Frequent loss of heterozygosity in CRISPR-Cas9-edited early human embryos.

CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4-20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

Genome sequence and genetic diversity of European ash trees.

Ash trees (genus Fraxinus, family Oleaceae) are widespread throughout the Northern Hemisphere, but are being devastated in Europe by the fungus Hymenoscyphus fraxineus, causing ash dieback, and in North America by the herbivorous beetle Agrilus planipennis. Here we sequence the genome of a low-heterozygosity Fraxinus excelsior tree from Gloucestershire, UK, annotating 38,852 protein-coding genes of which 25% appear ash specific when compared with the genomes of ten other plant species. Analyses of paralogous genes suggest a whole-genome duplication shared with olive (Olea europaea, Oleaceae). We also re-sequence 37 F. excelsior trees from Europe, finding evidence for apparent long-term decline in effective population size. Using our reference sequence, we re-analyse association transcriptomic data, yielding improved markers for reduced susceptibility to ash dieback. Surveys of these markers in British populations suggest that reduced susceptibility to ash dieback may be more widespread in Great Britain than in Denmark. We also present evidence that susceptibility of trees to H. fraxineus is associated with their iridoid glycoside levels. This rapid, integrated, multidisciplinary research response to an emerging health threat in a non-model organism opens the way for mitigation of the epidemic.

Resolving phylogeny and polyploid parentage using genus-wide genome-wide sequence data from birch trees.

Numerous plant genera have a history including frequent hybridisation and polyploidisation (allopolyploidisation), which means that their phylogeny is a network of reticulate evolution that cannot be accurately depicted as a bifurcating tree with a single tip per species. The genus Betula, which contains many ecologically important tree species, is a case in point. We generated genome-wide sequence reads for 27 diploid and 36 polyploid Betula species or subspecies using restriction site associated DNA (RAD) sequences. These reads were assembled into contigs with a mean length of 675 bp. We reconstructed the evolutionary relationships among diploid Betula species using both supermatrix (concatenation) and species tree methods. We identified the closest diploid relatives of the polyploids according to the relative rates at which reads from polyploids mapped to contigs from different diploid species within a concatenated reference sequence. By mapping reads from allopolyploids to their different putative diploid relatives we assembled contigs from the putative sub-genomes of allopolyploid taxa. We used these to build new phylogenies that included allopolyploid sub-genomes as separate tips. This approach yielded a highly evidenced phylogenetic hypothesis for the genus Betula, including the complex reticulate origins of the majority of its polyploid taxa. Our phylogeny divides the genus into two well supported clades, which, interestingly, differ in their seed-wing morphology. We therefore propose to split Betula into two subgenera.

Unidirectional diploid-tetraploid introgression among British birch trees with shifting ranges shown by restriction site-associated markers.

Hybridization may lead to introgression of genes among species. Introgression may be bidirectional or unidirectional, depending on factors such as the demography of the hybridizing species, or the nature of reproductive barriers between them. Previous microsatellite studies suggested bidirectional introgression between diploid Betula nana (dwarf birch) and tetraploid B. pubescens (downy birch) and also between B. pubescens and diploid B. pendula (silver birch) in Britain. Here, we analyse introgression among these species using 51 237 variants in restriction site-associated (RAD) markers in 194 individuals, called with allele dosages in the tetraploids. In contrast to the microsatellite study, we found unidirectional introgression into B. pubescens from both of the diploid species. This pattern fits better with the expected nature of the reproductive barrier between diploids and tetraploids. As in the microsatellite study, introgression into B. pubescens showed clear clines with increasing introgression from B. nana in the north and from B. pendula in the south. Unlike B. pendula alleles, introgression of B. nana alleles was found far from the current area of sympatry or allopatry between B. nana and B. pubescens. This pattern fits a shifting zone of hybridization due to Holocene reduction in the range of B. nana and expansion in the range of B. pubescens.

Generating CRISPR-Cas9-Mediated Null Mutations and Screening Targeting Efficiency in Human Pluripotent Stem Cells.

CRISPR-Cas9 mutagenesis facilitates the investigation of gene function in a number of developmental and cellular contexts. Human pluripotent stem cells (hPSCs), either embryonic or induced, are a tractable cellular model to investigate molecular mechanisms involved in early human development and cell fate decisions. hPSCs also have broad potential in regenerative medicine to model, investigate, and ameliorate diseases. Here, we provide an optimized protocol for efficient CRISPR-Cas9 genome editing of hPSCs to investigate the functional role of genes by engineering null mutations. We emphasize the importance of screening single guide RNAs (sgRNAs) to identify those with high targeting efficiency for generation of clonally derived null mutant hPSC lines. We provide important considerations for targeting genes that may have a role in hPSC maintenance. We also present methods to evaluate the on-target mutation spectrum and unintended karyotypic changes. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Selecting and ligating sgRNAs into expression plasmids Basic Protocol 2: Validation of sgRNA via in vitro transcription and cleavage assay Basic Protocol 3: Nucleofection of primed human embryonic stem cells Basic Protocol 4: MiSeq analysis of indel mutations Basic Protocol 5: Single cell cloning of targeted hPSCs Basic Protocol 6: Karyotyping of targeted hPSCs.

CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes.

Animals are essential genetic tools in scientific research and global resources in agriculture. In both arenas, a single sex is often required in surplus. The ethical and financial burden of producing and culling animals of the undesired sex is considerable. Using the mouse as a model, we develop a synthetic lethal, bicomponent CRISPR-Cas9 strategy that produces male- or female-only litters with one hundred percent efficiency. Strikingly, we observe a degree of litter size compensation relative to control matings, indicating that our system has the potential to increase the yield of the desired sex in comparison to standard breeding designs. The bicomponent system can also be repurposed to generate postnatal sex-specific phenotypes. Our approach, harnessing the technological applications of CRISPR-Cas9, may be applicable to other vertebrate species, and provides strides towards ethical improvements for laboratory research and agriculture.

Genomic assessment of local adaptation in dwarf birch to inform assisted gene flow.

When populations of a rare species are small, isolated and declining under climate change, some populations may become locally maladapted. Detecting this maladaptation may allow effective rapid conservation interventions, even if based on incomplete knowledge. Population maladaptation may be estimated by finding genome-environment associations (GEA) between allele frequencies and environmental variables across a local species range, and identifying populations whose allele frequencies do not fit with these trends. We can then design assisted gene flow strategies for maladapted populations, to adjust their allele frequencies, entailing lower levels of intervention than with undirected conservation action. Here, we investigate this strategy in Scottish populations of the montane plant dwarf birch (Betula nana). In genome-wide restriction site-associated single nucleotide polymorphism (SNP) data, we found 267 significant associations between SNP loci and environmental variables. We ranked populations by maladaptation estimated using allele frequency deviation from the general trends at these loci; this gave a different prioritization for conservation action than the Shapely Index, which seeks to preserve rare neutral variation. Populations estimated to be maladapted in their allele frequencies at loci associated with annual mean temperature were found to have reduced catkin production. Using an environmental niche modelling (ENM) approach, we found annual mean temperature (35%), and mean diurnal range (15%), to be important predictors of the dwarf birch distribution. Intriguingly, there was a significant correlation between the number of loci associated with each environmental variable in the GEA and the importance of that variable in the ENM. Together, these results suggest that the same environmental variables determine both adaptive genetic variation and species range in Scottish dwarf birch. We suggest an assisted gene flow strategy that aims to maximize the local adaptation of dwarf birch populations under climate change by matching allele frequencies to current and future environments.

ATR is a multifunctional regulator of male mouse meiosis.

Meiotic cells undergo genetic exchange between homologs through programmed DNA double-strand break (DSB) formation, recombination and synapsis. In mice, the DNA damage-regulated phosphatidylinositol-3-kinase-like kinase (PIKK) ATM regulates all of these processes. However, the meiotic functions of the PIKK ATR have remained elusive, because germline-specific depletion of this kinase is challenging. Here we uncover roles for ATR in male mouse prophase I progression. ATR deletion causes chromosome axis fragmentation and germ cell elimination at mid pachynema. This elimination cannot be rescued by deletion of ATM and the third DNA damage-regulated PIKK, PRKDC, consistent with the existence of a PIKK-independent surveillance mechanism in the mammalian germline. ATR is required for synapsis, in a manner genetically dissociable from DSB formation. ATR also regulates loading of recombinases RAD51 and DMC1 to DSBs and recombination focus dynamics on synapsed and asynapsed chromosomes. Our studies reveal ATR as a critical regulator of mouse meiosis.

From hybrids to hermaphrodites in population genetics.

A report on the 46th annual PopGroup conference, Glasgow, UK, December 18-21, 2012.