseases, especially for neurodegenerative diseases, because they have similar cerebral convolutions compared with human neocortex. Recently, CRISPR/Cas9 system shows enormous potential for engineering the pig genome. In this study, we expect to generate human Parkinson's disease pig model using CRISPR/Cas9 system by simultaneously targeting three distinct genomic loci, parkin/DJ-1/PINK1, in Bama miniature pigs. By co-injection of Cas9 mRNA and multiplexing single guide RNAs (sgRNAs) targeting parkin, DJ-1, and PINK1 genes, respectively, into in vivo derived pronuclear embryos, we simultaneously targeted three distinct genomic loci. The gene modified piglets remain healthy and display normal behavior at the age of 10 months. In addition, despite the high number of sgRNAs were employed in the present study, our trio-based whole-genome sequencing analysis suggested that the incidence of off-target events is low. Our results demonstrate that the simplicity, efficiency, and power of the CRISPR/Cas9 system to allow for the modification of multiple genes in pigs and yield results of high medical value.
| S-EPMC4746670 | biostudies-literature
Project description:The CRISPR/Cas9 system is a powerful, revolutionary tool for genome editing. However, it is not without limitations. There are PAM-free and CRISPR-tolerant regions that cannot be modified by the standard CRISPR/Cas9 system, and off-target activity impedes its broader applications. To avoid these drawbacks, we developed a very simple CRISPR/Cas9-assisted gRNA-free one-step (CAGO) genome editing technique which does not require the construction of a plasmid to express a specific gRNA. Instead, a universal N20 sequence with a very high targeting efficiency is inserted into the E. coli chromosome by homologous recombination, which in turn undergoes a double-stranded break by CRISPR/Cas9 and induces an intra-chromosomal recombination event to accomplish the editing process. This technique was shown to be able to edit PAM-free and CRISPR-tolerant regions with no off-target effects in Escherichia coli. When applied to multi-locus editing, CAGO was able to modify one locus in two days with a near 100% editing efficiency. Furthermore, modified CAGO was used to edit large regions of up to 100 kbp with at least 75% efficiency. Finally, genome editing by CAGO only requires a transformation procedure and the construction of a linear donor DNA cassette, which was further simplified by applying a modular design strategy. Although the technique was established in E. coli, it should be applicable to other organisms with only minor modifications.
| S-EPMC5709385 | biostudies-literature
Project description:Current methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.
| S-EPMC7925485 | biostudies-literature
Project description:The introduction of engineered site-specific DNA endonucleases has brought precise genome editing in many model organisms and human cells into the realm of possibility. In zebrafish, loss-of-function alleles have been successfully produced; however, germ line transmission of functional targeted knock-ins of protein tags or of SNP exchanges have not been reported. Here we show by phenotypic rescue that the CRISPR/Cas system can be used to target and repair a premature stop codon at the albino (alb) locus in zebrafish with high efficiency and precision. Using circular donor DNA containing CRISPR target sites we obtain close to 50% of larvae with precise homology-directed repair of the alb(b4) mutation, a small fraction of which transmitted the repaired allele in the germ line to the next generation (3/28 adult fish). The in vivo demonstration of germ line transmission of a precise SNP exchange in zebrafish underscores its suitability as a model for genetic research.
| S-EPMC4299274 | biostudies-literature
Project description:BackgroundTetracycline (Tet)-regulated expression system has become a widely applied tool to control gene activity. This study aimed to improve the Tet-on system with superior regulatory characteristics.ResultsBy comprehensively comparing factors of transactivators, Tet-responsive elements (TREs), orientations of induced expression cassette, and promoters controlling the transactivator, we developed an optimal Tet-on system with enhanced inducible efficiency and lower leakiness. With the system, we successfully performed effective inducible and reversible expression of microRNA, and presented a more precise and easily reproducible fine-tuning for confirming the target of a miRNA. Finally, the system was applied in CRISPR/Cas9-mediated knockout of nuclear factor of activated T cells-5 (NFAT5), a protective transcription factor in cellular osmoregulation.ConclusionsThis study established an improved Tet-on system for powerful and stringent gene regulation in functional genetic studies.
| S-EPMC6556963 | biostudies-literature
Project description:The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.
| S-EPMC5518993 | biostudies-literature
Project description:Genome engineering with designer nucleases is a rapidly progressing field, and the ability to correct human gene mutations in situ is highly desirable. We employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. We show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, because of its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates. To assess the off-target effects, we used both a predictive software platform to identify intragenic sequences of homology as well as a genome-wide screen utilizing linear amplification-mediated PCR. We observed no off-target activity and show RNA-guided endonuclease candidate sites that do not possess low sequence complexity function in a highly specific manner. Collectively, we provide proof of principle for precision genome editing in Fanconi anemia, a DNA repair-deficient human disorder.
| S-EPMC4326027 | biostudies-literature
Project description:BackgroundPrecise genetic modifications are preferred products of CRISPR-Cas9 mediated gene editing in mammalian cells but require the repair of induced double-strand breaks (DSB) through homology directed repair (HDR). Since HDR competes with the prevailing non-homologous end joining (NHEJ) pathway and depends on the presence of repair templates its efficiency is often limited and demands optimized methodology.ResultsFor the enhancement of HDR we redirect the DSB repair pathway choice by targeting the Ubiquitin mark for damaged chromatin at Histone H2A-K15. We used fusions of the Ubiquitin binding domain (UBD) of Rad18 or RNF169 with BRCA1 to promote HDR initiation and UBD fusions with DNA binding domains to attract donor templates and facilitate HDR processing. Using a traffic light reporter system in human HEK293 cells we found that the coexpression of both types of UBD fusion proteins promotes HDR, reduces NHEJ and shifts the HDR/NHEJ balance up to 6-fold. The HDR enhancing effect of UBD fusion proteins was confirmed at multiple endogenous loci.ConclusionsOur findings provide a novel efficient approach to promote precise gene editing in human cells.
| S-EPMC7585302 | biostudies-literature
Project description:Human induced pluripotent stem cells (hiPSCs) have been extensively used in the fields of developmental biology and disease modeling. CRISPR/Cas9 gene editing in iPSC lines often has a low frequency, which hampers its application in precise allele editing of disease-associated single nucleotide polymorphisms (SNPs), especially those in the noncoding parts of the genome. Here, we present a unique workflow to engineer isogenic iPSC lines by SNP editing from heterozygous to homozygous for disease risk alleles or non-risk alleles using a transient and straightforward transfection-based protocol. This protocol enables us to simultaneously obtain pure and clonal isogenic lines of all three possible genotypes of a SNP site within about 4 to 5 weeks.
| S-EPMC8260261 | biostudies-literature
Project description:The medicinal mushroom Cordyceps militaris contains abundant valuable bioactive ingredients that have attracted a great deal of attention in the pharmaceutical and cosmetic industries. However, the development of this valuable mushroom faces the obstacle of lacking powerful genomic engineering tools. Here, by excavating the endogenous tRNA-processed element, introducing the extrachromosomal plasmid and alongside with homologous template, we develop a marker-free CRISPR-Cas9-TRAMA genomic editing system to achieve the multiplex gene precise editing and large synthetic cluster deletion in C. militaris. We further operated editing in the synthetases of cordycepin and ergothioneine to demonstrate the application of Cas9-TRAMA system in protein modification, promoter strength evaluation and 10 kb metabolic synthetic cluster deletion. The Cas9-TRAMA system provides a scalable method for excavating the valuable metabolic resource of medicinal mushrooms and constructing a mystical cellular pathway to elucidate the complex cell behaviours of the edible mushroom.
| S-EPMC9733643 | biostudies-literature