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Cell Oct 2023SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by...
SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.
Topics: Animals; Humans; Mice; CRISPR-Cas Systems; Cryoelectron Microscopy; Gene Editing; Mutation; Genetic Therapy
PubMed: 37776859
DOI: 10.1016/j.cell.2023.08.031 -
Single-molecule genome-wide mutation profiles of cell-free DNA for non-invasive detection of cancer.Nature Genetics Aug 2023Somatic mutations are a hallmark of tumorigenesis and may be useful for non-invasive diagnosis of cancer. We analyzed whole-genome sequencing data from 2,511 individuals...
Somatic mutations are a hallmark of tumorigenesis and may be useful for non-invasive diagnosis of cancer. We analyzed whole-genome sequencing data from 2,511 individuals in the Pan-Cancer Analysis of Whole Genomes (PCAWG) study as well as 489 individuals from four prospective cohorts and found distinct regional mutation type-specific frequencies in tissue and cell-free DNA from patients with cancer that were associated with replication timing and other chromatin features. A machine-learning model using genome-wide mutational profiles combined with other features and followed by CT imaging detected >90% of patients with lung cancer, including those with stage I and II disease. The fixed model was validated in an independent cohort, detected patients with cancer earlier than standard approaches and could be used to monitor response to therapy. This approach lays the groundwork for non-invasive cancer detection using genome-wide mutation features that may facilitate cancer screening and monitoring.
Topics: Humans; Cell-Free Nucleic Acids; Prospective Studies; Mutation; Neoplasms; Mutation Rate; Lung Neoplasms
PubMed: 37500728
DOI: 10.1038/s41588-023-01446-3 -
Cell Reports Nov 2023H9N2 influenza viruses are globally endemic in birds, and a sharp increase in human infections with H9N2 occurred during 2021 to 2022. In this study, we assess the...
H9N2 influenza viruses are globally endemic in birds, and a sharp increase in human infections with H9N2 occurred during 2021 to 2022. In this study, we assess the antigenic and pathogenic impact of 23 hemagglutinin (HA) amino acid mutations. Our study reveals that three specific mutations, labeled R164Q, N166D, and I220T, are responsible for the binding of antibodies with escape mutations. Variants containing R164Q and I220T mutations increase viral replication in avian and mammalian cells. Furthermore, T150A and I220T mutations are found to enhance viral replication in mice, indicating that these mutations may have the potential to adapt mammals. Structure analysis reveals that residues 164 and 220 bearing R164Q and I220T mutations increase interactions with the surrounding residues. Our findings enrich current knowledge about the risk assessment regarding which predominant HA immune-escape mutations of H9N2 viruses may pose the greatest threat to the emergence of pandemics in birds and humans.
Topics: Humans; Animals; Mice; Hemagglutinins; Influenza A Virus, H9N2 Subtype; Influenza in Birds; Hemagglutinin Glycoproteins, Influenza Virus; Influenza, Human; Mutation; Birds; Chickens; Mammals
PubMed: 37948179
DOI: 10.1016/j.celrep.2023.113409 -
Cancer Cell Aug 2023Tumor mutational burden and heterogeneity has been suggested to fuel resistance to many targeted therapies. The cytosine deaminase APOBEC proteins have been implicated...
Tumor mutational burden and heterogeneity has been suggested to fuel resistance to many targeted therapies. The cytosine deaminase APOBEC proteins have been implicated in the mutational signatures of more than 70% of human cancers. However, the mechanism underlying how cancer cells hijack the APOBEC mediated mutagenesis machinery to promote tumor heterogeneity, and thereby foster therapy resistance remains unclear. We identify SYNCRIP as an endogenous molecular brake which suppresses APOBEC-driven mutagenesis in prostate cancer (PCa). Overactivated APOBEC3B, in SYNCRIP-deficient PCa cells, is a key mutator, representing the molecular source of driver mutations in some frequently mutated genes in PCa, including FOXA1, EP300. Functional screening identifies eight crucial drivers for androgen receptor (AR)-targeted therapy resistance in PCa that are mutated by APOBEC3B: BRD7, CBX8, EP300, FOXA1, HDAC5, HSF4, STAT3, and AR. These results uncover a cell-intrinsic mechanism that unleashes APOBEC-driven mutagenesis, which plays a significant role in conferring AR-targeted therapy resistance in PCa.
Topics: Male; Humans; Mutagenesis; Mutation; Prostatic Neoplasms; Receptors, Androgen; Chromosomal Proteins, Non-Histone; Heterogeneous-Nuclear Ribonucleoproteins; Cytidine Deaminase; Minor Histocompatibility Antigens; Polycomb Repressive Complex 1
PubMed: 37478850
DOI: 10.1016/j.ccell.2023.06.010 -
Microbiology (Reading, England) Nov 2023Genetic mutation, which provides the raw material for evolutionary adaptation, is largely a stochastic force. However, there is ample evidence showing that mutations can... (Review)
Review
Genetic mutation, which provides the raw material for evolutionary adaptation, is largely a stochastic force. However, there is ample evidence showing that mutations can also exhibit strong biases, with some mutation types and certain genomic positions mutating more often than others. It is becoming increasingly clear that mutational bias can play a role in determining adaptive outcomes in bacteria in both the laboratory and the clinic. As such, understanding the causes and consequences of mutation bias can help microbiologists to anticipate and predict adaptive outcomes. In this review, we provide an overview of the mechanisms and features of the bacterial genome that cause mutational biases to occur. We then describe the environmental triggers that drive these mechanisms to be more potent and outline the adaptive scenarios where mutation bias can synergize with natural selection to define evolutionary outcomes. We conclude by describing how understanding mutagenic genomic features can help microbiologists predict areas sensitive to mutational bias, and finish by outlining future work that will help us achieve more accurate evolutionary forecasts.
Topics: Mutation; Mutagenesis; Bacteria; Bias; Biological Evolution
PubMed: 37943288
DOI: 10.1099/mic.0.001404 -
Blood Advances Aug 2023TP53-mutated myeloid malignancies are associated with complex cytogenetics and extensive structural variants, which complicates detailed genomic analysis by conventional...
TP53-mutated myeloid malignancies are associated with complex cytogenetics and extensive structural variants, which complicates detailed genomic analysis by conventional clinical techniques. We performed whole-genome sequencing (WGS) of 42 acute myeloid leukemia (AML)/myelodysplastic syndromes (MDS) cases with paired normal tissue to better characterize the genomic landscape of TP53-mutated AML/MDS. WGS accurately determines TP53 allele status, a key prognostic factor, resulting in the reclassification of 12% of cases from monoallelic to multihit. Although aneuploidy and chromothripsis are shared with most TP53-mutated cancers, the specific chromosome abnormalities are distinct to each cancer type, suggesting a dependence on the tissue of origin. ETV6 expression is reduced in nearly all cases of TP53-mutated AML/MDS, either through gene deletion or presumed epigenetic silencing. Within the AML cohort, mutations of NF1 are highly enriched, with deletions of 1 copy of NF1 present in 45% of cases and biallelic mutations in 17%. Telomere content is increased in TP53-mutated AMLs compared with other AML subtypes, and abnormal telomeric sequences were detected in the interstitial regions of chromosomes. These data highlight the unique features of TP53-mutated myeloid malignancies, including the high frequency of chromothripsis and structural variation, the frequent involvement of unique genes (including NF1 and ETV6) as cooperating events, and evidence for altered telomere maintenance.
Topics: Humans; Chromothripsis; Mutation; Chromosome Aberrations; Leukemia, Myeloid, Acute; Myeloproliferative Disorders; Myelodysplastic Syndromes; Genomics; Tumor Suppressor Protein p53
PubMed: 37339484
DOI: 10.1182/bloodadvances.2023010156 -
Fertility and Sterility Nov 2023Approximately 80% of miscarriages happen within the first 12 weeks of gestation. More than half of early losses result from genetic defects, usually presenting as... (Review)
Review
Approximately 80% of miscarriages happen within the first 12 weeks of gestation. More than half of early losses result from genetic defects, usually presenting as abnormal chromosome numbers or gene rearrangements in the embryo. However, the impact of genetics on pregnancy loss goes well beyond embryonic aneuploidy. For example, the use of big data has recently led to the discovery of specific gene mutations that may be implicated in sporadic and recurrent miscarriages. Further, emerging data suggest that genetic factors play a role in conditions for which there is a causative association with recurrent pregnancy loss. Here, we summarize the evidence on the genetics of miscarriage and provide an overview of the diagnosis and prevention of genetic causes associated with sporadic and recurrent pregnancy loss.
Topics: Pregnancy; Female; Humans; Abortion, Habitual; Chromosome Aberrations; Aneuploidy; Mutation; Embryo, Mammalian
PubMed: 37648143
DOI: 10.1016/j.fertnstert.2023.08.952 -
Nature Medicine Dec 2023Gene therapy (GT) provides a potentially curative treatment option for patients with sickle cell disease (SCD); however, the occurrence of myeloid malignancies in GT...
Gene therapy (GT) provides a potentially curative treatment option for patients with sickle cell disease (SCD); however, the occurrence of myeloid malignancies in GT clinical trials has prompted concern, with several postulated mechanisms. Here, we used whole-genome sequencing to track hematopoietic stem cells (HSCs) from six patients with SCD at pre- and post-GT time points to map the somatic mutation and clonal landscape of gene-modified and unmodified HSCs. Pre-GT, phylogenetic trees were highly polyclonal and mutation burdens per cell were elevated in some, but not all, patients. Post-GT, no clonal expansions were identified among gene-modified or unmodified cells; however, an increased frequency of potential driver mutations associated with myeloid neoplasms or clonal hematopoiesis (DNMT3A- and EZH2-mutated clones in particular) was observed in both genetically modified and unmodified cells, suggesting positive selection of mutant clones during GT. This work sheds light on HSC clonal dynamics and the mutational landscape after GT in SCD, highlighting the enhanced fitness of some HSCs harboring pre-existing driver mutations. Future studies should define the long-term fate of mutant clones, including any contribution to expansions associated with myeloid neoplasms.
Topics: Humans; Hematopoiesis; Phylogeny; Mutation; Hematopoietic Stem Cells; Clone Cells; Anemia, Sickle Cell; Genetic Therapy; Neoplasms
PubMed: 37973947
DOI: 10.1038/s41591-023-02636-6 -
Cells Jan 2024Driver mutations are considered the cornerstone of cancer initiation. They are defined as mutations that convey a competitive fitness advantage, and hence, their... (Review)
Review
Driver mutations are considered the cornerstone of cancer initiation. They are defined as mutations that convey a competitive fitness advantage, and hence, their mutation frequency in premalignant tissue is expected to exceed the basal mutation rate. In old terms, that translates to "the survival of the fittest" and implies that a selective process underlies the frequency of cancer driver mutations. In that sense, each tissue is its own niche that creates a molecular selective pressure that may favor the propagation of a mutation or not. At the heart of this stands one of the biggest riddles in cancer biology: the tissue-predisposition to cancer driver mutations. The frequency of cancer driver mutations among tissues is non-uniform: for instance, mutations in are particularly frequent in colorectal cancer, and 99% of chronic myeloid leukemia patients harbor the driver fusion mutation, which is rarely found in solid tumors. Here, we provide a mechanistic framework that aims to explain how tissue-specific features, ranging from epigenetic underpinnings to the expression of viral transposable elements, establish a molecular basis for selecting cancer driver mutations in a tissue-specific manner.
Topics: Humans; Precancerous Conditions; Disease Susceptibility; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mutation; Mutation Rate
PubMed: 38247798
DOI: 10.3390/cells13020106 -
Scientific Reports Jul 2023Cleidocranial dysplasia (CCD) is a genetic disorder caused by mutations in the RUNX2 gene, affecting bone and teeth development. Previous studies focused on mutations in...
Cleidocranial dysplasia (CCD) is a genetic disorder caused by mutations in the RUNX2 gene, affecting bone and teeth development. Previous studies focused on mutations in the RUNX2 RHD domain, with limited investigation of mutations in the C-terminal domain. This study aimed to investigate the functional consequences of C-terminal mutations in RUNX2. Eight mutations were analyzed, and their effects on transactivation activity, protein expression, subcellular localization, and osteogenic potential were studied. Truncating mutations in the PST region and a missense mutation in the NMTS region resulted in increased transactivation activity, while missense mutations in the PST showed activity comparable to the control. Truncating mutations produced truncated proteins, while missense mutations produced normal-sized proteins. Mutant proteins were mislocalized, with six mutant proteins detected in both the nucleus and cytoplasm. CCD patient bone cells exhibited mislocalization of RUNX2, similar to the generated mutant. Mislocalization of RUNX2 and reduced expression of downstream genes were observed in MSCs from a CCD patient with the p.Ser247Valfs*3 mutation, leading to compromised osteogenic potential. This study provides insight into the functional consequences of C-terminal mutations in RUNX2, including reduced expression, mislocalization, and aberrant transactivation of downstream genes, contributing to the compromised osteogenic potential observed in CCD.
Topics: Humans; Core Binding Factor Alpha 1 Subunit; Mutation; Mutation, Missense; Cleidocranial Dysplasia
PubMed: 37500953
DOI: 10.1038/s41598-023-39293-1