Mammalian BTBD12 (SLX4) Protects against Genomic Instability during Mammalian Spermatogenesis

The mammalian ortholog of yeast Slx4, BTBD12, is an ATM substrate that functions as a scaffold for various DNA repair activities. Mutations of human BTBD12 have been reported in a new sub-type of Fanconi anemia patients. Recent studies have implicated the fly and worm orthologs, MUS312 and HIM-18, in the regulation of meiotic crossovers arising from double-strand break (DSB) initiating events and also in genome stability prior to meiosis. Using a Btbd12 mutant mouse, we analyzed the role of BTBD12 in mammalian gametogenesis. BTBD12 localizes to pre-meiotic spermatogonia and to meiotic spermatocytes in wildtype males. Btbd12 mutant mice have less than 15% normal spermatozoa and are subfertile. Loss of BTBD12 during embryogenesis results in impaired primordial germ cell proliferation and increased apoptosis, which reduces the spermatogonial pool in the early postnatal testis. During prophase I, DSBs initiate normally in Btbd12 mutant animals. However, DSB repair is delayed or impeded, resulting in persistent γH2AX and RAD51, and the choice of repair pathway may be altered, resulting in elevated MLH1/MLH3 focus numbers at pachynema. The result is an increase in apoptosis through prophase I and beyond. Unlike yeast Slx4, therefore, BTBD12 appears to function in meiotic prophase I, possibly during the recombination events that lead to the production of crossovers. In line with its expected regulation by ATM kinase, BTBD12 protein is reduced in the testis of Atm−/− males, and Btbd12 mutant mice exhibit increased genomic instability in the form of elevated blood cell micronucleus formation similar to that seen in Atm−/− males. Taken together, these data indicate that BTBD12 functions throughout gametogenesis to maintain genome stability, possibly by co-ordinating repair processes and/or by linking DNA repair events to the cell cycle via ATM

Mammalian genome innovation through transposon domestication

Since the discovery of transposons, their sheer abundance in host genomes has puzzled many. While historically viewed as largely harmless 'parasitic' DNAs during evolution, transposons are not a mere record of ancient genome invasion. Instead, nearly every element of transposon biology has been integrated into host biology. Here we review how host genome sequences introduced by transposon activities provide raw material for genome innovation and document the distinct evolutionary path of each species

Assessing heterogeneity among single embryos and single blastomeres using open microfluidic design.

The process by which a zygote develops from a single cell into a multicellular organism is poorly understood. Advances are hindered by detection specificity and sensitivity limitations of single-cell protein tools and by challenges in integrating multimodal data. We introduce an open microfluidic tool expressly designed for same-cell phenotypic, protein, and mRNA profiling. We examine difficult-to-study-yet critically important-murine preimplantation embryo stages. In blastomeres dissociated from less well-studied two-cell embryos, we observe no significant GADD45a protein expression heterogeneity, apparent at the four-cell stage. In oocytes, we detect differences in full-length versus truncated DICER-1 mRNA and protein, which are insignificant by the two-cell stage. Single-embryo analyses reveal intraembryonic heterogeneity, differences between embryos of the same fertilization event and between donors, and reductions in the burden of animal sacrifice. Open microfluidic design integrates with existing workflows and opens new avenues for assessing the cellular-to-molecular heterogeneity inherent to preimplantation embryo development

Multimodal detection of protein isoforms and nucleic acids from mouse pre-implantation embryos

Although mammalian embryo development depends on critical protein isoforms that arise from embryo-specific nucleic acid modifications, the role of these isoforms is not yet clear. Challenges arise in measuring protein isoforms and nucleic acids from the same single embryos and blastomeres. Here we present a multimodal technique for performing same-embryo nucleic acid and protein isoform profiling (single-embryo nucleic acid and protein profiling immunoblot, or snapBlot). The method integrates protein isoform measurement by fractionation polyacrylamide gel electrophoresis (fPAGE) with off-chip analysis of nucleic acids from the nuclei. Once embryos are harvested and cultured to the desired stage, they are sampled into the snapBlot device and subjected to fPAGE. After fPAGE, 'gel pallets' containing nuclei are excised from the snapBlot device for off-chip nucleic acid analyses. fPAGE and nuclei analyses are indexed to each starting sample, yielding same-embryo multimodal measurements. The entire protocol, including processing of samples and data analysis, takes 2-3 d. snapBlot is designed to help reveal the mechanisms by which embryo-specific nucleic acid modifications to both genomic DNA and messenger RNA orchestrate the growth and development of mammalian embryos

Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes*

The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared with microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a proof-of-principle experiment, we used CRISPR-EZ to target the tyrosinase (Tyr) gene, achieving 88% bi-allelic editing and 42% homology-directed repair-mediated precise sequence modification in live mice. Taken together, CRISPR-EZ is simple, economic, high throughput, and highly efficient with the potential to replace microinjection for in vivo genome editing in mice and possibly in other mammals

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. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. Here, we provide an optimized protocol covering sgRNA synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher with basic embryo-manipulation skills can obtain genetically modified mice in 6 weeks. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species