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The substitution rates of transitions are higher than expected by chance relative to those of transversions. Many have argued that selection disfavors transversions, as nonsynonymous transversions are less likely to conserve biochemical properties of the original amino acid. Only recently has it become feasible to directly test this selective hypothesis by comparing the fitness effects of a large number of transition and transversion mutations. For example, a recent study of six viruses and one beta-lactamase gene did not find evidence supporting the selective hypothesis. Here, we analyze the relative fitness effects of transition and transversion mutations from our recently published genome-wide study of mutational fitness effects in influenza virus. In contrast to prior work, we find that transversions are significantly more detrimental than transitions. Using what we believe to be an improved statistical framework, we also identify a similar trend in two HIV data sets. We further demonstrate a fitness difference in transition and transversion mutations using four deep mutational scanning data sets of influenza virus and HIV, which provided adequate statistical power. We find that three of the most commonly cited radical/conservative amino acid categories are predictive of fitness, supporting their utility in studies of positive selection and codon usage bias. We conclude that selection is a major contributor to the transition:transversion substitution bias in viruses and that this effect is only partially explained by the greater likelihood of transversion mutations to cause radical as opposed to conservative amino acid changes.

Abstract Nucleotides across a genome do not mutate at equal frequencies. Instead, specific nucleotide positions can exhibit much higher mutation rates than the genomic average due to their immediate nucleotide neighbors. These “mutational hotspots” can play a prominent role in adaptive evolution, yet we lack knowledge of which short nucleotide sequences drive hotspots. In this work, we employ a combination of experimental evolution with Pseudomonas fluorescens and bioinformatic analysis of various Salmonella species to characterize a short nucleotide motif (≥8 bp) that can drive T:A→G:C mutation rates >1000-fold higher than the baseline T→G rate in bacteria. First, we experimentally confirm previous analysis showing that homopolymeric tracts (≥3) of G with a 3′ T frequently mutate so that the T is replaced with a G, resulting in an extension of the guanine tract, i.e. GGGT → GGGG. We then demonstrate that the potency of this T:A→G:C hotspot is dependent on the nucleotides immediately flanking the GnT sequence. We find that the dinucleotide immediately 5′ to a G4 tract and the dinucleotide immediately 3′ to the T strongly affect the T:A→G:C mutation rate, which ranges from ∼5-fold higher than the typical rate to over 1000-fold higher depending on the flanking elements. GnT motifs are therefore comprised of several modular nucleotide components which each exert a significant, quantifiable effect on the mutation rate. This work advances our ability to accurately identify the position and quantify the mutagenicity of hotspot motifs predicated on short nucleotide sequences.

The year 2020 marks a decade since the first gene-edited plants were generated using homing endonucleases and zinc finger nucleases. The advent of CRISPR/Cas9 for gene-editing in 2012 was a major science breakthrough that revolutionized both basic and applied research in various organisms including plants and consequently honored with “The Nobel Prize in Chemistry, 2020.” CRISPR technology is a rapidly evolving field and multiple CRISPR-Cas derived reagents collectively offer a wide range of applications for gene-editing and beyond. While most of these technological advances are successfully adopted in plants to advance functional genomics research and development of innovative crops, others await optimization. One of the biggest bottlenecks in plant gene-editing has been the delivery of gene-editing reagents, since genetic transformation methods are only established in a limited number of species. Recently, alternative methods of delivering CRISPR reagents to plants are being explored. This review mainly focuses on the most recent advances in plant gene-editing including (1) the current Cas effectors and Cas variants with a wide target range, reduced size and increased specificity along with tissue specific genome editing tool kit (2) cytosine, adenine, and glycosylase base editors that can precisely install all possible transition and transversion mutations in target sites (3) prime editing that can directly copy the desired edit into target DNA by search and replace method and (4) CRISPR delivery mechanisms for plant gene-editing that bypass tissue culture and regeneration procedures including de novo meristem induction, delivery using viral vectors and prospects of nanotechnology-based approaches.

The effect of ubiquitous environmental conditions on mutational mechanisms, particularly loss of heterozygosity (LOH) remains poorly understood. Environment induced LOH can rapidly alter the genome and promote disease progression. Using mutation accumulation (MA) lines, we analysed the effect of ubiquitous environmental conditions on mutational mechanisms in a diploid hybrid (S288c/YJM789) baker’s yeast strain. These included blue light, low glucose (calorie restriction), oxidative stress (H 2 O 2 ), high temperature (37°C), ethanol, and salt (NaCl). The frequency of LOH increased significantly in all environments including calorie restriction relative to the control (YPD). Interestingly, the percentage of the genome covered by LOH varied significantly depending on the condition. For example, the LOH tracts seen in calorie restriction conditions were significantly shorter than those observed in blue light exposure that rapidly homozygotized the genome. We also report a unique mutational signature of blue light exposure comprising LOH, small indels, large deletions and transversion mutations (G:C > T:A; G:C > C:G), with the latter likely to result from the photooxidation of guanine bases. Our results suggest ubiquitous environmental conditions cause LOH but result in distinct mutational signatures due to the type of damage induced and the pathways used to repair them.

Molnupiravir is an antiviral, currently approved by the UK Medicines and Healthcare products Regulatory Agency (MHRA) for treating at-risk COVID-19 patients, that induces lethal error catastrophe in SARS-CoV-2. How this drug-induced mechanism of action might impact the emergence of resistance mutations is unclear. To investigate this, we used samples from the AGILE Candidate Specific Trial (CST)−2 (clinical trial number NCT04746183). The primary outcomes of AGILE CST-2 were to measure the drug safety and antiviral efficacy of molnupiravir in humans (180 participants randomised 1:1 with placebo). Here, we describe the pre-specified exploratory virological endpoint of CST-2, which was to determine the possible genomic changes in SARS-CoV-2 induced by molnupiravir treatment. We use high-throughput amplicon sequencing and minor variant analysis to characterise viral genomics in each participant whose longitudinal samples (days 1, 3 and 5 post-randomisation) pass the viral genomic quality criteria ( n  = 59 for molnupiravir and n  = 65 for placebo). Over the course of treatment, no specific mutations were associated with molnupiravir treatment. We find that molnupiravir significantly increased the transition:transversion mutation ratio in SARS-CoV-2, consistent with the model of lethal error catastrophe. This study highlights the utility of examining intra-host virus populations to strengthen the prediction, and surveillance, of potential treatment-emergent adaptations. Molnupiravir is an antiviral that forces lethal error catastrophe in SARS-CoV-2 RNAs. Here, the authors confirm the mechanism of action of molnupiravir in humans using samples obtained from the UK’s AGILE phase IIa clinical trial investigating the antiviral efficacy of the drug against SARS-CoV-2. No treatment-associated SARS-CoV-2 mutations were identified.

Background Chloroplast genome sequence data is very useful in studying/addressing the phylogeny of plants at various taxonomic ranks. However, there are no empirical observations on the patterns, directions, and mutation rates, which are the key topics in chloroplast genome evolution. In this study, we used Calycanthaceae as a model to investigate the evolutionary patterns, directions and rates of both nucleotide substitutions and structural mutations at different taxonomic ranks. Results There were 2861 polymorphic nucleotide sites on the five chloroplast genomes, and 98% of polymorphic sites were biallelic. There was a single-nucleotide substitution bias in chloroplast genomes. A → T or T → A (2.84%) and G → C or C → G (3.65%) were found to occur significantly less frequently than the other four transversion mutation types. Synonymous mutations kept balanced pace with nonsynonymous mutations, whereas biased directions appeared between transition and transversion mutations and among transversion mutations. Of the structural mutations, indels and repeats had obvious directions, but microsatellites and inversions were non-directional. Structural mutations increased the single nucleotide mutations rates. The mutation rates per site per year were estimated to be 0.14–0.34 × 10− 9 for nucleotide substitution at different taxonomic ranks, 0.64 × 10− 11 for indels and 1.0 × 10− 11 for repeats. Conclusions Our direct counts of chloroplast genome evolution events provide raw data for correctly modeling the evolution of sequence data for phylogenetic inferences.

Sickle-cell disease (SCD) is caused by an A·T-to-T·A transversion mutation in the β -globin gene ( HBB ). Here we show that prime editing can correct the SCD allele ( HBB S ) to wild type ( HBB A ) at frequencies of 15%–41% in haematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Seventeen weeks after transplantation into immunodeficient mice, prime-edited SCD HSPCs maintained HBB A levels and displayed engraftment frequencies, haematopoietic differentiation and lineage maturation similar to those of unedited HSPCs from healthy donors. An average of 42% of human erythroblasts and reticulocytes isolated 17 weeks after transplantation of prime-edited HSPCs from four SCD patient donors expressed HBB A , exceeding the levels predicted for therapeutic benefit. HSPC-derived erythrocytes carried less sickle haemoglobin, contained HBB A -derived adult haemoglobin at 28%–43% of normal levels and resisted hypoxia-induced sickling. Minimal off-target editing was detected at over 100 sites nominated experimentally via unbiased genome-wide analysis. Our findings support the feasibility of a one-time prime editing SCD treatment that corrects HBB S to HBB A , does not require any viral or non-viral DNA template and minimizes undesired consequences of DNA double-strand breaks. Prime editing can efficiently correct the sickle-cell allele to produce wild-type haemoglobin in patient haematopoietic stem cells that engraft efficiently in mice, yielding erythrocytes resistant to hypoxia-induced sickling.

In vivo genetic diversifiers have previously enabled efficient searches of genetic variant fitness landscapes for continuous directed evolution. However, existing genomic diversification modalities for mammalian genomic loci exclusively rely on deaminases to generate transition mutations within target loci, forfeiting access to most missense mutations. Here, we engineer CRISPR-guided error-prone DNA polymerases (EvolvR) to diversify all four nucleotides within genomic loci in mammalian cells. We demonstrate that EvolvR generates both transition and transversion mutations throughout a mutation window of at least 40 bp and implement EvolvR to evolve previously unreported drug-resistant MAP2K1 variants via substitutions not achievable with deaminases. Moreover, we discover that the nickase’s mismatch tolerance limits EvolvR’s mutation window and substitution biases in a gRNA-specific fashion. To compensate for gRNA-to-gRNA variability in mutagenesis, we maximize the number of gRNA target sequences by incorporating a PAM-flexible nickase into EvolvR. Finally, we find a strong correlation between predicted free energy changes underlying R-loop formation and EvolvR’s performance using a given gRNA. The EvolvR system diversifies all four nucleotides to enable the evolution of mammalian cells, while nuclease and gRNA-specific properties underlying nickase fidelity can be engineered to further enhance EvolvR’s mutation rates. Tools for diversifying genomic DNA in mammalian cells have long relied on base editors making C to T or A to G substitutions. Here, authors use RNA-guided DNA polymerases (EvolvR) to evolve mammalian cells using all twelve substitutions and show that nickase fidelity affects EvolvR’s mutation rates.

The mutant has a unique phenotype. It arrests sexual development when the fruiting bodies (perithecia) attain only 40% of their normal diameter, regardless of whether the mutant participates in a cross with the wild type ( x ) as the male or female parent. I first learnt about when this journal invited me to review ' ' by D. D. Perkins, A. Radford and M. S. Sachs ( 80: 53-54, 2001). The compendium also informed me that the first Neurospora genetic map was published here ( 32, 243-256, 1936). The mutant was discovered and characterized by T. E. Johnson, who also localized the mutation to a chromosome 1 segment that spanned more than 3.3 Mb DNA ( 92, 1107-1120, 1979). The second paper came 30 years later from my laboratory. We mapped the mutation to a single base pair, a T:A to A:T transversion mutation, and thus identified the altered gene ( 88: 33-39, 2009). To map , we leveraged our expertise in making strains bearing chromosome segment duplications. The strains were generated in crosses of the wild type with translocation strains ( x ). A translocation transfers a segment of one chromosome into another. Mapping with s localized to a 330 kbp segment. Conventional mapping with crossovers and selection against noncrossovers subsequently localized it to a 33 kbp segment. This interval was small enough to pick up the mutation by sequencing its DNA. The Fmf-1 protein activates genes required for mating pheromone signalling. The male gametes (conidia) fail to secrete the pheromone that attracts receptors on the female sexual structures (protoperithecia). Conversely, protoperithecia do not express the cognate receptor for the pheromone from the conidia. Consequently, the x cross fails to fertilize protoperithecia and arrests their maturation into perithecia. Genetic mapping, especially mapping, fails to impress many nongeneticists these days. How do x crosses produce progeny? Why are s and crossovers even needed? Why select against noncrossovers? Why not just sequence the genomes of the wild type and mutant, identify genes whose DNA is altered in the mutant, and then test them one by one? Many forget that DNA sequencing, especially of 'hard to access' centromeric sequences, was not as easy and inexpensive then. Isolating offered us the possibility of enriching for RIP-defective mutants. RIP is a mutational process that occurs during a sexual cross and induces multiple G:C to A:T transition mutations in all copies of any DNA sequences duplicated in the otherwise haploid Neurospora genome. It is the most mutagenic process known in biology. Reputedly, linked duplications were 'mutated at frequencies of 95% or more' ( 75: 313-324, 1996). My student, Srividhya Iyer, created a linked duplication of by inserting a second copy of it within 5 kbp of the endogenous gene. Most progeny from duplication-homozygous crosses would inherit a RIP-mutated allele, rendering them infertile. If the f progeny are germinated en masse, and allowed to randomly inter-cross, then only crosses between the minor fraction of non-RIPed progeny can generate the f . Likewise, for the f , f , etc. Later generations, hence, become progressively enriched for RIP-defective mutants. In the f progeny examined by Iyer, the RIP-induced mutant fraction was not 95%, but 'merely' 85%, a lesser enrichment efficiency than we desired. Therefore, the enrichment attempt was abandoned. This is not for the first time, nor the last, that a beautiful strategy was killed by an ugly fact.