Whole-genome mapping of APOBEC mutagenesis in metastatic urothelial carcinoma identifies driver hotspot mutations and a novel mutational signature.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) enzymes mutate specific DNA sequences and hairpin-loop structures, challenging the distinction between passenger and driver hotspot mutations. Here, we characterized 115 whole genomes of metastatic urothelial carcinoma (mUC) to identify APOBEC mutagenic hotspot drivers. APOBEC-associated mutations were detected in 92% of mUCs and were equally distributed across the genome, while APOBEC hotspot mutations (ApoHMs) were enriched in open chromatin. Hairpin loops were frequent targets of didymi (twins in Greek), two hotspot mutations characterized by the APOBEC SBS2 signature, in conjunction with an uncharacterized mutational context (Ap[C>T]). Next, we developed a statistical framework that identified ApoHMs as drivers in coding and non-coding genomic regions of mUCs. Our results and statistical framework were validated in independent cohorts of 23 non-metastatic UCs and 3,744 samples of 17 metastatic cancers, identifying cancer-type-specific drivers. Our study highlights the role of APOBEC in cancer development and may contribute to developing novel targeted therapy options for APOBEC-driven cancers.

CRISPR/Cas9-mediated мultiplexed multi-allelic mutagenesis of genes located on A, B and R subgenomes of hexaploid triticale.

KEY MESSAGE: The first-time generation of hexaploid triticale plants harbouring variable panels of novel mutations in gene families involved in starch biosynthesis has been achieved by the subgenome-independent multiplexed CRISPR/Cas9-mediated editing.

Cell cycle gene alterations associate with a redistribution of mutation risk across chromosomal domains in human cancers.

Mutations in human cells exhibit increased burden in heterochromatic, late DNA replication time (RT) chromosomal domains, with variation in mutation rates between tissues mirroring variation in heterochromatin and RT. We observed that regional mutation risk further varies between individual tumors in a manner independent of cell type, identifying three signatures of domain-scale mutagenesis in >4,000 tumor genomes. The major signature reflects remodeling of heterochromatin and of the RT program domains seen across tumors, tissues and cultured cells, and is robustly linked with higher expression of cell proliferation genes. Regional mutagenesis is associated with loss of activity of the tumor-suppressor genes RB1 and TP53, consistent with their roles in cell cycle control, with distinct mutational patterns generated by the two genes. Loss of regional heterogeneity in mutagenesis is associated with deficiencies in various DNA repair pathways. These mutation risk redistribution processes modify the mutation supply towards important genes, diverting the course of somatic evolution.

CRISPR/Cas9 as a Mutagenic Factor.

The discovery of the CRISPR/Cas9 microbial adaptive immune system has revolutionized the field of genetics, by greatly enhancing the capacity for genome editing. CRISPR/Cas9-based editing starts with DNA breaks (or other lesions) predominantly at target sites and, unfortunately, at off-target genome sites. DNA repair systems differing in accuracy participate in establishing desired genetic changes but also introduce unwanted mutations, that may lead to hereditary, oncological, and other diseases. New approaches to alleviate the risks associated with genome editing include attenuating the off-target activity of editing complex through the use of modified forms of Cas9 nuclease and single guide RNA (sgRNA), improving delivery methods for sgRNA/Cas9 complex, and directing DNA lesions caused by the sgRNA/Cas9 to non-mutagenic repair pathways. Here, we have described CRISPR/Cas9 as a new powerful mutagenic factor, discussed its mutagenic properties, and reviewed factors influencing the mutagenic activity of CRISPR/Cas9.

Reassessing the contribution of TOMATO AGAMOUS-LIKE1 to fruit ripening by CRISPR/Cas9 mutagenesis.

KEY MESSAGE: CRISPR/Cas9-edited TOMATO AGAMOUS-LIKE1 (TAGL1) provided new insights into fruit ripening. TOMATO AGAMOUS LIKE 1 (TAGL1) has been identified as playing a key role in the process of tomato fruit development and ripening. We have re-evaluated the functions of TAGL1 using CRISPR/Cas9 mutagenesis. Three KO mutants contained frameshift mutations resulting in premature termination codons due to a 1 bp insertion. TAGL1-KO mutants exhibited dark immature fruits and orange ripening fruits. The fruit shape was characterized by a prominent pointed tip at the end and the pericarp thickness was significantly thinner. TAGL1-KO mutants showed reduced ethylene biosynthesis, increased firmness, and delayed onset of ripening. The chlorophyll content of TAGL1-KO mutants was higher in the mature green stage and the lycopene content of TAGL1-KO mutants in the ripening stage was lower compared to the WT. ACS2, ACS4, ACO1, ACO3, PG2a, PL, PME, EXP1, and PSY1 in the mutants were significantly down-regulated during ripening. Ripening fruits in the double mutant of rin and tagl1 showed a more extreme phenotype than the rin mutant suggesting that the double mutation acts synergistically during ripening. TAGL1-targeted mutagenesis by CRISPR/Cas9 strengthens its regulatory functions controlling ripening parameters and provides new insights into fruit ripening.

CRISPR base editor-based targeted random mutagenesis (BE-TRM) toolbox for directed evolution.

Directed evolution (DE) of desired locus by targeted random mutagenesis (TRM) tools is a powerful approach for generating genetic variations with novel or improved functions, particularly in complex genomes. TRM-based DE involves developing a mutant library of targeted DNA sequences and screening the variants for the desired properties. However, DE methods have for a long time been confined to bacteria and yeasts. Lately, CRISPR/Cas and DNA deaminase-based tools that circumvent enduring barriers such as longer life cycle, small library sizes, and low mutation rates have been developed to facilitate DE in native genetic environments of multicellular organisms. Notably, deaminase-based base editing-TRM (BE-TRM) tools have greatly expanded the scope and efficiency of DE schemes by enabling base substitutions and randomization of targeted DNA sequences. BE-TRM tools provide a robust platform for the continuous molecular evolution of desired proteins, metabolic pathway engineering, creation of a mutant library of desired locus to evolve novel functions, and other applications, such as predicting mutants conferring antibiotic resistance. This review provides timely updates on the recent advances in BE-TRM tools for DE, their applications in biology, and future directions for further improvements. [BMB Reports 2024; 57(1): 30-39].

Genomics of predictive radiation mutagenesis in oilseed rape: modifying seed oil composition.

Rapeseed is a crop of global importance but there is a need to broaden the genetic diversity available to address breeding objectives. Radiation mutagenesis, supported by genomics, has the potential to supersede genome editing for both gene knockout and copy number increase, but detailed knowledge of the molecular outcomes of radiation treatment is lacking. To address this, we produced a genome re-sequenced panel of 1133 M2 generation rapeseed plants and analysed large-scale deletions, single nucleotide variants and small insertion-deletion variants affecting gene open reading frames. We show that high radiation doses (2000 Gy) are tolerated, gamma radiation and fast neutron radiation have similar impacts and that segments deleted from the genomes of some plants are inherited as additional copies by their siblings, enabling gene dosage decrease. Of relevance for species with larger genomes, we showed that these large-scale impacts can also be detected using transcriptome re-sequencing. To test the utility of the approach for predictive alteration of oil fatty acid composition, we produced lines with both decreased and increased copy numbers of Bna.FAE1 and confirmed the anticipated impacts on erucic acid content. We detected and tested a 21-base deletion expected to abolish function of Bna.FAD2.A5, for which we confirmed the predicted reduction in seed oil polyunsaturated fatty acid content. Our improved understanding of the molecular effects of radiation mutagenesis will underpin genomics-led approaches to more efficient introduction of novel genetic variation into the breeding of this crop and provides an exemplar for the predictive improvement of other crops.

Base-editing mutagenesis maps alleles to tune human T cell functions.

CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1-6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency7,8. Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules targeting base editors to protein-coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base-editing screens in primary immune cells thus provide biochemical insights with the potential to accelerate immunotherapy design.

A PCR-Induced Mutagenesis-Restriction Fragment Length Polymorphism Method for the Detection of CRISPR-Induced Indels.

As CRISPR-based technologies are widely used for knocking out genes in cell lines and organisms, there is a need for the development of reliable, cost-effective, and fast methods that identify fully mutated clones. In this context, we present a novel strategy named PCR-induced mutagenesis-restriction fragment length polymorphism (PIM-RFLP), which is based on the well-documented robustness and simplicity of the classical PCR-RFLP approach. PIM-RFLP allows the assessment of the editing efficiency in pools of edited cells and the effective identification of fully mutated single-cell clones. It is based on the creation by mutagenic PCR of a restriction enzyme degenerate cleavage site in the PCR product of the wild-type allele, which can then be distinguished from the indel-containing alleles following the standard RFLP procedure. PIM-RFLP is highly accessible, can be executed in a single day, and appears to outperform Sanger sequencing deconvolution algorithms in the detection of fully mutated clones.

Random mutagenesis as a tool for industrial strain improvement for enhanced production of antibiotics: a review.

Secondary metabolites are produced by microbes in minimal quantities in the natural environment out of necessity. However, in the pharmaceutical industry, their overproduction becomes essential. To achieve higher yields, genetic modifications are employed to create strains that surpass the productivity of the initially isolated strains. While rational screening and genetic engineering have emerged as valuable practices in recent years, the cost-effective technique of mutagenesis and selection, known as "random screening," remains a preferred method for efficient short-term strain development. This review aims to comprehensively explore all aspects of strain improvement, focusing on why random mutagenesis continues to be widely adopted.

Clonality, trafficking, and molecular alterations among Hprt mutant T lymphocytes isolated from control mice versus mice treated with N-ethyl-N-nitrosourea.

Mutations in T lymphocytes (T-cells) are informative quantitative markers for environmental mutagen exposures, but risk extrapolations from rodent models to humans also require an understanding of how T-cell development and proliferation kinetics impact mutagenic outcomes. Rodent studies have shown that patterns in chemical-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene of T-cells differ between lymphoid organs. The current work was performed to obtain knowledge of the relationships between maturation events during T-cell development and changes in chemical-induced mutant frequencies over time in differing immune compartments of a mouse model. A novel reverse transcriptase-polymerase chain reaction based method was developed to determine the specific T-cell receptor beta (Tcrb) gene mRNA expressed in mouse T-cell isolates, enabling sequence analysis of the PCR product that then identifies the specific hypervariable CDR3 junctional region of the expressed Tcrb gene for individual isolates. Characterization of spontaneous Hprt mutant isolates from the thymus, spleen, and lymph nodes of control mice for their Tcrb gene expression found evidence of in vivo clonal amplifications of Hprt mutants and their trafficking between tissues in the same animal. Concurrent analyses of Hprt mutations and Tcrb gene rearrangements in different lymphoid tissues of control versus N-ethyl-N-nitrosourea-exposed mice permitted elucidation of the localization and timing of mutational events in T-cells, establishing that mutagenesis occurs primarily in the pre-rearrangement replicative period in pre-thymic/thymic populations. These findings demonstrate that chemical-induced mutagenic burden is determined by the combination of mutagenesis and T-cell clonal expansion, processes with roles in immune function and in the pathogenesis of autoimmune disease and cancer.

Genome-wide direct quantification of in vivo mutagenesis using high-accuracy paired-end and complementary consensus sequencing.

Error-corrected next-generation sequencing (ecNGS) is an emerging technology for accurately measuring somatic mutations. Here, we report paired-end and complementary consensus sequencing (PECC-Seq), a high-accuracy ecNGS approach for genome-wide somatic mutation detection. We characterize a novel 2-aminoimidazolone lesion besides 7,8-dihydro-8-oxoguanine and the resulting end-repair artifacts originating from NGS library preparation that obscure the sequencing accuracy of NGS. We modify library preparation protocol for the enzymatic removal of end-repair artifacts and improve the accuracy of our previously developed duplex consensus sequencing method. Optimized PECC-Seq shows an error rate of <5 × 10-8 with consensus bases compressed from approximately 25 Gb of raw sequencing data, enabling the accurate detection of low-abundance somatic mutations. We apply PECC-Seq to the quantification of in vivo mutagenesis. Compared with the classic gpt gene mutation assay using gpt delta transgenic mice, PECC-Seq exhibits high sensitivity in quantitatively measuring dose-dependent mutagenesis induced by Aristolochic acid I (AAI). Moreover, PECC-Seq specifically characterizes the distinct genome-wide mutational signatures of AAI, Benzo[a]pyrene, N-Nitroso-N-ethylurea and N-nitrosodiethylamine and reveals the mutational signature of Quinoline in common mouse models. Overall, our findings demonstrate that high-accuracy PECC-Seq is a promising tool for genome-wide somatic mutagenesis quantification and for in vivo mutagenicity testing.

The chromatin network helps prevent cancer-associated mutagenesis at transcription-replication conflicts.

Genome instability is a feature of cancer cells, transcription being an important source of DNA damage. This is in large part associated with R-loops, which hamper replication, especially at head-on transcription-replication conflicts (TRCs). Here we show that TRCs trigger a DNA Damage Response (DDR) involving the chromatin network to prevent genome instability. Depletion of the key chromatin factors INO80, SMARCA5 and MTA2 results in TRCs, fork stalling and R-loop-mediated DNA damage which mostly accumulates at S/G2, while histone H3 Ser10 phosphorylation, a mark of chromatin compaction, is enriched at TRCs. Strikingly, TRC regions show increased mutagenesis in cancer cells with signatures of homologous recombination deficiency, transcription-coupled nucleotide excision repair (TC-NER) and of the AID/APOBEC cytidine deaminases, being predominant at head-on collisions. Thus, our results support that the chromatin network prevents R-loops and TRCs from genomic instability and mutagenic signatures frequently associated with cancer.

Mutagenesis-based plant breeding approaches and genome engineering: A review focused on tomato.

Breeding is the most important and efficient method for crop improvement involving repeated modification of the genetic makeup of a plant population over many generations. In this review, various accessible breeding approaches, such as conventional breeding and mutation breeding (physical and chemical mutagenesis and insertional mutagenesis), are discussed with respect to the actual impact of research on the economic improvement of tomato agriculture. Tomatoes are among the most economically important fruit crops consumed worldwide because of their high nutritional content and health-related benefits. Additionally, we summarize mutation-based mapping approaches, including Mutmap and MutChromeSeq, for the efficient mapping of several genes identified by random indel mutations that are beneficial for crop improvement. Difficulties and challenges in the adaptation of new genome editing techniques that provide opportunities to demonstrate precise mutations are also addressed. Lastly, this review focuses on various effective and convenient genome editing tools, such as RNA interference (RNAi), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), and their potential for the improvement of numerous desirable traits to allow the development of better varieties of tomato and other horticultural crops.

Evolutionary safety of lethal mutagenesis driven by antiviral treatment.

Nucleoside analogs are a major class of antiviral drugs. Some act by increasing the viral mutation rate causing lethal mutagenesis of the virus. Their mutagenic capacity, however, may lead to an evolutionary safety concern. We define evolutionary safety as a probabilistic assurance that the treatment will not generate an increased number of mutants. We develop a mathematical framework to estimate the total mutant load produced with and without mutagenic treatment. We predict rates of appearance of such virus mutants as a function of the timing of treatment and the immune competence of patients, employing realistic assumptions about the vulnerability of the viral genome and its potential to generate viable mutants. We focus on the case study of Molnupiravir, which is an FDA-approved treatment against Coronavirus Disease-2019 (COVID-19). We estimate that Molnupiravir is narrowly evolutionarily safe, subject to the current estimate of parameters. Evolutionary safety can be improved by restricting treatment with this drug to individuals with a low immunological clearance rate and, in future, by designing treatments that lead to a greater increase in mutation rate. We report a simple mathematical rule to determine the fold increase in mutation rate required to obtain evolutionary safety that is also applicable to other pathogen-treatment combinations.

Comparative de novo transcriptome analysis and random UV mutagenesis: application in high biomass and astaxanthin production enhancement for Haematococcus pluvialis.

BACKGROUND: Astaxanthin is a natural carotenoid with strong antioxidant capacity. The high demand on astaxanthin by cosmetic, food, pharmaceutical and aquaculture industries promote its value in the biotechnological research. Haematococcus pluvialis Flotow 1844 has been characterized as one of the most promising species for natural astaxanthin biosynthesis. Even though H. pluvialis as an advantage in producing astaxanthin, its slow grow-yield limits usage of the species for large-scale production. METHODS AND RESULTS: In this study we generated mutated H. pluvialis strain by using one-step random UV mutagenesis approach for higher biomass production in the green flagellated period and in turn higher astaxanthin accumulation in red stage per unit algae harvest. Isolated mutant strains were tested for the astaxanthin accumulation and yield of biomass. Among tested strains only mutant strain designated as only MT-3-7-2 showed a consistent and higher growth pattern, the rest had shown a fluctuated and then decreased growth rate than wild type. To demonstrate the phenotypical changes in MT-3-7-2 is associated with transcriptome, we carried out comparative analysis of transcriptome profiles between MT-3-7-2 and the wild type strains. De novo assembly was carried out to obtain the transcripts. Differential expression levels for the transcripts were evaluated by functional annotation analysis. CONCLUSIONS: Data showed that increased biomass for the MT-3-7-2 strain was different from wild type with expression of transcripts upregulated in carbohydrate metabolism and downregulated in lipid metabolisms. Our data suggests a switching mechanism is enrolled between carbohydrate and lipid metabolism to regulate cell proliferation and stress responses.

Targeted In Vivo Mutagenesis in Yeast Using CRISPR/Cas9 and Hyperactive Cytidine and Adenine Deaminases.

Directed evolution is a preferred strategy to improve the function of proteins such as enzymes that act as bottlenecks in metabolic pathways. Common directed evolution approaches rely on error-prone PCR-based libraries where the number of possible variants is usually limited by cellular transformation efficiencies. Targeted in vivo mutagenesis can advance directed evolution approaches and help to overcome limitations in library generation. In the current study, we aimed to develop a high-efficiency time-controllable targeted mutagenesis toolkit in the yeast Saccharomyces cerevisiae by employing the CRISPR/Cas9 technology. To that end, we fused the dCas9 protein with hyperactive variants of adenine and cytidine deaminases aiming to create an inducible CRISPR-based mutagenesis tool targeting a specific DNA sequence in vivo with extended editing windows and high mutagenesis efficiency. We also investigated the effect of guide RNA multiplexing on the mutagenesis efficiency both phenotypically and on the DNA level.

Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editing.

It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (NaVs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling NaV1.2 to identify more than 100 gRNAs that change NaV1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in NaV1.2.

CRISPR-Associated Transposase for Targeted Mutagenesis in Diverse Proteobacteria.

Genome editing tools, through the disruption of an organism's native genetic material or the introduction of non-native DNA, facilitate functional investigations to link genotypes to phenotypes. Transposons have been instrumental genetic tools in microbiology, enabling genome-wide, randomized disruption of genes and insertions of new genetic elements. Due to this randomness, identifying and isolating particular transposon mutants (i.e., those with modifications at a genetic locus of interest) can be laborious, often requiring one to sift through hundreds or thousands of mutants. Programmable, site-specific targeting of transposons became possible with recently described CRISPR-associated transposase (CASTs) systems, allowing the streamlined recovery of desired mutants in a single step. Like other CRISPR-derived systems, CASTs can be programmed by guide-RNA that is transcribed from short DNA sequence(s). Here, we describe a CAST system and demonstrate its function in bacteria from three classes of Proteobacteria. A dual plasmid strategy is demonstrated: (i) CAST genes are expressed from a broad-host-range replicative plasmid and (ii) guide-RNA and transposon are encoded on a high-copy, suicidal pUC plasmid. Using our CAST system, single-gene disruptions were performed with on-target efficiencies approaching 100% in Beta- and Gammaproteobacteria (Burkholderia thailandensis and Pseudomonas putida, respectively). We also report a peak efficiency of 45% in the Alphaproteobacterium Agrobacterium fabrum. In B. thailandensis, we performed simultaneous co-integration of transposons at two different target sites, demonstrating CAST's utility in multilocus strategies. The CAST system is also capable of high-efficiency large transposon insertion totaling over 11 kbp in all three bacteria tested. Lastly, the dual plasmid system allowed for iterative transposon mutagenesis in all three bacteria without loss of efficiency. Given these iterative capabilities and large payload capacity, this system will be helpful for genome engineering experiments across several fields of research.