Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. We generated isogenic CRISPR–Cas9 knockouts (∆) of 43 genes in human induced pluripotent stem cells, cultured them in the absence of added DNA damage and performed whole-genome sequencing of 173 subclones. ∆OGG1, ∆UNG, ∆EXO1, ∆RNF168, ∆MLH1, ∆MSH2, ∆MSH6, ∆PMS1 and ∆PMS2 produced marked mutational signatures indicative of them being critical mitigators of endogenous DNA modifications. Detailed analyses revealed mutational mechanistic insights, including how 8-oxo-2′-deoxyguanosine elimination is sequence context specific while uracil clearance is sequence context independent. Mismatch repair (MMR) deficiency signatures are engendered by oxidative damage (C > A transversions) and differential misincorporation by replicative polymerases (T > C and C > T transitions), and we propose a reverse template slippage model for T > A transversions. ∆MLH1, ∆MSH6 and ∆MSH2 signatures were similar to each other but distinct from ∆PMS2. Finally, we developed a classifier, MMRDetect, where application to 7,695 whole-genome-sequenced cancers showed enhanced detection of MMR-deficient tumors, with implications for responsiveness to immunotherapies. Nik-Zainal and colleagues leverage CRISPR–Cas9 and whole-genome sequencing to examine mutational patterns following knockout of 42 human DNA repair genes. They further develop and validate a clinically relevant tool to detect mismatch repair-deficient tumors.

The International Mouse Phenotyping Consortium reports the generation of new mouse mutant strains for more than 5,000 genes, including 2,850 novel null, 2,987 novel conditional-ready and 4,433 novel reporter alleles.

Abstract Cancer whole-genome sequencing has revealed characteristic mutational signatures associated with defective DNA repair that underpin human genetic diseases. To define the direct mutagenic effects of DNA repair deficiency at the genome-wide level, we investigate mutational signatures generated by CRISPR-Cas9-based knockouts of 42 genes involved in DNA repair/replication using a human-induced pluripotent stem cell line. Knockouts (Δ) of nine DNA repair genes reveal substitution/indel mutational signatures. Notably, dissection of signatures of defective mismatch repair (MMR) uncovers gene-specific characteristics including distinguishing features of ΔMLH1, ΔMSH2, and ΔMSH6 from ΔPMS2. This gene-specificity is also exhibited by hIPSCs derived from patients with autosomal recessive Constitutional Mismatch Repair Deficiency (CMMRD) that carry biallelic germline mutations of MMR genes. Furthermore, gene-specificity manifests in whole genome sequenced primary human cancers. Additionally, detailed analyses reveal putative sources of endogenous DNA damage that contribute to MMR signatures, including guanine oxidation, errors of DNA polymerases and reversed template slippage or double slippage. Finally, we find that using all mutational signatures of MMR-deficiency as identified in this study results in improved sensitivity and specificity in classifying MMR-deficient tumors, critical for accurate patient stratification for therapeutic intervention. Citation Format: Xueqing Zou, Gene Koh, Scott Nanda, Andrea Degasperi, Katie Urgo, Wendy Bushell, Chukwuma Agu, Vanesa Perez-Alonso, Daniel Rueda, Julia Foreman, Rebecca Harris, Josef Jiricny, Bill Skarnes, Serena Nik-Zainal. Direct mutational consequences of CRISPR-cas9 gene-edited DNA repair genes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4887.

Asymptomatic and obligatory liver stage (LS) infection of Plasmodium parasites presents an attractive target for antimalarial vaccine and drug development. Lack of robust cellular models to study LS infection has hindered the discovery and validation of host genes essential for intrahepatic parasite development. Here, we present a chemically differentiated mouse embryonic stem cell (ESC)-based LS model, which supports complete development of Plasmodium berghei exoerythrocytic forms (EEFs) and can be used to define new host-parasite interactions. Using our model, we established that host Pnpla2, coding for adipose triglyceride lipase, is dispensable for P. berghei EEF development. In addition, we also evaluated in-vitro-differentiated human hepatocyte-like cells (iHLCs) to study LS of P. berghei and found it to be a sub-optimal infection model. Overall, our results present a new mouse ESC-based P. berghei LS infection model that can be utilized to study the impact of host genetic variation on parasite development.

Chlamydia trachomatis remains a leading cause of bacterial sexually transmitted infections and preventable blindness worldwide. There are, however, limited in vitro models to study the role of host genetics in the response of macrophages to this obligate human pathogen. Here, we describe an approach using macrophages derived from human induced pluripotent stem cells (iPSdMs) to study macrophage– Chlamydia interactions in vitro . We show that iPSdMs support the full infectious life cycle of C. trachomatis in a manner that mimics the infection of human blood-derived macrophages. Transcriptomic and proteomic profiling of the macrophage response to chlamydial infection highlighted the role of the type I interferon and interleukin 10-mediated responses. Using CRISPR/Cas9 technology, we generated biallelic knockout mutations in host genes encoding IRF5 and IL-10RA in iPSCs, and confirmed their roles in limiting chlamydial infection in macrophages. This model can potentially be extended to other pathogens and tissue systems to advance our understanding of host-pathogen interactions and the role of human genetics in influencing the outcome of infections.

The International Knockout Mouse Consortium (IKMC) developed high throughput gene trapping and gene targeting pipelines that produced mostly conditional mutations of more than 18,500 genes in C57BL/6N mouse embryonic stem (ES) cells which have been archived and are freely available to the research community as a frozen resource. From this unprecedented resource more than 6,000 mutant mouse strains have been produced by the IKMC and mostly the International Mouse Phenotyping Consortium (IMPC). In addition, a cre-driver resource was established including 250 inducible cre-driver mouse strains in a C57BL/6 background. Complementing the cre-driver resource, a collection of comprising 27 cre-driver rAAVs has also been produced. The resources can be easily accessed at the IKMC/IMPC web portal (www.mousephenotype.org). The IKMC/IMPC resource is a standardized reference library of mouse models with defined genetic backgrounds that enables the analysis of gene-disease associations in mice of different genetic makeup and should therefore have a major impact on biomedical research.

The clinical complications of malaria are caused by the parasite expansion in the blood. Invasion of erythrocytes is a complex process that depends on multiple receptor-ligand interactions. Identification of host receptors is paramount for fighting the disease as it could reveal new intervention targets, but the enucleated nature of erythrocytes makes genetic approaches impossible and many receptors remain unknown. Host-parasite interactions evolve rapidly and are therefore likely to be species-specific. As a results, understanding of invasion receptors outside the major human pathogen Plasmodium falciparum is very limited. Here we use mouse embryonic stem cells (mESCs) that can be genetically engineered and differentiated into erythrocytes to identify receptors for the rodent malaria parasite Plasmodium berghei. Two proteins previously implicated in human malaria infection: glycophorin C (GYPC) and Band-3 (Slc4a1) were deleted in mESCs to generate stable cell lines, which were differentiated towards erythropoiesis. In vitro infection assays revealed that while deletion of Band-3 has no effect, absence of GYPC results in a dramatic decrease in invasion, demonstrating the crucial role of this protein for P. berghei infection. This stem cell approach offers the possibility of targeting genes that may be essential and therefore difficult to disrupt in whole organisms and has the potential to be applied to a variety of parasites in diverse host cell types.

In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.

Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.