Genomic manipulation projects at GEC this year are from the following scientific areas: 1) Human disease modeling: It is often desirable to have mutations of human genetic conditions replicated in mouse so that the diseases can be modeled. In the last year, several of such models were developed at GEC in collaboration with a few investigators at NEI. Mutations in the Cep290 locus can lead to Leber's congenital amaurosis (LCA) in human, which is often manifested as vision loss. Two frequently encountered disease-generating point mutations in human were engineered into the Cep290 locus in mouse to generate potential models for the disease, which were created through the CRISPR/Cas9 technology by directly modifying the mouse genome in oocytes with no ES cell manipulation necessary. In collaboration with Dr. J. Fielding Hejtmancik's laboratory at NEI, we also produced three point mutations of the Kcnj13 gene, a candidate for dominant and recessive forms of snowflake vitreoretinal degeneration in human. 2) Reverse genetic confirmation of disease candidate genes: The core supports the disease gene discovery programs by making knockout and knockin models of the candidate genes to establish direct linkage between the genes identified in forward genetic screens and the phenotypic manifestations against which the screens were conducted. During the past year, one project was conducted in this area, generating the knockin mutation CDC14a C278S for a human inherited defenses disease. 3) Functional genomic studies of genes predicted to be important in physiology and pathology: Most of the current genome editing projects aimed at simply understanding the functions of various genes relevant to NEI, as well as other participating IC research programs. Work in this area currently includes the characterization of the miR183 cluster knockout, which is involved in sensory neuron function, and deletion of which has displayed phenotypes of retinal dysfunctions; completion of the construction of the conditional knockin of the human CRH enhancer element into the mouse CRH locus, and many other targets for indel mutagenesis. During the past year, we have worked on 50 different gene targeting projects at various stages. In order to achieve mutagenesis goals of all the projects above, we have conducted the following experimental procedures: *Provided 78 consultations for genome editing and other projects by meeting the investigators from NEI and many other labs on campus * Designed and constructed 58 recombinant DNA clones through Gibson assembly for gene targeting in ES cells, CRISPR/Cas9-mediated gene knockin in mouse zygotes, and lentiviral construction. *Cloned 254 guide RNA constructs for T7 in vitro transcription reactions * Produced and characterized 136 guide RNAs by in vitro transcription assays for CRISPR-mediated genome editing. * performed numerous Surveyor assays for 136 guide RNAs for pre-injection screening * performed numerous in vitro Cas9-guIde RNA mediated DNA double strand DNA break assays for 136 guide RNAs to evaluate efficiency of guide RNA-Cas9 interactions prior to injection *Designed and Performed 2858 genotyping assays by PCR to identify F0 founders of genome edited mice * Made 70 endotoxin-free, large scale DNA preparations of DNA constructs. * Conducted over 200 transfection assays on 11 gene targeting projects in ES cells, picked 7176 ES clones, expanded and cryopreserved over 8208 individual ES clones, expanded 52 positive ES clones and in addition, conducted at least 6144 genomic DNA extractions and the same number of genomic PCR reactions for screening of targeted mutations. * Made 70 endotoxin-free, large scale DNA preparations of DNA constructs. * Conducted over 200 transfection assays on 11 gene targeting projects in ES cells, picked 7176 ES clones, expanded and cryopreserved over 8208 individual ES clones, expanded 52 positive ES clones and in addition, conducted at least 6144 genomic DNA extractions and the same number of genomic PCR reactions for screening of targeted mutations. * Improved methodology of CRISPR reagent preparation for microinjection to reduce RNA precipitation and increase success rate for injection; prepared 133 CRISPR samples for injection. * Optimized CRISPR-mediated genome editing in mouse zygotes with CRSIPR recombinant protein based RNP methodology *Adapted and improved single strand DNA oligo-mediated homologous recombination for point mutation and small tag engineering directly in mouse zygotes by testing the asymmetric cargo methodology and reached high efficiency of HDR by 70% or better, greatly reduced the time required for the tasks engineering point mutations or small tag knockin * Adapted digital PRC technology and performed 10 dPCR assays to detect point mutations, genomic deletions and indel mutation to evaluate efficiencies of various strategies of genome editing * Established a Tet-inducible Cas9-GFP knockin allele in R1 ES cells at the Rosa26 locus to use as a efficient tool for genome editing * Established and finalized a novel approach to introduce knockin alleles such as a reporter allele or an epitope tag in R1 ES cells without the need of a selection cassette with CRISPR technology. Manuscript in preparation. * Designed and engineered a novel and convenient cloning vector for guide RNA cloning and synthesis, and distributed the vector to labs of several institutes. * Provided training to several graduate students and post doctoral fellows from NEI and other institutes over the subjects of genome editing, cellular reprogramming and Lentviral technology. *Participated in teaching of 3 workshops on CRISPR technology. *Collaborated with 4 transgenic core facility on campus and outside NIH on microinjection tasks by sending additional constructs to those facilities for microinjection * microinjected 6 ES cell lines into mouse embryos to generate 7 chimeric mice * microinjected 22 CRISPR/Cas9 constructs into fertilized mouse oocytes for 84 injection sessions to produce 103 mutant mice * isolated DNA from 14,205 mouse tail biopsy samples * performed 10,332 PCR reactions to genotype mice in the facility * set up 3,257 matings to propagate mouse lines * completed or oversaw weaning, tagging, and tail biopsy of 12,402 mice born in the facility * made 687 mouse deliveries to researchers' labs * provided 162 consultations to researchers on breeding strategies * rederived 9 mouse lines * worked on cryopreservation of 76 mouse lines freezing 6602 mouse embryos at the two cell stage, and 800 straws of sperm * cryopreserved 2 lines of zebrafish as frozen sperm and validated 2 lines. Cryopreserved testes from 1 lines of zebrafish * performed assisted reproduction to save 20 mouse lines from extinction and/or reconstitute mouse lines from frozen stock * validated 47 lines of frozen mouse germplasm (sperm and embryos) These services and collaborative services were performed for 17 PIs from 5 NEI labs (LI, LRCMB, N-NRL, OGVFB, OSD), plus 8 PIs from 5 other institutes at NIH (NHLBI, NINDS, NICHD, NIMH, and NCCIH) and 1 investigator at Jon Hopkins University.
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