Tyrosinase is the rate-limiting enzyme for the production of melanin pigmentation. In the mouse and other animals, homozygous null mutations in the Tyrosinase gene (Tyr) result in the absence of pigmentation, i.e. albinism. Here we used the CRISPR/Cas9 system to generate mono- and bi-allelic null mutations in the Tyr locus by zygote injection of two single-guide and Cas9 RNAs. Injection into C57BL/6N wild-type embryos resulted in one completely albino founder carrying two different Tyr mutations. In addition, three pigmentation mosaics and fully pigmented littermates were obtained that transmitted new mutant Tyr alleles to progeny in test crosses with albinos. Injection into Tyr heterozygous (B6CBAF1/J × FVB/NJ) zygotes resulted in the generation of numerous albinos and also mice with a graded range of albino mosaicism. Deep sequencing revealed that the majority of the albinos and the mosaics had more than two new mutant alleles. These visual phenotypes and molecular genotypes highlight the somatic mosaicism and allele complexity in founders that occurs for targeted genes during CRISPR/Cas9-mediated mutagenesis by zygote injection in mice.

Abstract. We have identified three DNase I-hypersensitive sites in chromatin between 15 and 17 kb upstream of the mouse proa2(I) collagen gene. These sites were detected in cells that produce type I collagen but not in cells that do not express these genes. A construction containing the sequences from -17 kb to +54 bp of the mouse proa2(I) collagen gene, cloned upstream of either the Escherichia coli 13-galactosidase or the firefly luciferase reporter gene, showed strong enhancer activity in transgenic mice when compared with the levels seen previously in animals harboring shorter promoter fragments. Especially high levels of expression of the reporter gene were seen in dermis, fascia, and the fibrous layers of many internal organs. High levels of expression could also be detected in some osteoblastic cells. When various fragments of the 5' flanking sequences were cloned upstream of the 350-bp proximal proet2(I) collagen promoter linked to the lacZ gene, the cis-acting elements responsible for enhancement were localized in the region between -13.5 and -19.5 kb, the same region that contains the three DNase I-hypersensitive sites. Moreover, the DNA segment from -13.5 to -19.5 kb was also able to drive the cell-specific expression of a 220-bp mouse proal(I) collagen promoter, which is silent in transgenic mice. Hence, our data suggest that a far-upstream enhancer element plays a role in regulating high levels of expression of the mouse pro~2(I) collagen gene.T HE genetic programs that control the differentiation of fibroblasts and osteoblasts are still poorly understood. We are using the type I collagen genes as markers for these cell lineages to better understand the mechanisms by which they express their phenotypes. The type I collagen genes are expressed at high levels in osteoblasts, in odontoblasts, and in fibroblasts of tendons and skin, whereas in other tissues, their expression is considerably lower and is due mainly to the presence of fibroblasts and mesenchymal cells in these tissues. Type I collagen belongs to the fibrillar class of collagens and is composed of two al(I) chains and one a2(I) chain (Cole, 1994;Philajaniemi and Rehn, 1995, and references therein for review of collagen types and functions). Excessive amounts of these proteins along with other extracellular matrix proteins are found in fibrotic diseases including cirrhosis, glomerulosclerosis, and scleroderma.

The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are still poorly understood. We have used the gene for a chondrocyte marker, the pro␣1(II) collagen gene (Col2a1), as a model to delineate a minimal sequence needed for chondrocyte expression and identify chondrocyte-specific proteins binding to this sequence. We previously localized a cartilage-specific enhancer to 156 bp of the mouse Col2a1 intron 1. We show here that four copies of a 48-bp subsegment strongly increased promoter activity in transiently transfected rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not in 10T1/2 fibroblasts. They also directed cartilage specificity in transgenic mouse embryos. These 48 bp include two 11-bp inverted repeats with only one mismatch. Tandem copies of an 18-bp element containing the 3 repeat strongly enhanced promoter activity in RCS cells and chondrocytes but not in fibroblasts. Transgenic mice harboring 12 copies of this 18-mer expressed luciferase in ribs and vertebrae and in isolated chondrocytes but not in noncartilaginous tissues except skin and brain. In gel retardation assays, an RCS cell-specific protein and another closely related protein expressed only in RCS cells and primary chondrocytes bound to a 10-bp sequence within the 18-mer. Mutations in these 10 bp abolished activity of the multimerized 18-bp enhancer, and deletion of these 10 bp abolished enhancer activity of 465-and 231-bp intron 1 segments. This sequence contains a low-affinity binding site for POU domain proteins, and competition experiments with a high-affinity POU domain binding site strongly suggested that the chondrocyte proteins belong to this family. Together, our results indicate that an 18-bp sequence in Col2a1 intron 1 controls chondrocyte expression and suggest that RCS cells and chondrocytes contain specific POU domain proteins involved in enhancer activity.Acquisition of the chondrocyte phenotype by mesenchymal cells is one of the major pathways of differentiation of these cells. Chondrocytes form several types of cartilages including the growth plate cartilages essential to skeletal formation and cartilages that have supporting roles and persist throughout adult life such as the articular cartilages and the cartilages of the nose, ear, and trachea. Chondrocyte differentiation presumably involves first the commitment of undifferentiated mesenchymal cells to the chondrocyte lineage (1). Cell condensation and further maturation lead to a fully differentiated phenotype characterized by the synthesis of cartilage extracellular matrix proteins, including collagen types II, IX, and XI, the large proteoglycan aggrecan, the link protein, and the cartilage oligomeric protein (24). Recent molecular and biochemical studies with cell culture, gene inactivation experiments with mice, and the identification of genes responsible for mouse and human skeletal abnormalities have documented the importance of growth and differentiation factors, extracellular matrix proteins, signaling mediators, and tr...

Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis , an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models. Electronic supplementary material The online version of this article (10.1186/s13059-019-1776-2) contains supplementary material, which is available to authorized users.

Cartilage collagen fibrils, which are characterized by their thin, uniform diameters, are formed of a multicomponent system of three collagen types (H1, IX, and XI) and interacting proteoglycans. We have used a genetic approach to test whether the proper assembly of this multiprotein structure was altered by overexpression of one of its normal components. Here we show that in transgenic mice in which the normal mouse al(II) collagen is overexpressed, thick abnormal collagen fibrils are generated. Mice that showed the highest expression of the transgene also displayed a larger proportion of abnormal fibrils and died at birth. We propose that an imbalance among the constituents of the cartilage collagen fibrils disrupts the mechanism that controls their assembly. The results show the applicability of the transgenic mice system to studies of complex multicomponent protein assemblies in intact animals.Collagens form a variety of supramolecular structures that have different functions in extracellular matrices (1, 2). In cartilages, collagen fibrils have specific properties characterized by their nearly uniform, thin diameter of -10-40 nm (3, 4) and their multicomponent nature consisting of three different collagens (types II, IX, and XI). Type II collagen, the major structural component of these fibrils, is a homotrimer of three al(II) chains. Heterotrimeric type XI collagen, which is a minor component, is thought to reside in the central portion of the fibril (4), whereas type IX collagen, another heterotrimeric minor component, associates laterally on the fibril surface (5). Given the complexity of most collagen fibrils, the role of each component in fibril assembly is difficult to evaluate by using in vitro reconstitution experiments. We have therefore chosen a genetic approach to examine the role of specific components in fibrillogenesis. Specifically, we hypothesized that the increased expression of one of the normal components of the fibrils would alter the proper assembly of this multiprotein structure. To test this hypothesis we generated transgenic mice that harbored a normal polymorphic variant of the mouse al(II) procollagen gene (Col2a-1), and we show that increased expression of normal type II collagen disrupts the mechanism that controls the growth of cartilage fibrils to generate giant collagen fibrils. MATERIALS AND METHODSIntroduction of a Silent Mutation in Col2a-1. The oligonucleotide used for mutagenesis (5'-TGCAGGGCCCTATGG-GACCCCGTGG) corresponds to part of the sequence of exon 7 and contains an Apa I recognition site (underlined) and a single base mutation (in boldface type) that changes the Nco I site CCATGG to CTATGG. The PCR product was digested with Apa I, and the 150-bp Apa I-Apa I fragment was first cloned into a Pst I-EcoRI subclone, which was subsequently cloned into a 2.2-kb Kpn I-Cla I genomic subclone, from which a 1.9-kb Xho I-Xho I fragment was derived and inserted into a 13-kb clone p8045. The reconstruction of the gene was completed by addition of the 28-kb Cla I fragment...