Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity.

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for "correcting" variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S.

On the potential role of globins in brown adipose tissue: a novel conceptual model and studies in myoglobin knockout mice.

Myoglobin (Mb) regulates O bioavailability in muscle and heart as the partial pressure of O (Po) drops with increased tissue workload. Globin proteins also modulate cellular NO pools, "scavenging" NO at higher Po and converting NO to NO as Po falls. Myoglobin binding of fatty acids may also signal a role in fat metabolism.

Metabolic physiology and skeletal muscle phenotypes in male and female myoglobin knockout mice.

Myoglobin (Mb) is a regulator of O bioavailability in type I muscle and heart, at least when tissue O levels drop. Mb also plays a role in regulating cellular nitric oxide (NO) pools. Robust binding of long-chain fatty acids and long-chain acylcarnitines to Mb, and enhanced glucose metabolism in hearts of Mb knockout (KO) mice, suggest additional roles in muscle intermediary metabolism and fuel selection.

A resource of targeted mutant mouse lines for 5,061 genes.

The International Mouse Phenotyping Consortium reports the generation of new mouse mutant strains for over 5,000 genes, including 2,850 novel null, 2,987 novel conditional- ready, and 4,433 novel reporter alleles.

Generation of desminopathy in rats using CRISPR-Cas9.

Background: Desminopathy is a clinically heterogeneous muscle disease caused by over 60 different mutations in desmin. The most common mutation with a clinical phenotype in humans is an exchange of arginine to proline at position 350 of desmin leading to p.R350P.

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.

Efficient mouse genome engineering by CRISPR-EZ technology.

CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions.

Abnormal Mammary Development in 129:STAT1-Null Mice is Stroma-Dependent.

Female 129:Stat1-null mice (129S6/SvEvTac-Stat1(tm1Rds) homozygous) uniquely develop estrogen-receptor (ER)-positive mammary tumors. Herein we report that the mammary glands (MG) of these mice have altered growth and development with abnormal terminal end buds alongside defective branching morphogenesis and ductal elongation. We also find that the 129:Stat1-null mammary fat pad (MFP) fails to sustain the growth of 129S6/SvEv wild-type and Stat1-null epithelium.

Rescue of germline transmission from chimeras by IVF after sperm analysis.

Successful production of genetically modified mouse lines is dependent on germline transmission (GLT) of mutant alleles from chimeras. When natural mating fails to achieve GLT due to male infertility, sickness, or other problems, sperm can be harvested from chimeras and used for assisted reproductive technologies such as in vitro fertilization (IVF) to attempt to "rescue" GLT. However, a rational, evidence-based approach to determine if such extraordinary efforts should be attempted on a chimera has not been established.

Assessment of three generations of mice derived by ICSI using freeze-dried sperm.

Although the derivation of mice by intracytoplasmic sperm injection (ICSI) using freeze-dried sperm has been demonstrated previously, a comprehensive analysis of their viability, health, and fertility has not. The purpose of the present study was to determine the extent to which ICSI using freeze-dried sperm stored at 4 degrees C for 1-2 months from mice on either an inbred (C57BL/6J) or hybrid (B6D2F1/J) genetic background results in genomic instability and/or phenotypic abnormality in mice and two generations of their progeny. Fertilization rates (number of 2-cells per injected oocytes) using ICSI of fresh and freeze-dried sperm were similar within and between mouse strains, although fewer freeze-dried sperm-derived embryos than fresh sperm-derived embryos developed to blastocysts in vitro (C57BL/6J and B6D2F1/J) and liveborn pups in vivo (B6D2F1/J only).