CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Even for the laboratory mouse, a model that has thrived under the benefits of embryonic stem (ES) cell knockout capabilities for nearly three decades, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 technology enables one to manipulate the genome with unprecedented simplicity and speed. It allows generation of null, conditional, precisely mutated, reporter, or tagged alleles in mice. Moreover, it holds promise for other applications beyond genome editing. The crux of this system is the efficient and targeted introduction of DNA breaks that are repaired by any of several pathways in a predictable but not entirely controllable manner. Thus, further optimizations and improvements are being developed. Here, we summarize current applications and provide a practical guide to use the CRISPR/Cas9 system for mouse mutagenesis, based on published reports and our own experiences. We discuss critical points and suggest technical improvements to increase efficiency of RNA-guided genome editing in mouse embryos and address practical problems such as mosaicism in founders, which complicates genotyping and phenotyping. We describe a next-generation sequencing strategy for simultaneous characterization of on- and off-target editing in mice derived from multiple CRISPR experiments. Additionally, we report evidence that elevated frequency of precise, homology-directed editing can be achieved by transient inhibition of the Ligase IV-dependent nonhomologous end-joining pathway in one-celled mouse embryos.

Eggs Well Done: Germ cells can endure extensive DNA damage during their development. Programmed meiotic double-strand breaks (DSBs) are essential for proper segregation of chromosomes to oocytes and sperm. However, incomplete DSB repair by recombination activates a checkpoint that triggers cell death. Exogenous DNA damage is also lethal to oocytes via a highly sensitive checkpoint. Bolcun-Filas et al. (p. 533 ) show that the CHK2 kinase is a key component of both checkpoints in mouse oocytes. Deletion of Chk2 restored fertility to females that would otherwise be sterile because of a meiotic recombination mutation or radiation exposure.

In mammalian meiosis, homologous chromosome synapsis is coupled with recombination. As in most eukaryotes, mammalian meiocytes have checkpoints that monitor the fidelity of these processes. We report that the mouse ortholog (Trip13) of pachytene checkpoint 2 (PCH2), an essential component of the synapsis checkpoint in Saccharomyces cerevisiae and Caenorhabditis elegans, is required for completion of meiosis in both sexes. TRIP13-deficient mice exhibit spermatocyte death in pachynema and loss of oocytes around birth. The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA. These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes. Double mutant analysis demonstrated that the recombination and synapsis genes Spo11, Mei1, Rec8, and Dmc1 are all epistatic to Trip13, suggesting that TRIP13 does not have meiotic checkpoint function in mice. Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers. To our knowledge, this is the first model to separate recombination defects from asynapsis in mammalian meiosis, and provides the first evidence that unrepaired DNA damage alone can trigger the pachytene checkpoint response in mice. It is critical that the chromosomes carried by sperm and eggs contain faithful representations of the genome of the individual that produced them. During the process of meiosis, the maternal and paternal copies of each chromosome “synapse” with each other (become tightly associated), exchange genetic material via the process of recombination, then separate into daughter cells in the first of two meiotic cell divisions. The intricate chromosome behavior is subject to errors, so most organisms have evolved meiotic “checkpoints” that monitor fidelity of chromosome synapsis and repair of DNA damage. These checkpoints cause defective cells to self destruct rather than generate defective sperm or eggs. We studied the effects of deleting mouse Trip13, a gene that in distant organisms plays a key role in meiotic checkpoint control. These experiments revealed that instead of having a checkpoint role, Trip13 is required for one of the two major classes of recombination in meiosis that is required for repairing broken DNA molecules. The chromosomes still synapsed normally, but animals were sterile due to massive death of oocytes and spermatocytes. These results indicate that, in addition to a checkpoint that responds to failed synapsis, one exists to specifically detect unrepaired DNA damage that is due to failed recombination.

The transcriptional regulation of mammalian meiosis is poorly characterized, owing to few genetic and ex vivo models. From a genetic screen, we identify the transcription factor MYBL1 as a male-specific master regulator of several crucial meiotic processes. Spermatocytes bearing a novel separation-of-function allele (Mybl1^repro9) had subtle defects in autosome synapsis in pachynema, a high incidence of unsynapsed sex chromosomes, incomplete double-strand break repair on synapsed pachytene chromosomes and a lack of crossing over. MYBL1 protein appears in pachynema, and its mutation caused specific alterations in expression of diverse genes, including some translated postmeiotically. These data, coupled with chromatin immunoprecipitation (ChIP-chip) experiments and bioinformatic analysis of promoters, identified direct targets of MYBL1 regulation. The results reveal that MYBL1 is a master regulator of meiotic genes that are involved in multiple processes in spermatocytes, particularly those required for cell cycle progression through pachynema.

chaos1 (for chromosome aberrations occurring spontaneously 1) is a recessive mutation that was originally identified in a phenotype-based screen for chromosome instability mutants in mice. Mutant animals exhibit significantly higher frequencies of spontaneous and radiation- or mitomycin C-induced micronucleated erythrocytes, indicating a potential defect in homologous recombination or interstrand cross-link repair. The chaos1 allele was genetically associated with a missense mutation in Polq, which encodes DNA polymerase θ. We demonstrate here that chaos1 is a mutant allele of Polq by using two genetic approaches: chaos1 mutant phenotype correction by a bacterial artificial chromosome carrying wild-type Polq and a failed complementation test between chaos1 and a Polq-disrupted allele generated by gene targeting. To investigate the potential involvement of Polq in DNA double-strand break repair, we introduced chaos1 into an Atm (for ataxia telangiectasia mutated)-deficient background. The majority (∼90%) of double-homozygous mice died during the neonatal period. Surviving double mutants exhibited synergistic phenotypes such as severe growth retardation and enhanced chromosome instability. However, remarkably, double mutants had delayed onset of thymic lymphoma, significantly increasing life span. These data suggest a unique role of Polq in maintaining genomic integrity, which is probably distinctive from the major homologous recombination pathway regulated by ATM.