SummarySomatic LINE-1 (L1) retrotransposition during neurogenesis is a potential source of genotypic variation among neurons. As a neurogenic niche, the hippocampus supports pronounced L1 activity. However, the basal parameters and biological impact of L1-driven mosaicism remain unclear. Here, we performed single-cell retrotransposon capture sequencing (RC-seq) on individual human hippocampal neurons and glia, as well as cortical neurons. An estimated 13.7 somatic L1 insertions occurred per hippocampal neuron and carried the sequence hallmarks of target-primed reverse transcription. Notably, hippocampal neuron L1 insertions were specifically enriched in transcribed neuronal stem cell enhancers and hippocampus genes, increasing their probability of functional relevance. In addition, bias against intronic L1 insertions sense oriented relative to their host gene was observed, perhaps indicating moderate selection against this configuration in vivo. These experiments demonstrate pervasive L1 mosaicism at genomic loci expressed in hippocampal neurons.

Highlights d Single-cell genomic analysis of hippocampal neurons reveals a somatic L1 insertion d The donor L1 is slightly 5ʹ truncated and lacks a conserved YY1 binding site d Young L1s with truncated or mutated YY1 binding sites are globally hypomethylated d L1 is able to mobilize in the brain because of locus-specific exceptions to repression

LINE-1 (L1) retrotransposons are a noted source of genetic diversity and disease in mammals. To expand its genomic footprint, L1 must mobilize in cells that will contribute their genetic material to subsequent generations. Heritable L1 insertions may therefore arise in germ cells and in pluripotent embryonic cells, prior to germline specification, yet the frequency and predominant developmental timing of such events remain unclear. Here, we applied mouse retrotransposon capture sequencing (mRC-seq) and whole-genome sequencing (WGS) to pedigrees of C57BL/6J animals, and uncovered an L1 insertion rate of ≥1 event per eight births. We traced heritable L1 insertions to pluripotent embryonic cells and, strikingly, to early primordial germ cells (PGCs). New L1 insertions bore structural hallmarks of target-site primed reverse transcription (TPRT) and mobilized efficiently in a cultured cell retrotransposition assay. Together, our results highlight the rate and evolutionary impact of heritable L1 retrotransposition and reveal retrotransposition-mediated genomic diversification as a fundamental property of pluripotent embryonic cells in vivo.