BackgroundConditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient.ResultsHere we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live offspring.Conclusions Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1220-4) contains supplementary material, which is available to authorized users.

Osteoclasts are unique cells that resorb bone, and are involved in not only bone remodeling but also pathological bone loss such as osteoporosis and rheumatoid arthritis. The regulation of osteoclasts is based on a number of molecules but full details of these molecules have not yet been understood. MicroRNAs are produced by Dicer cleavage an emerging regulatory system for cell and tissue function. Here, we examine the effects of Dicer deficiency in osteoclasts on osteoclastic activity and bone mass in vivo. We specifically knocked out Dicer in osteoclasts by crossing Dicer flox mice with cathepsin K-Cre knock-in mice. Dicer deficiency in osteoclasts decreased the number of osteoclasts (N.Oc/BS) and osteoclast surface (Oc.S/BS) in vivo. Intrinsically, Dicer deficiency in osteoclasts suppressed the levels of TRAP positive multinucleated cell development in culture and also reduced NFATc1 and TRAP gene expression. MicroRNA analysis indicated that expression of miR-155 was suppressed by RANKL treatment in Dicer deficient cells. Dicer deficiency in osteoclasts suppressed osteoblastic activity in vivo including mineral apposition rate (MAR) and bone formation rate (BFR) and also suppressed expression of genes encoding type I collagen, osteocalcin, Runx2, and Efnb2 in vivo. Dicer deficiency in osteoclasts increased the levels of bone mass indicating that the Dicer deficiency-induced osteoclastic suppression was dominant over Dicer deficiency-induced osteoblastic suppression. On the other hand, conditional Dicer deletion in osteoblasts by using 2.3 kb type I collagen-Cre did not affect bone mass. These results indicate that Dicer in osteoclasts controls activity of bone resorption in vivo.

Collagen VI-related myopathies are disorders of connective tissue presenting with an overlap phenotype combining clinical involvement from the muscle and from the connective tissue. Not all patients displaying related overlap phenotypes between muscle and connective tissue have mutations in collagen VI. Here, we report a homozygous recessive loss of function mutation and a de novo dominant mutation in collagen XII (COL12A1) as underlying a novel overlap syndrome involving muscle and connective tissue. Two siblings homozygous for a loss of function mutation showed widespread joint hyperlaxity combined with weakness precluding independent ambulation, while the patient with the de novo missense mutation was more mildly affected, showing improvement including the acquisition of walking. A mouse model with inactivation of the Col12a1 gene showed decreased grip strength, a delay in fiber-type transition and a deficiency in passive force generation while the muscle seems more resistant to eccentric contraction induced force drop, indicating a role for a matrix-based passive force-transducing elastic element in the generation of the weakness. This new muscle connective tissue overlap syndrome expands on the emerging importance of the muscle extracellular matrix in the pathogenesis of muscle disease.

Type XII collagen–null mice have fragile bones with disorganized collagen fiber arrangement, decreased bone matrix formation, and delayed osteoblast differentiation.

Tendons are composed of fibroblasts and collagen fibrils. The fibrils are organized uniaxially and grouped together into fibers. Collagen VI is a non-fibrillar collagen expressed in developing and adult tendons. Human collagen VI mutations result in muscular dystrophy, joint hyperlaxity and contractures. The purpose of this study is to determine the functional roles of collagen VI in tendon matrix assembly. During tendon development, collagen VI was expressed throughout the extracellular matrix, but enriched around fibroblasts and their processes. To analyze the functional roles of collagen VI a mouse model with a targeted inactivation of Col6a1 gene was utilized. Ultrastructural analysis of Col6a1−/− versus wild type tendons demonstrated disorganized extracellular micro-domains and collagen fibers in the Col6a1−/− tendon. In the col6a1−/− tendon, fibril structure and diameter distribution was abnormal compared to wild type controls. Col6a1−/− fibrils had smaller diameters and the diameter distributions were shifted significantly toward the smaller diameters. An analysis of fibril density (number/μm2) demonstrated an ~2.5 fold increase in the Col6a1−/− versus wild type tendons. In addition, the fibril arrangement and structure was aberrant in the peri-cellular regions of Col6a1−/− tendons with frequent very large fibrils and twisted fibrils observed restricted to this region. The biomechanical properties were analyzed in mature tendons. A significant decrease in cross sectional area was observed. The percent relaxation, maximum load, maximum stress, stiffness and modulus were analyzed and Col6a1−/− tendons demonstrated a significant reduction in maximum load and stiffness compared to wild type tendons. An increase in matrix metalloproteinase activity was suggested in the absence of collagen VI. This suggests alterations in tenocyte expression due to disruption of cell-matrix interactions. The changes in expression may result in alterations in the peri-cellular environment. In addition, the absence of collagen VI may alter the sequestering of regulatory molecules such as leucine rich proteoglycans. These changes would result in dysfunctional regulation of tendon fibrillogenesis indirectly mediated by collagen VI.

On page 51726, we misleadingly stated that the central acidic domain in Dsl1p is essential for viability. Evidence for this was presented in Fig. 3C. This figure shows that a mutant carrying two Trp-to-Ala replacements in this region did not support growth of a dsl1 deletion mutant. However, we recently created a GAL-regulated TAP-tagged version of DSL1 carrying five Trp-to-Ala substitutions in this region to use as a negative control in pulldown experiments. Surprisingly, this mutant complemented the dsl1 knock-out. Even a single-copy untagged version of this allele could replace the wild-type gene. However, these cells grow poorly at all temperatures tested and show phenotypes similar to those of Dsl1p-depleted cells.Sequencing showed that the plasmid used for the growth assay in our previous work contained a C-terminal truncation in addition to the Trp-to-Ala substitutions at positions 413 and 455. A mutant clone with an intact C terminus supported growth of a dsl1 deletion mutant.Our recent data are still consistent with the notion that the outer tryptophan motifs in the acidic domain of Dsl1p ( ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.orgWe suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.

Renin angiotensin system (RAS) regulates circulating blood volume and blood pressure systemically, whereas RAS also plays a role in the local milieu. Previous in vitro studies suggested that RAS may be involved in the regulation of bone cells. However, it was not known whether molecules involved in RAS are present in bone in vivo. In this study, we examined the presence of RAS components in adult bone and the effects of angiotensin II type 2 (AT2) receptor blocker on bone mass. Immunohistochemistry revealed that AT2 receptor protein was expressed in both osteoblasts and osteoclasts. In addition, renin and angiotensin II-converting enzyme were expressed in bone cells in vivo. Treatment with AT2 receptor blocker significantly enhanced the levels of bone mass, and this effect was based on the enhancement of osteoblastic activity as well as the suppression of osteoclastic activity in vivo. These results indicate that RAS components are present in adult bone and that blockade of AT2 receptor results in alteration in bone mass.Osteoporosis is one of the major diseases associated with aging. This disease is based on the imbalance between the two major activities, i.e. bone formation and bone resorption. Systemic signals such as parathyroid hormone (PTH) 3 and vitamin D are the major regulators of the maintenance of bone mass and blood calcium (1-3). In addition, bone mass levels are also determined by a central nervous control through the activities of sympathetic tone on both bone formation and resorption sides (4 -7). In the local milieu of bone, two major types of cells, osteoblasts and osteoclasts, are located in close proximity, exchange their signals, and coordinately resorb and form bone matrix (8). Such events are controlled by molecules present in the local microenvironment. These include cytokines, their modulators, and matrix proteins secreted by osteoblasts and osteoclasts (9 -11). However, the bone environment is quite heterogeneous, and there are also cells other than these two types. Microvasculatures are serving as a root to supply osteoclast progenitors, which are derived from hematopoietic lineage cells. Vasculatures in bone are also considered to give rise to progenitors for osteoblastic cells from their perivascular regions (12). In addition to the anatomical relationship between the vascular cells and bone cells, these cells may be functionally involved in the coordinate regulation of bone mass.Recent clinical studies indicated that beta blockers and antihypertension drugs would reduce the risk of bone fractures in the elderly populations (13,14). This suggests a possible link between vascular and skeletal systems. Renin angiotensin system (RAS) is operating not only systemically but also locally in several tissues, and bone microenvironments have been studied in this regard (15,16). Osteoblasts and osteoclasts express angiotensin II type 1 receptor in cell cultures (17-19), suggesting the existence of local RAS in bone. However, whether RAS components are expressed in bone in vivo is not known.A...

BackgroundAlthough CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining.ResultsHere, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes.ConclusionsOur results provide a technical platform for high-throughput knock-in.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3331-9) contains supplementary material, which is available to authorized users.