Andrew J. Modzelewski

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.

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.

SFMBT1 functions with LSD1 to regulate expression of canonical histone genes and chromatin-related factors

SFMBT1 (Scm [Sex comb on midleg] with four MBT [malignant brain tumor] domains 1) is a poorly characterized mammalian MBT domain-containing protein homologous to Drosophila SFMBT, a Polycomb group protein involved in epigenetic regulation of gene expression. Here, we show that SFMBT1 regulates transcription in somatic cells and during spermatogenesis through the formation of a stable complex with LSD1 and CoREST. When bound to its gene targets, SFMBT1 recruits its associated proteins and causes chromatin compaction and transcriptional repression. SFMBT1, LSD1, and CoREST share a large fraction of target genes, including those encoding replication-dependent histones. Simultaneous occupancy of histone genes by SFMBT1, LSD1, and CoREST is regulated during the cell cycle and correlates with the loss of RNA polymerase II at these promoters during G2, M, and G1. The interplay between the repressive SFMBT1-LSD1-CoREST complex and RNA polymerase II contributes to the timely transcriptional regulation of histone genes in human cells. SFMBT1, LSD1, and CoREST also form a stable complex in germ cells, and their chromatin binding activity is regulated during spermatogenesis.

The roles of microRNAs and siRNAs in mammalian spermatogenesis

MicroRNAs and siRNAs, both of which are AGO-bound small RNAs, are essential for mammalian spermatogenesis. Although their precise germline roles remain largely uncharacterized, recent discoveries suggest that they function in mechanisms beyond microRNA-mediated post-transcriptional control, playing roles in DNA repair and transcriptional regulation within the nucleus. Here, we discuss the latest findings regarding roles for AGO proteins and their associated small RNAs in the male germline. We integrate genetic, clinical and genomics data, and draw upon findings from non-mammalian models, to examine potential roles for AGO-bound small RNAs during spermatogenesis. Finally, we evaluate the emerging and differing roles for AGOs and AGO-bound small RNAs in the male and female germlines, suggesting potential reasons for these sexual dimorphisms. Summary: This Review article summarizes the latest findings regarding roles for AGO proteins and their associated small RNAs in the male germline, with a particular focus on spermatogenesis.

Dgcr8 and Dicer are essential for sex chromosome integrity during meiosis in males

ABSTRACT Small RNAs play crucial roles in regulating gene expression during mammalian meiosis. To investigate the function of microRNAs (miRNAs) and small interfering RNAs (siRNAs) during meiosis in males, we generated germ-cell-specific conditional deletions of Dgcr8 and Dicer in mice. Analysis of spermatocytes from both conditional knockout lines revealed that there were frequent chromosomal fusions during meiosis, always involving one or both sex chromosomes. RNA sequencing indicates upregulation of Atm in spermatocytes from miRNA-deficient mice, and immunofluorescence imaging demonstrates an increased abundance of activated ATM kinase and mislocalization of phosphorylated MDC1, an ATM phosphorylation substrate. The Atm 3′UTR contains many potential microRNA target sites, and, notably, target sites for several miRNAs depleted in both conditional knockout mice were highly effective at promoting repression. RNF8, a telomere-associated protein whose localization is controlled by the MDC1–ATM kinase cascade, normally associates with the sex chromosomes during pachytene, but in both conditional knockouts redistributed to the autosomes. Taken together, these results suggest that Atm dysregulation in microRNA-deficient germ lines contributes to the redistribution of proteins involved in chromosomal stability from the sex chromosomes to the autosomes, resulting in sex chromosome fusions during meiotic prophase I. Highlighted Article: miRNA-deficient spermatocytes display frequent sex chromosome fusions and fail to progress through meiosis in a process that is probably mediated by dysregulation of Atm.

Transcriptome profiling of the developing male germ line identifies the miR-29 family as a global regulator during meiosis

ABSTRACT MicroRNAs are essential for spermatogenesis. However, the stage-specific requirements for particular miRNAs in the male mammalian germ line remain largely uncharacterized. The miR-34 family is, to date, the only miRNA proven to be necessary for the production of sperm in mammals, though its germline roles are poorly understood. Here, we generate and analyze paired small RNA and mRNA profiles across different stages of germline development in male mice, focusing on time points shortly before and during meiotic prophase I. We show that in addition to miR-34, miR-29 also mediates widespread repression of mRNA targets during meiotic prophase I in the male mouse germline. Furthermore, we demonstrate that predicted miR-29 target mRNAs in meiotic cells are largely distinct from those of miR-34, indicating that miR-29 performs a regulatory function independent of miR-34. Prior to this work, no germline role has been attributed to miR-29. To begin to understand roles for miR-29 in the germ line, we identify targets of miR-29 undergoing post transcriptional downregulation during meiotic prophase I, which likely correspond to the direct targets of miR-29. Interestingly, candidate direct targets of miR-29 are enriched in transcripts encoding extracellular matrix components. Our results implicate the miR-29 family as an important regulatory factor during male meiosis.

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.

Single-embryo and single-blastomere immunoblotting reports protein expression heterogeneity in early-stage preimplantation embryos

Understanding how a zygote develops from a single cell into a multicellular organism has benefitted from single-cell tools, including RNA sequencing (RNA-Seq) and immunofluorescence (IF). However, scrutinizing inter- and intra-embryonic phenotypic variation is hindered by two fundamental limitations; the loose correlation between transcription and translation and the cross-reactivity of immunoreagents. To address these challenges, we describe a high-specificity microfluidic immunoblot optimized to quantify protein expression from all stages of mouse preimplantation development. Despite limited availability of isoform-specific immunoreagents, the immunoblot resolves inter-embryonic heterogeneity of embryo-specific isoforms (i.e., DICER-1). We observed significantly higher DICER-1 isoform expression in oocytes when compared to two-cell embryos, and further find that protein expression levels follow the same trend as mRNA for both the full-length and truncated DICER-1 isoforms. At the morula stage, we assayed both whole and disaggregated embryos for loading controls (β-tubulin, GAPDH) and markers that regulate cell fate decisions (CDX-2, SOX-2). In disaggregated morula, we found that cell volume showed positive, linear correlation with expression of β-tubulin and SOX-2. In dissociated two-cell and four-cell blastomeres, we detect significant inter-blastomeric variation in GADD45a expression, corroborating suspected cellular heterogeneity even in the earliest multicellular stage of preimplantation embryos. As RNA-Seq and other transcript-centric approaches continue to further probe preimplantation development, the demand for companion protein-based techniques rises. The reported microfluidic immunoblot serves as an essential tool for understanding mammalian development by providing high-specificity and direct measurements of protein targets at single-embryo and single-blastomere resolution.

Reproductive and Developmental Genomics Retreat at Cornell University, 2012

On June 14–15th, the Wiley journal Molecular Reproduction and Development partnered with the Cornell University’s Center for Reproductive Genomics and Vertebrate Genomics, along with NICHD-funded training grants, to host a retreat on Cornell’s Ithaca, New York campus. The event brought together Cornell scientists including NIH-trainees, and researchers from many other institutions, possessing varied expertise and utilizing diverse model systems. The goal was to facilitate multidisciplinary interaction amongst colleagues and foster a perspective of comparative biology towards a shared interest in genomics-based research.