Mouse genomic editing 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 Ormd locus can lead to age related macula degeneration (AMD) in human, which is often manifested as vision loss at older age. A deletion mutation of the gene Ormd was engineered in mouse to generate potential model for the disease, which was created through the CRISPR/Cas9 technology by directly modifying the mouse genome in fertilized oocytes. In collaboration with Thomas Friedman in NIDCD we also developed point mutation knockin mutants for modeling human syndromic hearing loss conditions. 2) 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 knockout of the Sept2 gene, and many other targets for indel or deletion mutagenesis. During the past year, we have worked on 53 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 87 consultations for genome editing and other projects to investigators from NEI and many other labs on campus NIH wide * Designed and constructed 120 recombinant DNA clones through Gibson assembly for gene targeting in ES cells, CRISPR/Cas9-mediated gene knockin in mouse zygotes, and lentiviral construction. * Produced and characterized 114 guide RNAs by in vitro transcription assays for CRISPR-mediated genome editing. * performed 61 Surveyor assays for 16 chromosomal loci in pre-injection screening * performed 26 in vitro Cas9-guIde RNA mediated DNA double strand DNA break assays to evaluate efficiency of guide RNA-Cas9 interactions prior to injection *Designed and Performed 2800 genotyping assays by PCR to identify F0 founders of genome edited mice * Made 45 endotoxin-free, large scale DNA preparations of DNA constructs. * Conducted over 40 transfection assays on 16 gene targeting projects in ES cells, picked 8256 ES clones, expanded and cryopreserved over 8256 individual ES clones, expanded 42 positive ES clones and in addition, conducted at least 8256 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 128 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 128 reactions for 20 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, and achieved efficient germline transmission. * Fine-tuned our navel 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 submitted. * Provided training to several graduate students and postdoctoral fellows from NEI and other institutes over the subjects of genome editing and mouse ES cell technology. *Participated in teaching of 3 workshops on CRISPR technology and 1 workshop on mouse ES cell technology. *Collaborated with 2 transgenic core facilities on campus and outside NIH on various genome editing projects * microinjected 5 ES cell lines into mouse embryos to generate 92 chimeric mice * microinjected 48 CRISPR/Cas9 constructs into fertilized mouse oocytes for 109 injection sessions to produce 127 mutant mice (67 knockin and 60 knockout) out of 906 F0 founder mice. * isolated DNA from 13,759 mouse tail biopsy samples * performed 8,041 PCR reactions to genotype mice in the facility * set up 2,975 matings to propagate mouse lines * completed or oversaw weaning, tagging, and tail biopsy of 12,601 mice born in the facility * made 655 mouse deliveries to researchers' labs * provided 184 consultations to researchers on breeding strategies * rederived 4 mouse lines * worked on cryopreservation of 40 mouse lines freezing 3,572 mouse embryos at the two cell stage, and 480 straws of sperm * cryopreserved 9 lines of zebrafish as frozen sperm and validated 7 lines. Cryopreserved testes from 2 lines of zebrafish * performed assisted reproduction to save 23 mouse lines from extinction and/or reconstitute mouse lines from frozen stock * validated 38 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 (NINDS, NICHD, NIDCD, NIAMS and NCI) and 1 investigator at Jon Hopkins University.
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