The modern human aryl hydrocarbon receptor is more active when ancestralized by genome editing.

The aryl hydrocarbon receptor (AHR) is a transcription factor that has many functions in mammals. Its best known function is that it binds aromatic hydrocarbons and induces the expression of cytochrome P450 genes, which encode enzymes that metabolize aromatic hydrocarbons and other substrates. All present-day humans carry an amino acid substitution at position 381 in the AHR that occurred after the divergence of modern humans from Neandertals and Denisovans. Previous studies that have expressed the ancestral and modern versions of AHR from expression vectors have yielded conflicting results with regard to their activities. Here, we use genome editing to modify the endogenous AHR gene so that it encodes to the ancestral, Neandertal-like AHR protein in human cells. In the absence of exogenous ligands, the expression of AHR target genes is higher in cells expressing the ancestral AHR than in cells expressing the modern AHR, and similar to the expression in chimpanzee cells. Furthermore, the modern human AHR needs higher doses of three ligands than the ancestral AHR to induce the expression of target genes. Thus, the ability of AHR to induce the expression of many of its target genes is reduced in modern humans.

Efficient high-precision homology-directed repair-dependent genome editing by HDRobust.

Homology-directed repair (HDR), a method for repair of DNA double-stranded breaks can be leveraged for the precise introduction of mutations supplied by synthetic DNA donors, but remains limited by low efficiency and off-target effects. In this study, we report HDRobust, a high-precision method that, via the combined transient inhibition of nonhomologous end joining and microhomology-mediated end joining, resulted in the induction of point mutations by HDR in up to 93% (median 60%, s.e.m. 3) of chromosomes in populations of cells. We found that, using this method, insertions, deletions and rearrangements at the target site, as well as unintended changes at other genomic sites, were largely abolished. We validated this approach for 58 different target sites and showed that it allows efficient correction of pathogenic mutations in cells derived from patients suffering from anemia, sickle cell disease and thrombophilia.

A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing.

A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000 year-old Neandertal individual with 8341 mtDNA sequences identified among 4.8 Gb of DNA generated from approximately 0.3 g of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs, and allows an estimate of the divergence date between the two mtDNA lineages of 660,000 +/- 140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared with other primate lineages, suggesting that the effective population size of Neandertals was small.

Simultaneous precise editing of multiple genes in human cells.

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.

deML: robust demultiplexing of Illumina sequences using a likelihood-based approach.

MOTIVATION: Pooling multiple samples increases the efficiency and lowers the cost of DNA sequencing. One approach to multiplexing is to use short DNA indices to uniquely identify each sample. After sequencing, reads must be assigned in silico to the sample of origin, a process referred to as demultiplexing. Demultiplexing software typically identifies the sample of origin using a fixed number of mismatches between the read index and a reference index set. This approach may fail or misassign reads when the sequencing quality of the indices is poor. RESULTS: We introduce deML, a maximum likelihood algorithm that demultiplexes Illumina sequences. deML computes the likelihood of an observed index sequence being derived from a specified sample. A quality score which reflects the probability of the assignment being correct is generated for each read. Using these quality scores, even very problematic datasets can be demultiplexed and an error threshold can be set. AVAILABILITY AND IMPLEMENTATION: deML is freely available for use under the GPL (http://bioinf.eva.mpg.de/deml/).

Genetic history of an archaic hominin group from Denisova Cave in Siberia.

Using DNA extracted from a finger bone found in Denisova Cave in southern Siberia, we have sequenced the genome of an archaic hominin to about 1.9-fold coverage. This individual is from a group that shares a common origin with Neanderthals. This population was not involved in the putative gene flow from Neanderthals into Eurasians; however, the data suggest that it contributed 4-6% of its genetic material to the genomes of present-day Melanesians. We designate this hominin population 'Denisovans' and suggest that it may have been widespread in Asia during the Late Pleistocene epoch. A tooth found in Denisova Cave carries a mitochondrial genome highly similar to that of the finger bone. This tooth shares no derived morphological features with Neanderthals or modern humans, further indicating that Denisovans have an evolutionary history distinct from Neanderthals and modern humans.

A recent evolutionary change affects a regulatory element in the human FOXP2 gene.

The FOXP2 gene is required for normal development of speech and language. By isolating and sequencing FOXP2 genomic DNA fragments from a 49,000-year-old Iberian Neandertal and 50 present-day humans, we have identified substitutions in the gene shared by all or nearly all present-day humans but absent or polymorphic in Neandertals. One such substitution is localized in intron 8 and affects a binding site for the transcription factor POU3F2, which is highly conserved among vertebrates. We find that the derived allele of this site is less efficient than the ancestral allele in activating transcription from a reporter construct. The derived allele also binds less POU3F2 dimers than POU3F2 monomers compared with the ancestral allele. Because the substitution in the POU3F2 binding site is likely to alter the regulation of FOXP2 expression, and because it is localized in a region of the gene associated with a previously described signal of positive selection, it is a plausible candidate for having caused a recent selective sweep in the FOXP2 gene.

Response to Comment on "Human TKTL1 implies greater neurogenesis in frontal neocortex of modern humans than Neanderthals".

Herai et al. discuss the known fact that a low percentage of modern humans who lack any overt phenotypes carry the ancestral TKTL1 allele. Our paper demonstrates that the amino acid substitution in TKTL1 increases neural progenitor cells and neurogenesis in the developing brain. It is another question if, and to what extent, this has consequences for the adult brain.

Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage.

The first step in CRISPR-Cas9-mediated genome editing is the cleavage of target DNA sequences that are complementary to so-called spacer sequences in CRISPR guide RNAs (gRNAs). However, some DNA sequences are refractory to CRISPR-Cas9 cleavage, which is at least in part due to gRNA misfolding. To overcome this problem, we have engineered gRNAs with highly stable hairpins in their constant parts and further enhanced their stability by chemical modifications. The 'Genome-editing Optimized Locked Design' (GOLD)-gRNA increases genome editing efficiency up to around 1000-fold (from 0.08 to 80.5%) with a mean increase across different other targets of 7.4-fold. We anticipate that this improved gRNA will allow efficient editing regardless of spacer sequence composition and will be especially useful if a desired genomic site is difficult to edit.

Longer metaphase and fewer chromosome segregation errors in modern human than Neanderthal brain development.

Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals.

Human TKTL1 implies greater neurogenesis in frontal neocortex of modern humans than Neanderthals.

Neanderthal brains were similar in size to those of modern humans. We sought to investigate potential differences in neurogenesis during neocortex development. Modern human transketolase-like 1 (TKTL1) differs from Neanderthal TKTL1 by a lysine-to-arginine amino acid substitution. Using overexpression in developing mouse and ferret neocortex, knockout in fetal human neocortical tissue, and genome-edited cerebral organoids, we found that the modern human variant, hTKTL1, but not the Neanderthal variant, increases the abundance of basal radial glia (bRG) but not that of intermediate progenitors (bIPs). bRG generate more neocortical neurons than bIPs. The hTKTL1 effect requires the pentose phosphate pathway and fatty acid synthesis. Inhibition of these metabolic pathways reduces bRG abundance in fetal human neocortical tissue. Our data suggest that neocortical neurogenesis in modern humans differs from that in Neanderthals.

Point-of-care bulk testing for SARS-CoV-2 by combining hybridization capture with improved colorimetric LAMP.

Efforts to contain the spread of SARS-CoV-2 have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can be applied to large numbers of people. However, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation and sophisticated laboratory equipment to achieve throughputs that allow whole communities to be tested on a regular basis. Here we present Cap-iLAMP (capture and improved loop-mediated isothermal amplification) which combines a hybridization capture-based RNA extraction of gargle lavage samples with an improved colorimetric RT-LAMP assay and smartphone-based color scoring. Cap-iLAMP is compatible with point-of-care testing and enables the detection of SARS-CoV-2 positive samples in less than one hour. In contrast to direct addition of the sample to improved LAMP (iLAMP), Cap-iLAMP prevents false positives and allows single positive samples to be detected in pools of 25 negative samples, reducing the reagent cost per test to ~1 Euro per individual.

Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic knock-out adipocyte cell lines.

Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out.

Comment on "Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment".

Trujillo et al. (Research Articles, 12 February 2021, eaax2537) conclude that the reintroduction of an ancestral amino acid substitution in the protein NOVA1 drastically alters the development of brain organoids. We show that cell lines used by the authors carry heterozygous deletions of the target DNA sequence, providing another plausible explanation for the effects observed.

Reduced purine biosynthesis in humans after their divergence from Neandertals.

We analyze the metabolomes of humans, chimpanzees, and macaques in muscle, kidney and three different regions of the brain. Although several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution contributes to much or all of the reduction of de novo synthesis of purines in humans.

From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing.

Current efforts to recover the Neandertal and mammoth genomes by 454 DNA sequencing demonstrate the sensitivity of this technology. However, routine 454 sequencing applications still require microgram quantities of initial material. This is due to a lack of effective methods for quantifying 454 sequencing libraries, necessitating expensive and labour-intensive procedures when sequencing ancient DNA and other poor DNA samples. Here we report a 454 sequencing library quantification method based on quantitative PCR that effectively eliminates these limitations. We estimated both the molecule numbers and the fragment size distributions in sequencing libraries derived from Neandertal DNA extracts, SAGE ditags and bonobo genomic DNA, obtaining optimal sequencing yields without performing any titration runs. Using this method, 454 sequencing can routinely be performed from as little as 50 pg of initial material without titration runs, thereby drastically reducing costs while increasing the scope of sample throughput and protocol development on the 454 platform. The method should also apply to Illumina/Solexa and ABI/SOLiD sequencing, and should therefore help to widen the accessibility of all three platforms.

A Neanderthal Sodium Channel Increases Pain Sensitivity in Present-Day Humans.

The sodium channel Nav1.7 is crucial for impulse generation and conduction in peripheral pain pathways [1]. In Neanderthals, the Nav1.7 protein carried three amino acid substitutions (M932L, V991L, and D1908G) relative to modern humans. We expressed Nav1.7 proteins carrying all combinations of these substitutions and studied their electrophysiological effects. Whereas the single amino acid substitutions do not affect the function of the ion channel, the full Neanderthal variant carrying all three substitutions, as well as the combination of V991L with D1908G, shows reduced inactivation, suggesting that peripheral nerves were more sensitive to painful stimuli in Neanderthals than in modern humans. We show that, due to gene flow from Neanderthals, the three Neanderthal substitutions are found in ∼0.4% of present-day Britons, where they are associated with heightened pain sensitivity.

A direct RT-qPCR approach to test large numbers of individuals for SARS-CoV-2.

SARS-CoV-2 causes substantial morbidity and mortality in elderly and immunocompromised individuals, particularly in retirement homes, where transmission from asymptomatic staff and visitors may introduce the infection. Here we present a cheap and fast screening method based on direct RT-qPCR to detect SARS-CoV-2 in single or pooled gargle lavages ("mouthwashes"). This method detects individuals with large viral loads (Ct≤29) and we use it to test all staff at a nursing home daily over a period of three weeks in order to reduce the risk that the infection penetrates the facility. This or similar approaches can be implemented to protect hospitals, nursing homes and other institutions in this and future viral epidemics.

Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands.

To increase the yield of DNA sequence generated by the 454 technology from small amounts of starting DNA, we investigated the efficiency of each step in the 454 library preparation process. We find that the last step, when the single-stranded library is released by NaOH, is inefficient and highly variable. When this step is replaced with heat treatment, library amounts dramatically increase. Furthermore, when sequencing templates are first isolated by NaOH treatment and subsequently by heat treatment, the sequences of both strands of individual template DNA molecules can be determined. Using this approach, we confirm that C/G base pairs observed as T/A base pairs in Neanderthal DNA sequences are due to a modification of the cytosine rather than guanine residues.