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Single-Cell Sperm Sequencing Reveals New Details About Recombination

NEW YORK (GenomeWeb) – With the help of single-cell sequencing of mouse sperm cells, a research team led by investigators at the University of Oxford has gained a higher-resolution view of the sequence and chromosome features that influence mammalian meiotic recombination events. 

"This large study of crossovers localized at a fine scale in a mammal provides a resource for understanding crossover formation genome-wide," senior author Peter Donnelly, director of the Wellcome Trust Centre for Human Genetics and professor of statistical science at the University of Oxford, and his colleagues wrote in a paper published in Science this week.

There, the researchers outlined their efforts to tease out the factors affecting crossover-based DNA swaps — an analysis that relied on a kilobase-resolution map of genome-wide crossover events assembled from single-cell, whole genome sequences for more than 200 individual sperm cells from a mouse hybrid born to B6 and CAST strain parents.

"We have developed a method for amplifying and sequencing DNA from single cells," the authors explained, "and have applied it to sperm to identify crossovers with high resolution."

Past studies have highlighted the importance of the histone methyltransferase enzyme PRDM9 in meiotic recombination in mice, humans, and other animals, the team explained. PRDM9 binds to specific DNA sites, and a subset of these undergo programmed double-strand breaks that are repaired in a manner that leads to homolog crossovers, with help from a protein called DMC1.

Because double-strand DNA breaks appear to be more common than full-blown crossover events in mammalian cells, the researchers took a closer look at the full range of features that portend meiotic recombination in the hybrid mouse model.

"Although it is clear that not all [double-strand breaks] are equally likely to resolve as crossovers," they wrote, "the factors affecting this decision remain largely unknown."

Using a single-cell sequencing approach that involved mechanical cell isolation, RNA random priming, and Klenow fragment extension-based DNA amplification, the team established genome sequences on 217 individual sperm cells from a B6-CAST hybrid mouse that was also assessed by bulk sperm DNA sequencing.

They also profiled recombination hotspots, PRDM9 binding sites, and histone marks in testis tissue from the same animal or from an animal with the same genetic background, using a combination of chromatin immunoprecipitation sequencing and micrococcal nuclease sequencing.

After mapping 2,649 crossover events throughout the genome based on the single-cell sperm sequences, the team identified more than 1,600 recombination hotspots across the autosomal chromosomes.

By folding in ChIP-seq and other data, meanwhile, the investigators got a look at the varied factors that seemed to influence crossover events at sites with different recombination frequencies — from the PRDM9 allele present to the underlying nucleotide composition of a recombination site sequence, nucleosome positions, and distance from telomeres. 

"Combining this map with molecular assays measuring stages of recombination, we identified factors that affect crossover probability, including PRDM9 binding on the non-initiating template homolog and telomere proximity," the authors wrote. "These factors also influence the time for sites of recombination-initiating DNA double-strand breaks to find and engage their homologs, with rapidly engaging sites more likely to form crossovers."