• Cas9-free line
• lincRNA
• loss of function
• soybean
• β-conglycinin deficiency

• Seed Storage Proteins/genetics
• Seed Storage Proteins/chemistry
• Globulins/genetics
• Globulins/metabolism
• Globulins/chemistry
• Glycine max/genetics
• Glycine max/metabolism
• Antigens, Plant/genetics
• Antigens, Plant/chemistry
• Soybean Proteins/genetics
• Soybean Proteins/metabolism
• Soybean Proteins/chemistry
• CRISPR-Cas Systems
• Gene Editing
• RNA, Long Noncoding/genetics
• Gene Expression Regulation, Plant
• Seeds/genetics
• Seeds/metabolism
• Seeds/chemistry

• 5hmC
• DNA methylation
• cell cycle
• chromatin
• immunofluorescence
• mouse ESCs

• 5-Methylcytosine
• Animals
• DNA
• DNA Methylation
• Demethylation
• Embryonic Development
• Female
• Mice
• Mouse Embryonic Stem Cells
• Pregnancy

• PJT-165992 CIHR

• Animals
• Bone Development/genetics
• CRISPR-Cas Systems
• Cells, Cultured
• Gene Expression Regulation, Developmental
• Gene Targeting/methods
• Genetic Testing/methods
• Haploidy
• Hematopoietic Cell Growth Factors/genetics
• Hematopoietic Cell Growth Factors/metabolism
• Homeodomain Proteins/genetics
• Homeodomain Proteins/metabolism
• Lectins, C-Type/genetics
• Lectins, C-Type/metabolism
• Mice
• Mice, Knockout
• Mouse Embryonic Stem Cells/metabolism
• Relaxin/genetics
• Relaxin/metabolism
• Transcription Factors/genetics
• Transcription Factors/metabolism

• Chinese Academy of Sciences
• National Natural Science Foundation of China
• Shanghai Municipal Commission for Science and Technology
• Chinese Ministry of Science and Technology
• Strategic Priority Research Program of the Chinese Academy of Sciences

In Japan, it is possible to generate chimeric animals from specified embryos by combining animal blastocysts with human pluripotent stem (PS) cells (animal-human PS chimera). However, the production of animal-human PS chimeras has been restricted because of ethical concerns, such as the development of human-like intelligence and formation of humanized gametes in the animals, owing to the contributions of human PS cells to the brain and reproductive organs. To solve these problems, we established a novel blastocyst complementation technology that does not contribute to the gametes or the brain. First, we established GFP-expressing mouse embryonic stem cells (G-mESCs) in which the Prdm14 and Otx2 genes were knocked out and generated chimeric mice by injecting them into PDX-1-deficient blastocysts. The results showed that the G-mESCs did not contribute to the formation of gametes and the brain. Therefore, in the PDX-1-deficient mice complemented by G-mESCs without the Prdm14 and Otx2 genes, the germline was not transmitted to the next generations. This approach could address concerns regarding the development of both human gametes and a human-like brain upon mouse blastocyst complementation using human stem cells.

• Otx2
• Prdm14
• blastocyst complementation

Genome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. We present and characterize a robust method for rapid, scarless introduction or correction of disease-associated variants in hPSCs using CRISPR/Cas9. Utilizing non-integrated plasmid vectors that express a puromycin N-acetyl-transferase (PAC) gene, whose expression and translation is linked to that of Cas9, we transiently select for cells based on their early levels of Cas9 protein. Under optimized conditions, co-delivery with single-stranded donor DNA enabled isolation of clonal cell populations containing both heterozygous and homozygous precise genome edits in as little as 2 weeks without requiring cell sorting or high-throughput sequencing. Edited cells isolated using this method did not contain any detectable off-target mutations and displayed expected functional phenotypes after directed differentiation. We apply the approach to a variety of genomic loci in five hPSC lines cultured using both feeder and feeder-free conditions.

•

Stringent transient puromycin selection enriches for hPSCs with scarless genome edits

• CRISPR/Cas9
• disease modeling
• genome editing
• human pluripotent stem cells
• puromycin
• scarless
• transient selection

The combination of genome-edited human embryonic stem cells (hESCs) and subsequent neural differentiation is a powerful tool to study neurodevelopmental disorders. Since the naïve state of pluripotency has favourable characteristics for efficient genome-editing, we optimized a workflow for the CRISPR/Cas9 system in these naïve stem cells. Editing efficiencies of respectively 1.3–8.4% and 3.8–19% were generated with the Cas9 nuclease and the D10A Cas9 nickase mutant. Next to this, wildtype and genome-edited naïve hESCs were successfully differentiated to neural progenitor cells. As a proof-of-principle of our workflow, two monoclonal genome-edited naïve hESCs colonies were obtained for TUNA, a long non-coding RNA involved in pluripotency and neural differentiation. In these genome-edited hESCs, an effect was seen on expression of TUNA, although not on neural differentiation potential. In conclusion, we optimized a genome-editing workflow in naïve hESCs that can be used to study candidate genes involved in neural differentiation and/or functioning.

• Diploidization
• Genetic screening
• Haploid
• Pluripotency

In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a β₂m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β₂m knockout (KO) clones did not give rise to tumors in syngeneic mice (C57BL/6N), unless NK cells were depleted, suggesting that outgrowth of the β₂m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IA^b molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.

• Animals
• Cell Line, Tumor
• Clustered Regularly Interspaced Short Palindromic Repeats
• Enzyme-Linked Immunosorbent Assay
• Major Histocompatibility Complex/immunology
• Mice
• Mice, Inbred C57BL
• Mice, Knockout
• beta 2-Microglobulin/genetics

• Deutsches Krebsforschungszentrum (DE)

• Adult
• Aged
• Animals
• Cartilage, Articular/cytology
• Cartilage, Articular/metabolism
• Cartilage, Articular/pathology
• Cells, Cultured
• Chondrocytes/cytology
• Chondrocytes/metabolism
• DNA Methylation
• Fibroblasts/cytology
• Fibroblasts/metabolism
• Humans
• Matrix Metalloproteinase 3/genetics
• Matrix Metalloproteinase 3/metabolism
• Mice
• Middle Aged
• Nuclear Proteins/genetics
• Nuclear Proteins/metabolism
• Osteoarthritis, Knee/metabolism
• Osteoarthritis, Knee/pathology
• Promoter Regions, Genetic
• Proto-Oncogene Proteins/antagonists & inhibitors
• Proto-Oncogene Proteins/genetics
• Proto-Oncogene Proteins/metabolism
• RNA Interference
• RNA, Small Interfering/metabolism
• Tumor Necrosis Factor-alpha/pharmacology
• Twist-Related Protein 1/genetics
• Twist-Related Protein 1/metabolism
• Up-Regulation/drug effects

• R56 AR050631 NIAMS NIH HHS
• R01 AG049617 NIA NIH HHS
• R01 AR050631 NIAMS NIH HHS
• R01 AR065379 NIAMS NIH HHS
• P01 AG007996 NIA NIH HHS

• Animals
• Apoptosis Regulatory Proteins
• CRISPR-Cas Systems
• Chimera/genetics
• Embryonic Stem Cells/metabolism
• Female
• Gene Deletion
• Gene Editing/methods
• Gene Knock-In Techniques
• Genes, Lethal
• HSP40 Heat-Shock Proteins/genetics
• Hydrocephalus/genetics
• Hydrocephalus/pathology
• INDEL Mutation
• Male
• Mice
• Mice, Inbred C57BL
• Mice, Inbred DBA
• Mice, Inbred ICR
• Mice, Transgenic
• Molecular Chaperones
• Point Mutation
• Pregnancy
• RNA, Guide, CRISPR-Cas Systems/genetics
• Sperm Motility/genetics
• Spermatozoa/abnormalities

• CRISPR/Cas9
• Genetic modification
• Haploid embryonic stem cells
• Mouse

• Animals
• Base Sequence
• CRISPR-Cas Systems/genetics
• Embryonic Stem Cells/metabolism
• Gene Editing/methods
• Haploidy
• Humans
• Mice

Vertebrate genomes are highly methylated at cytosine residues in CpG sequences. CpG methylation plays an important role in epigenetic gene silencing and genome stability. Compared with other epigenetic modifications, CpG methylation is thought to be relatively stable; however, it is sometimes affected by environmental changes, leading to epigenetic instability and disease. CpG methylation is reversible and regulated by DNA methyltransferases and demethylases including ten-eleven translocation. Here, we discuss CpG methylation instability and the regulation of CpG methylation by DNA methyltransferases and ten-eleven translocation in pluripotent stem cells.

• CRISPR/Cas9
• Clonal selection
• Dot blot
• Screening

• Base Sequence
• CRISPR-Cas Systems
• Cell Line
• ELAV-Like Protein 1/chemistry
• ELAV-Like Protein 1/genetics
• Gene Knockout Techniques
• Gene Targeting/methods
• Genotype
• Humans
• Immunoblotting
• Molecular Sequence Data
• Mutation
• RNA, Guide, CRISPR-Cas Systems/chemistry
• RNA, Guide, CRISPR-Cas Systems/genetics
• Sequence Alignment

• S10 OD016290 NIH HHS
• UCR initial complement funds
• California Alliance for Minority Participation

• engineered nucleases
• gene correction
• genome editing
• inversion
• short nucleotide repeat
• translocation

• Animals
• Biological Science Disciplines/trends
• Clustered Regularly Interspaced Short Palindromic Repeats/physiology
• Disease Models, Animal
• Gene Editing/history
• Gene Editing/methods
• Gene Editing/trends
• Gene Targeting/methods
• Genetic Therapy/history
• Genetic Therapy/methods
• Genetic Therapy/trends
• History, 21st Century
• Humans
• Medicine/trends

• Animals
• CRISPR-Cas Systems
• Embryonic Stem Cells/cytology
• Embryonic Stem Cells/enzymology
• Embryonic Stem Cells/metabolism
• Gene Editing/trends
• Gene Knock-In Techniques/trends
• Gene Knockout Techniques/trends
• Humans
• Induced Pluripotent Stem Cells/cytology
• Induced Pluripotent Stem Cells/enzymology
• Induced Pluripotent Stem Cells/metabolism
• Mice
• Mice, Knockout/genetics
• Mice, Knockout/metabolism
• Models, Genetic
• Mosaicism
• Pluripotent Stem Cells/cytology
• Pluripotent Stem Cells/enzymology
• Pluripotent Stem Cells/metabolism

Haploid cells are useful for studying gene functions because disruption of a single allele can cause loss-of-function phenotypes. Recent success in generating haploid embryonic stem cells (ESCs) in mice, rats, and monkeys provides a new platform for simple genetic manipulation of the mammalian genome. Use of haploid ESCs enhances the genome-editing potential of the CRISPR/Cas system. For example, CRISPR/Cas was used in haploid ESCs to generate multiple knockouts and large deletions at high efficiency. In addition, genome-wide screening is facilitated by haploid cell lines containing gene knockout libraries.

• CRISPR/Cas
• embryonic stem cell
• haploid

• Alleles
• Animals
• Base Sequence
• CRISPR-Cas Systems/genetics
• Cloning, Organism
• DNA Methylation/genetics
• Gene Library
• Genetic Testing
• Haploidy
• Heterozygote
• Mice
• Mice, Inbred C57BL
• Mice, Knockout
• Mice, Mutant Strains
• Molecular Sequence Data
• Mouse Embryonic Stem Cells/metabolism
• RNA, Guide, CRISPR-Cas Systems/metabolism

• 095606 Wellcome Trust
• GM051279 NIGMS NIH HHS

CRISPR system is a powerful defense mechanism in bacteria and archaea to provide immunity against viruses. Recently, this process found a new application in intended targeting of the genomes. CRISPR-mediated genome editing is performed by two main components namely single guide RNA and Cas9 protein. Despite the enormous data generated in this area, there is a dearth of high throughput resource. Therefore, we have developed CrisprGE, a central hub of CRISPR/Cas-based genome editing. Presently, this database holds a total of 4680 entries of 223 unique genes from 32 model and other organisms. It encompasses information about the organism, gene, target gene sequences, genetic modification, modifications length, genome editing efficiency, cell line, assay, etc. This depository is developed using the open source LAMP (Linux Apache MYSQL PHP) server. User-friendly browsing, searching facility is integrated for easy data retrieval. It also includes useful tools like BLAST CrisprGE, BLAST NTdb and CRISPR Mapper. Considering potential utilities of CRISPR in the vast area of biology and therapeutics, we foresee this platform as an assistance to accelerate research in the burgeoning field of genome engineering.

Database URL: http://crdd.osdd.net/servers/crisprge/.

• embryonic stem cells
• genetic screening
• haploid cells
• transgenic mice

The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been successfully used for genome editing in a variety of organisms. Here, we report the use of dual sgRNA-guided Cas9 nuclease to generate knockout mutants of protein coding genes, noncoding genes, and repetitive sequences in C. elegans. Co-injection of C. elegans with dual sgRNAs results in the removal of the interval between two sgRNAs and the loss-of-function phenotype of targeted genes. We sought to determine how large an interval can be eliminated and found that at least a 24 kb chromosome segment can be deleted using this dual sgRNA/Cas9 strategy. The deletion of large chromosome segments facilitates mutant screening by PCR and agarose electrophoresis. Thus, the use of the CRISPR/Cas9 system in combination with dual sgRNAs provides a powerful platform with which to easily generate gene knockout mutants in C. elegans. Our data also suggest that encoding multiple sgRNA sequences into a single CRISPR array to simultaneously edit several sites within the genome may cause the off-target deletion of chromosome sequences.

Near-haploid human cell lines are instrumental for genetic screens and genome engineering as gene inactivation is greatly facilitated by the absence of a second gene copy. However, no completely haploid human cell line has been described, hampering the genetic accessibility of a subset of genes. The near-haploid human cell line HAP1 contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15. This large fragment encompasses 330 genes and is integrated on the long arm of Chromosome 19. Here, we employ a CRISPR/Cas9-based genome engineering strategy to excise this sizeable chromosomal fragment and to efficiently and reproducibly derive clones that retain their haploid state. Importantly, spectral karyotyping and single-nucleotide polymorphism (SNP) genotyping revealed that engineered-HAPloid (eHAP) cells are fully haploid with no gross chromosomal aberrations induced by Cas9. Furthermore, whole-genome sequence and transcriptome analysis of the parental HAP1 and an eHAP cell line showed that transcriptional changes are limited to the excised Chromosome 15 fragment. Together, we demonstrate the feasibility of efficiently engineering megabase deletions with the CRISPR/Cas9 technology and report the first fully haploid human cell line.

• Disease model
• Gene editing
• Induced pluripotent stem cells
• Regenerative medicine
• Reprogramming technique

• Animals
• Cellular Reprogramming
• Drug Evaluation, Preclinical
• Gene Targeting
• Humans
• Induced Pluripotent Stem Cells/cytology
• Induced Pluripotent Stem Cells/transplantation
• Mice
• Models, Biological
• Regenerative Medicine

Recently, an epoch-making genome engineering technology using clustered regularly at interspaced short palindromic repeats (CRISPR) and CRISPR associated (Cas) nucleases, was developed. Previous technologies for genome manipulation require the time-consuming design and construction of genome-engineered nucleases for each target and have, therefore, not been widely used in mouse research where standard techniques based on homologous recombination are commonly used. The CRISPR/Cas system only requires the design of sequences complementary to a target locus, making this technology fast and straightforward. In addition, CRISPR/Cas can be used to generate mice carrying mutations in multiple genes in a single step, an achievement not possible using other methods. Here, we review the uses of this technology in genetic analysis and manipulation, including achievements made possible to date and the prospects for future therapeutic applications.

• Clustered regularly at interspaced short palindromic repeats
• Clustered regularly at interspaced short palindromic repeats associated 9
• Genome engineering
• Double-strand breaks
• Non-homologous end joining
• Homology-directed repair

• CRISPR-Cas9 system
• CRISPR-Cas9-induced indels
• CRISPR-Cas9-mediated HR
• Co-CRISPR
• blasticidin selection

The CRISPR/Cas system, in which the Cas9 endonuclease and a guide RNA complementary to the target are sufficient for RNA-guided cleavage of the target DNA, is a powerful new approach recently developed for targeted gene disruption in various animal models. However, there is little verification of microinjection methods for generating knockout mice using this approach. Here, we report the verification of microinjection methods of the CRISPR/Cas system. We compared three methods for injection: (1) injection of DNA into the pronucleus, (2) injection of RNA into the pronucleus, and (3) injection of RNA into the cytoplasm. We found that injection of RNA into the cytoplasm was the most efficient method in terms of the numbers of viable blastocyst stage embryos and full-term pups generated. This method also showed the best overall knockout efficiency.