Quantifying allele-specific CRISPR editing activity with CRISPECTOR2.0.

Off-target effects present a significant impediment to the safe and efficient use of CRISPR-Cas genome editing. Since off-target activity is influenced by the genomic sequence, the presence of sequence variants leads to varying on- and off-target profiles among different alleles or individuals. However, a reliable tool that quantifies genome editing activity in an allelic context is not available. Here, we introduce CRISPECTOR2.0, an extended version of our previously published software tool CRISPECTOR, with an allele-specific editing activity quantification option. CRISPECTOR2.0 enables reference-free, allele-aware, precise quantification of on- and off-target activity, by using de novo sample-specific single nucleotide variant (SNV) detection and statistical-based allele-calling algorithms. We demonstrate CRISPECTOR2.0 efficacy in analyzing samples containing multiple alleles and quantifying allele-specific editing activity, using data from diverse cell types, including primary human cells, plants, and an original extensive human cell line database. We identified instances where an SNV induced changes in the protospacer adjacent motif sequence, resulting in allele-specific editing. Intriguingly, differential allelic editing was also observed in regions carrying distal SNVs, hinting at the involvement of additional epigenetic factors. Our findings highlight the importance of allele-specific editing measurement as a milestone in the adaptation of efficient, accurate, and safe personalized genome editing.

Nicotinamide-Expanded Allogeneic Natural Killer Cells with CD38 Deletion, Expressing an Enhanced CD38 Chimeric Antigen Receptor, Target Multiple Myeloma Cells.

Natural killer (NK) cells are a vital component of cancer immune surveillance. They provide a rapid and potent immune response, including direct cytotoxicity and mobilization of the immune system, without the need for antigen processing and presentation. NK cells may also be better tolerated than T cell therapy approaches and are susceptible to various gene manipulations. Therefore, NK cells have become the focus of extensive translational research. Gamida Cell's nicotinamide (NAM) platform for cultured NK cells provides an opportunity to enhance the therapeutic potential of NK cells. CD38 is an ectoenzyme ubiquitously expressed on the surface of various hematologic cells, including multiple myeloma (MM). It has been selected as a lead target for numerous monoclonal therapeutic antibodies against MM. Monoclonal antibodies target CD38, resulting in the lysis of MM plasma cells through various antibody-mediated mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis, significantly improving the outcomes of patients with relapsed or refractory MM. However, this therapeutic strategy has inherent limitations, such as the anti-CD38-induced depletion of CD38-expressing NK cells, thus hindering ADCC. We have developed genetically engineered NK cells tailored to treat MM, in which CD38 was knocked-out using CRISPR-Cas9 technology and an enhanced chimeric antigen receptor (CAR) targeting CD38 was introduced using mRNA electroporation. This combined genetic approach allows for an improved cytotoxic activity directed against CD38-expressing MM cells without self-inflicted NK-cell-mediated fratricide. Preliminary results show near-complete abolition of fratricide with a 24-fold reduction in self-lysis from 19% in mock-transfected and untreated NK cells to 0.8% of self-lysis in CD38 knock-out CAR NK cells. Furthermore, we have observed significant enhancements in CD38-mediated activity in vitro, resulting in increased lysis of MM target cell lines. CD38 knock-out CAR NK cells also demonstrated significantly higher levels of NK activation markers in co-cultures with both untreated and αCD38-treated MM cell lines. These NAM-cultured NK cells with the combined genetic approach of CD38 knockout and addition of CD38 CAR represent a promising immunotherapeutic tool to target MM.

CRISPR/Cas9 ß-globin gene targeting in human haematopoietic stem cells.

The ß-haemoglobinopathies, such as sickle cell disease and ß-thalassaemia, are caused by mutations in the ß-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure ß-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult ß-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for ß-haemoglobinopathies.

The cell line A-to-I RNA editing catalogue.

Adenosine-to-inosine (A-to-I) RNA editing is a common post transcriptional modification. It has a critical role in protecting against false activation of innate immunity by endogenous double stranded RNAs and has been associated with various regulatory processes and diseases such as autoimmune and cardiovascular diseases as well as cancer. In addition, the endogenous A-to-I editing machinery has been recently harnessed for RNA engineering. The study of RNA editing in humans relies heavily on the usage of cell lines as an important and commonly-used research tool. In particular, manipulations of the editing enzymes and their targets are often developed using cell line platforms. However, RNA editing in cell lines behaves very differently than in normal and diseased tissues, and most cell lines exhibit low editing levels, requiring over-expression of the enzymes. Here, we explore the A-to-I RNA editing landscape across over 1000 human cell lines types and show that for almost every editing target of interest a suitable cell line that mimics normal tissue condition may be found. We provide CLAIRE, a searchable catalogue of RNA editing levels across cell lines available at http://srv00.recas.ba.infn.it/atlas/claire.html, to facilitate rational choice of appropriate cell lines for future work on A-to-I RNA editing.

A Comprehensive TALEN-Based Knockout Library for Generating Human-Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases.

RATIONALE: Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome. OBJECTIVE: The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro. METHODS AND RESULTS: By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development. CONCLUSIONS: Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.

Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells.

Genome-editing technologies are currently being translated to the clinic. However, cellular effects of the editing machinery have yet to be fully elucidated. Here, we performed global microarray-based gene expression measurements on human CD34+ hematopoietic stem and progenitor cells that underwent editing. We probed effects of the entire editing process as well as each component individually, including electroporation, Cas9 (mRNA or protein) with chemically modified sgRNA, and AAV6 transduction. We identified differentially expressed genes relative to control treatments, which displayed enrichment for particular biological processes. All editing machinery components elicited immune, stress, and apoptotic responses. Cas9 mRNA invoked the greatest amount of transcriptional change, eliciting a distinct viral response and global transcriptional downregulation, particularly of metabolic and cell cycle processes. Electroporation also induced significant transcriptional change, with notable downregulation of metabolic processes. Surprisingly, AAV6 evoked no detectable viral response. We also found Cas9/sgRNA ribonucleoprotein treatment to be well tolerated, in spite of eliciting a DNA damage signature. Overall, this data establishes a benchmark for cellular tolerance of CRISPR/Cas9-AAV6-based genome editing, ensuring that the clinical protocol is as safe and efficient as possible.

Therapeutic Genome Editing and its Potential Enhancement through CRISPR Guide RNA and Cas9 Modifications.

Genome editing with engineered nucleases is a rapidly growing field thanks to transformative technologies that allow researchers to precisely alter genomes for numerous applications including basic research, biotechnology, and human gene therapy. The genome editing process relies on creating a site-specific DNA double-strand break (DSB) by engineered nucleases and then allowing the cell's repair machinery to repair the break such that precise changes are made to the DNA sequence. The recent development of CRISPR-Cas systems as easily accessible and programmable tools for genome editing accelerates the progress towards using genome editing as a new approach to human therapeutics. Here we review how genome editing using engineered nucleases works and how using different genome editing outcomes can be used as a tool set for treating human diseases. We then review the major challenges of therapeutic genome editing and we discuss how its potential enhancement through CRISPR guide RNA and Cas9 protein modifications could resolve some of these challenges.

Nuclease-mediated gene editing by homologous recombination of the human globin locus.

Tal-effector nucleases (TALENs) are engineered proteins that can stimulate precise genome editing through specific DNA double-strand breaks. Sickle cell disease and ß-thalassemia are common genetic disorders caused by mutations in ß-globin, and we engineered a pair of highly active TALENs that induce modification of 54% of human ß-globin alleles near the site of the sickle mutation. These TALENS stimulate targeted integration of therapeutic, full-length beta-globin cDNA to the endogenous ß-globin locus in 19% of cells prior to selection as quantified by single molecule real-time sequencing. We also developed highly active TALENs to human γ-globin, a pharmacologic target in sickle cell disease therapy. Using the ß-globin and γ-globin TALENs, we generated cell lines that express GFP under the control of the endogenous ß-globin promoter and tdTomato under the control of the endogenous γ-globin promoter. With these fluorescent reporter cell lines, we screened a library of small molecule compounds for their differential effect on the transcriptional activity of the endogenous ß- and γ-globin genes and identified several that preferentially upregulate γ-globin expression.

DNA damage bypass operates in the S and G2 phases of the cell cycle and exhibits differential mutagenicity.

Translesion DNA synthesis (TLS) employs low-fidelity DNA polymerases to bypass replication-blocking lesions, and being associated with chromosomal replication was presumed to occur in the S phase of the cell cycle. Using immunostaining with anti-replication protein A antibodies, we show that in UV-irradiated mammalian cells, chromosomal single-stranded gaps formed in S phase during replication persist into the G2 phase of the cell cycle, where their repair is completed depending on DNA polymerase ζ and Rev1. Analysis of TLS using a high-resolution gapped-plasmid assay system in cell populations enriched by centrifugal elutriation for specific cell cycle phases showed that TLS operates both in S and G2. Moreover, the mutagenic specificity of TLS in G2 was different from S, and in some cases overall mutation frequency was higher. These results suggest that TLS repair of single-stranded gaps caused by DNA lesions can lag behind chromosomal replication, is separable from it, and occurs both in the S and G2 phases of the cell cycle. Such a mechanism may function to maintain efficient replication, which can progress despite the presence of DNA lesions, with TLS lagging behind and patching regions of discontinuity.

PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells.

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.

Targeting Tumor-Associated Sialic Acids Using Chimeric Switch Receptors Based on Siglec-9 Enhances the Antitumor Efficacy of Engineered T Cells.

Cancer exploits different mechanisms to escape T-cell immunosurveillance, including overexpression of checkpoint ligands, secretion of immunosuppressive molecules, and aberrant glycosylation. Herein, we report that IFNγ, a potent immunomodulator secreted in the tumor microenvironment, can induce α2,6 hypersialylation in cancer cell lines derived from various histologies. We focused on Siglec-9, a receptor for sialic acid moieties, and demonstrated that the Siglec-9+ T-cell population displayed reduced effector function. We speculated that Siglec-9 in primary human T cells can act as a checkpoint molecule and demonstrated that knocking out Siglec-9 using a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system enhanced the functionality of primary human T cells. Finally, we aimed to augment cancer-specific T-cell activity by taking advantage of tumor hypersialylation. Thus, we designed several Siglec-9-based chimeric switch receptors (CSR), which included an intracellular moiety derived from costimulatory molecules (CD28/41BB) and different hinge regions. In an antigen-specific context, T cells transduced with Siglec-9 CSRs demonstrated increased cytokine secretions and upregulation of activation markers. Moreover, T cells equipped with specific Siglec-9 CSRs mediated robust antitumor activity in a xenograft model of human tumors. Overall, this work sheds light on tumor evasion mechanisms mediated by sialylated residues and exemplifies an approach to improve engineered T cell-based cancer treatment. See related Spotlight by Abken, p. 1310.

Homology-Directed-Repair-Based Genome Editing in HSPCs for the Treatment of Inborn Errors of Immunity and Blood Disorders.

Genome engineering via targeted nucleases, specifically CRISPR-Cas9, has revolutionized the field of gene therapy research, providing a potential treatment for diseases of the blood and immune system. While numerous genome editing techniques have been used, CRISPR-Cas9 homology-directed repair (HDR)-mediated editing represents a promising method for the site-specific insertion of large transgenes for gene knock-in or gene correction. Alternative methods, such as lentiviral/gammaretroviral gene addition, gene knock-out via non-homologous end joining (NHEJ)-mediated editing, and base or prime editing, have shown great promise for clinical applications, yet all possess significant drawbacks when applied in the treatment of patients suffering from inborn errors of immunity or blood system disorders. This review aims to highlight the transformational benefits of HDR-mediated gene therapy and possible solutions for the existing problems holding the methodology back. Together, we aim to help bring HDR-based gene therapy in CD34+ hematopoietic stem progenitor cells (HSPCs) from the lab bench to the bedside.

CRISPR-Cas9 engineering of the RAG2 locus via complete coding sequence replacement for therapeutic applications.

RAG2-SCID is a primary immunodeficiency caused by mutations in Recombination-activating gene 2 (RAG2), a gene intimately involved in the process of lymphocyte maturation and function. ex-vivo manipulation of a patient's own hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9/rAAV6 gene editing could provide a therapeutic alternative to the only current treatment, allogeneic hematopoietic stem cell transplantation (HSCT). Here we show an innovative RAG2 correction strategy that replaces the entire endogenous coding sequence (CDS) for the purpose of preserving the critical endogenous spatiotemporal gene regulation and locus architecture. Expression of the corrective transgene leads to successful development into CD3+TCRαß+ and CD3+TCRγδ+ T cells and promotes the establishment of highly diverse TRB and TRG repertoires in an in-vitro T-cell differentiation platform. Thus, our proof-of-concept study holds promise for safer gene therapy techniques of tightly regulated genes.

Multiplex HDR for disease and correction modeling of SCID by CRISPR genome editing in human HSPCs.

Severe combined immunodeficiency (SCID) is a group of disorders caused by mutations in genes involved in the process of lymphocyte maturation and function. CRISPR-Cas9 gene editing of the patient's own hematopoietic stem and progenitor cells (HSPCs) ex vivo could provide a therapeutic alternative to allogeneic hematopoietic stem cell transplantation, the current gold standard for treatment of SCID. To eliminate the need for scarce patient samples, we engineered genotypes in healthy donor (HD)-derived CD34+ HSPCs using CRISPR-Cas9/rAAV6 gene-editing, to model both SCID and the therapeutic outcomes of gene-editing therapies for SCID via multiplexed homology-directed repair (HDR). First, we developed a SCID disease model via biallelic knockout of genes critical to the development of lymphocytes; and second, we established a knockin/knockout strategy to develop a proof-of-concept single-allelic gene correction. Based on these results, we performed gene correction of RAG2-SCID patient-derived CD34+ HSPCs that successfully developed into CD3+ T cells with diverse TCR repertoires in an in vitro T cell differentiation platform. In summary, we present a strategy to determine the optimal configuration for CRISPR-Cas9 gene correction of SCID using HD-derived CD34+ HSPCs, and the feasibility of translating this gene correction approach in patient-derived CD34+ HSPCs.

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi.

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.

Microfluidic tool for rapid functional characterization of CRISPR complexes.

RNA guided nucleases are regarded as the future genome editing technologies. As such, they need to meet strong safety margins. Two major challenges in incorporating CRISPR technologies into the clinical world are off-target activity and editing efficiency. The common way to tackle such issues is to measure the binding and cleavage kinetics of the CRISPR enzyme. This can be challenging since, for example, DNA is not released from the CAS9 protein post cleavage. Here a promising new microfluidic approach to characterizing Enzymatic Interaction and Function of CRISPR complexes on a microfluidic platform (EnzyMIF) is presented. The method can rapidly detect the kd, koff, km and kcat for various RNA guided nucleases. In this work, two single guide RNAs with significantly different in-cell cleavage efficiency, RAG2 and RAG1, are used as proof-of-concept. The EnzyMIF assay results provide biochemical characterization of these guide RNAs that can explain the difference in cleavage using both wild type (WT) CAS9 and HiFi CAS9. Notably, it is shown that EnzyMIF characterization correlates with cell culture genomic editing efficiency results. It is suggested that EnzyMIF can predict the quality of cleavage rapidly and quantitatively.

High-Throughput Imaging of CRISPR- and Recombinant Adeno-Associated Virus-Induced DNA Damage Response in Human Hematopoietic Stem and Progenitor Cells.

CRISPR-Cas technology has revolutionized gene editing, but concerns remain due to its propensity for off-target interactions. This, combined with genotoxicity related to both CRISPR-Cas9-induced double-strand breaks and transgene delivery, poses a significant liability for clinical genome-editing applications. Current best practice is to optimize genome-editing parameters in preclinical studies. However, quantitative tools that measure off-target interactions and genotoxicity are costly and time-consuming, limiting the practicality of screening large numbers of potential genome-editing reagents and conditions. Here, we show that flow-based imaging facilitates DNA damage characterization of hundreds of human hematopoietic stem and progenitor cells per minute after treatment with CRISPR-Cas9 and recombinant adeno-associated virus serotype 6. With our web-based platform that leverages deep learning for image analysis, we find that greater DNA damage response is observed for guide RNAs with higher genome-editing activity, differentiating even single on-target guide RNAs with different levels of off-target interactions. This work simplifies the characterization and screening process of genome-editing parameters toward enabling safer and more effective gene-therapy applications.

Different hotspot p53 mutants exert distinct phenotypes and predict outcome of colorectal cancer patients.

The TP53 gene is mutated in approximately 60% of all colorectal cancer (CRC) cases. Over 20% of all TP53-mutated CRC tumors carry missense mutations at position R175 or R273. Here we report that CRC tumors harboring R273 mutations are more prone to progress to metastatic disease, with decreased survival, than those with R175 mutations. We identify a distinct transcriptional signature orchestrated by p53R273H, implicating activation of oncogenic signaling pathways and predicting worse outcome. These features are shared also with the hotspot mutants p53R248Q and p53R248W. p53R273H selectively promotes rapid CRC cell spreading, migration, invasion and metastasis. The transcriptional output of p53R273H is associated with preferential binding to regulatory elements of R273 signature genes. Thus, different TP53 missense mutations contribute differently to cancer progression. Elucidation of the differential impact of distinct TP53 mutations on disease features may make TP53 mutational information more actionable, holding potential for better precision-based medicine.

Lessons Learned From Five Years of Newborn Screening for Severe Combined Immunodeficiency in Israel.

BACKGROUND: Implementation of newborn screening (NBS) programs for severe combined immunodeficiency (SCID) have advanced the diagnosis and management of affected infants and undoubtedly improved their outcomes. Reporting long-term follow-up of such programs is of great importance. OBJECTIVE: We report a 5-year summary of the NBS program for SCID in Israel. METHODS: Immunologic and genetic assessments, clinical analyses, and outcome data from all infants who screened positive were evaluated and summarized. RESULTS: A total of 937,953 Guthrie cards were screened for SCID. A second Guthrie card was requested on 1,169 occasions (0.12%), which resulted in 142 referrals (0.015%) for further validation tests. Flow cytometry immune-phenotyping, T cell receptor excision circle measurement in peripheral blood, and expression of TCRVß repertoire for the validation of positive cases revealed a specificity and sensitivity of 93.7% and 75.9%, respectively, in detecting true cases of SCID. Altogether, 32 SCID and 110 non-SCID newborns were diagnosed, making the incidence of SCID in Israel as high as 1:29,000 births. The most common genetic defects in this group were associated with mutations in DNA cross-link repair protein 1C and IL-7 receptor α (IL-7Rα) genes. No infant with SCID was missed during the study time. Twenty-two SCID patients underwent hematopoietic stem cell transplantation, which resulted in a 91% survival rate. CONCLUSIONS: Newborn screening for SCID should ultimately be applied globally, specifically to areas with high rates of consanguineous marriages. Accumulating data from follow-up studies on NBS for SCID will improve diagnosis and treatment and enrich our understanding of immune development in health and disease.

Stepwise-edited, human melanoma models reveal mutations' effect on tumor and microenvironment.

Establishing causal relationships between genetic alterations of human cancers and specific phenotypes of malignancy remains a challenge. We sequentially introduced mutations into healthy human melanocytes in up to five genes spanning six commonly disrupted melanoma pathways, forming nine genetically distinct cellular models of melanoma. We connected mutant melanocyte genotypes to malignant cell expression programs in vitro and in vivo, replicative immortality, malignancy, rapid tumor growth, pigmentation, metastasis, and histopathology. Mutations in malignant cells also affected tumor microenvironment composition and cell states. Our melanoma models shared genotype-associated expression programs with patient melanomas, and a deep learning model showed that these models partially recapitulated genotype-associated histopathological features as well. Thus, a progressive series of genome-edited human cancer models can causally connect genotypes carrying multiple mutations to phenotype.