Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation.

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.

The mitophagy receptor Bcl-2-like protein 13 stimulates adipogenesis by regulating mitochondrial oxidative phosphorylation and apoptosis in mice.

Metabolic programming of bone marrow stromal cells (BMSCs) could influence the function of progenitor osteoblasts or adipocytes and hence determine skeletal phenotypes. Adipocytes predominantly utilize oxidative phosphorylation, whereas osteoblasts use glycolysis to meet ATP demand. Here, we compared progenitor differentiation from the marrow of two inbred mouse strains, C3H/HeJ (C3H) and C57BL6J (B6). These strains differ in both skeletal mass and bone marrow adiposity. We hypothesized that genetic regulation of metabolic programs controls skeletal stem cell fate. Our experiments identified Bcl-2-like protein 13 (Bcl2l13), a mitochondrial mitophagy receptor, as being critical for adipogenic differentiation. We also found that Bcl2l13 is differentially expressed in the two mouse strains, with C3H adipocyte progenitor differentiation being accompanied by a >2-fold increase in Bcl2l13 levels relative to B6 marrow adipocytes. Bcl2l13 expression also increased during adipogenic differentiation in mouse ear mesenchymal stem cells (eMSCs) and the murine preadipocyte cell line 3T3-L1. The higher Bcl2l13 expression correlated with increased mitochondrial fusion and biogenesis. Importantly, Bcl2l13 knockdown significantly impaired adipocyte differentiation in both 3T3-L1 cells and eMSCs. Mechanistically, Bcl2l13 knockdown reprogrammed cells to rely more on glycolysis to meet ATP demand in the face of impaired oxidative phosphorylation. Bcl2l13 knockdown in eMSCs increased mitophagy. Moreover, Bcl2l13 prevented apoptosis during adipogenesis. Our findings indicate that the mitochondrial receptor Bcl2l13 promotes adipogenesis by increasing oxidative phosphorylation, suppressing apoptosis, and providing mitochondrial quality control through mitophagy. We conclude that genetic programming of metabolism may be important for lineage determination and cell function within the bone marrow.