To date the only Neandertal genome that has been sequenced to high quality is from an individual found in Southern Siberia. We sequenced the genome of a female Neandertal from ~50 thousand years ago from Vindija Cave, Croatia to ~30-fold genomic coverage. She carried 1.6 differences per ten thousand base pairs between the two copies of her genome, fewer than present-day humans, suggesting that Neandertal populations were of small size. Our analyses indicate that she was more closely related to the Neandertals that mixed with the ancestors of present-day humans living outside of sub-Saharan Africa than the previously sequenced Neandertal from Siberia, allowing 10-20% more Neandertal DNA to be identified in present-day humans, including variants involved in LDL cholesterol levels, schizophrenia and other diseases.

We sequenced the genome of a Neandertal from Chagyrskaya Cave in the Altai Mountains, Russia, to 27-fold genomic coverage. We show that this Neandertal was a female and that she was more related to Neandertals in western Eurasia [Prüfer et al., Science 358, 655–658 (2017); Hajdinjak et al., Nature 555, 652–656 (2018)] than to Neandertals who lived earlier in Denisova Cave [Prüfer et al., Nature 505, 43–49 (2014)], which is located about 100 km away. About 12.9% of the Chagyrskaya genome is spanned by homozygous regions that are between 2.5 and 10 centiMorgans (cM) long. This is consistent with the fact that Siberian Neandertals lived in relatively isolated populations of less than 60 individuals. In contrast, a Neandertal from Europe, a Denisovan from the Altai Mountains, and ancient modern humans seem to have lived in populations of larger sizes. The availability of three Neandertal genomes of high quality allows a view of genetic features that were unique to Neandertals and that are likely to have been at high frequency among them. We find that genes highly expressed in the striatum in the basal ganglia of the brain carry more amino-acid-changing substitutions than genes expressed elsewhere in the brain, suggesting that the striatum may have evolved unique functions in Neandertals.

When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.

We sequenced the genome of a Neandertal from Chagyrskaya Cave in the Altai Mountains, Russia, to 27-fold genomic coverage. We estimate that this individual lived ~80,000 years ago and was more closely related to Neandertals in western Eurasia (1,2) than to Neandertals who lived earlier in Denisova Cave (3), which is located about 100 km away. About 12.9% of the Chagyrskaya genome is spanned by homozygous regions that are between 2.5 and 10 centiMorgans (cM) long. This is consistent with that Siberian Neandertals lived in relatively isolated populations of less than 60 individuals. In contrast, a Neandertal from Europe, a Denisovan from the Altai Mountains and ancient modern humans seem to have lived in populations of larger sizes. The availability of three Neandertal genomes of high quality allows a first view of genetic features that were unique to Neandertals and that are likely to have been at high frequency among them. We find that genes highly expressed in the striatum in the basal ganglia of the brain carry more amino acid-changing substitutions than genes expressed elsewhere in the brain, suggesting that the striatum may have evolved unique functions in Neandertals.

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