Improvements in Gene Editing Technology Boost Its Applications in Livestock

Accelerated development of novel CRISPR/Cas9-based genome editing techniques provides a feasible approach to introduce a variety of precise modifications in the mammalian genome, including introduction of multiple edits simultaneously, efficient insertion of long DNA sequences into specific targeted loci as well as performing nucleotide transitions and transversions. Thus, the CRISPR/Cas9 tool has become the method of choice for introducing genome alterations in livestock species. The list of new CRISPR/Cas9-based genome editing tools is constantly expanding. Here, we discuss the methods developed to improve efficiency and specificity of gene editing tools as well as approaches that can be employed for gene regulation, base editing, and epigenetic modifications. Additionally, advantages and disadvantages of two primary methods used for the production of gene-edited farm animals: somatic cell nuclear transfer (SCNT or cloning) and zygote manipulations will be discussed. Furthermore, we will review agricultural and biomedical applications of gene editing technology.

Trends of Mutation Accumulation Across Global SARS-CoV-2 Genomes: Implications for the Evolution of the Novel Coronavirus

To understand SARS-CoV-2 microevolution, this study explored the genome-wide frequency, gene-wise distribution, and molecular nature of all point-mutations detected across its 71,703 RNA-genomes deposited in the GISAID repository, till 21 August 2020. Globally, nsp1/nsp2/nsp3/ nsp11 and orf7a/orf3a/S were the most mutation-ridden non-structural and structural genes respectively. Phylogeny based on 4,618 spatiotemporally-representative genomes revealed that entities belonging to the early lineages are mostly spread over Asian countries (including India, the biggest hotspot of the pandemic) whereas the recently-derived lineages are more globally distributed. Of the total 16,602 polymorphism-bearing sites in the pan-genome, 11,037 and 4,965 involved transitions and transversions, which in turn were predominated by cytidine-to-uridine and guanosine-to-uridine conversions, respectively. Positive selection of nonsynonymous mutations (dN/dS >1) in most of the structural, but not non-structural, genes indicated that SARS-CoV-2 has already harmonized its replication/transcription machineries with the host’s metabolic system, while it is still redefining virulence/transmissibility strategies at the molecular level.

Comparative Analysis of Nucleotide Variability of the mtDNA cyt b Gene of tench (Tinca tinca L.) in Populations of the Western and Eastern Parts of Eurasia

Линь (Tinca tinca L.) – один из древнейших сохранившихся представителей семейства карповых (Cyprinidae) в водоёмах Евразии, выделяемый в отдельное монотипическое подсемейство Tincinae. Ранее были выявлены филогеографические группы, разделяющиеся на западные (Англия, Испания, Польша) и восточные (Болгария, Иран, Китай) популяции Евразии. Была поставлена задача установить, к какой филогруппе будут относиться популяции северо-восточной части России, в частности, Ярославской области, а также оценить максимальное правдоподобие нуклеотидных замещений по гену cyt b. Проведён сравнительный анализ полученных данных с данными по линю западной и восточной частей Евразии. Оказалось, что общее смещение транзиции/трансверсии в российских популяциях составляет R = 168,236, в западных популяциях – R = 488,667. В популяциях линя Рыбинского водохранилища в значительной степени трансзиции преобладают над трансверсиями. Это свидетельствует о высоком генетическом разнообразии, устойчивости и адаптационных характеристиках линя из Рыбинского водохранилища. В западных популяциях количество трансзиций и трансверсий по своим значениям близки, наблюдается небольшое преобладание трансзиций над трансверсиями. В основном западные популяции линя имеют не естественное, а искусственное происхождение, являясь результатом направленного антропогенного скрещивания. Соответственно значительно снижаются показатели устойчивости и адаптации, а также повышается риск возникновения вредных (летальных) спонтанных мутаций, которые могут быстро закрепиться и повлиять на состояние западных популяций линя. Это подтверждается коэффициентами скорости транзиции/трансверсии: в западных популяциях они практически одинаковы k1 = 985.029 (пурины) и k2 = 1000 (пиримидины), в российских популяциях коэффициенты скорости транзиции/трансверсии составляют k1 = 1000 (пурины) и k2 = 1 (пиримидины). Проведённый анализ подтверждает существование двух филогеографических групп, но в связи с недостатком данных по российским популяциям необходимо продолжение исследования, тем более, что российская (в частности ярославская) популяция занимает особое положение по соотношению транзиций и трансверсий. Tench (Tinca tinca L.) is one of the oldest surviving representatives of the cyprinid family (Cyprinidae) in water bodies of Eurasia distinguished into a separate monotypic subfamily Tincinae. Earlier phylogeographic groups were identified divided into western (England, Spain, Poland) and eastern (Bulgaria, Iran, China) populations of Eurasia. The task was to establish which phylogroup the populations of the northeastern part of Russia, in particular, the Yaroslavl region, would belong to, as well as to assess the maximum likelihood of nucleotide substitutions on the cyt b gene. A comparative analysis of the obtained data was carried out with data on the line of the western and eastern parts of Eurasia. It turned out that the total displacement of transition / transversion in Russian populations is R = 168.236, in western populations – R = 488.667. In the tench populations of the Rybinsk reservoir, to a large extent, transitions prevail over transversions. This indicates a high genetic diversity, resistance and adaptive characteristics of tench from the Rybinsk Reservoir. In western populations the number of transitions and transversions are close in their values, there is a small predominance of transitions over transversions. Basically, the western populations of tench are not of natural but artificial origin, being a result of directed anthropogenic crossing. Accordingly, the indicators of resistance and adaptation are significantly reduced and the risk of harmful (lethal) spontaneous mutations which can quickly gain a foothold and affect the state of western tench populations increases. This is confirmed by the coefficients of the rate of transition / transversion: in western populations they are practically the same k1 = 985.029 (purines) and k2 = 1000 (pyrimidines), in Russian populations the rates of transition / transversion are k1 = 1000 (purines) and k2 = 1 (pyrimidines). The performed analysis confirms the existence of two phylogeographic groups, but due to the lack of data on Russian populations, it is necessary to continue the research especially since the Russian (in particular Yaroslavl) population occupies a special position in terms of the ratio of transitions and transversions.

Population dynamics of GC-changing mutations in humans and great apes

BackgroundThe nucleotide composition of the genome is a balance between origin and fixation rates of different mutations. For example, it is well-known that transitions occur more frequently than transversions, particularly at CpG sites. Differences in fixation rates of mutation types are less explored. Specifically, recombination-associated GC-biased gene conversion (gBGC) may differentially impact GC-changing mutations, due to differences in their genomic distributions and efficiency of mismatch repair mechanisms. Given that recombination evolves rapidly across species, we explore gBGC of different mutation types across human populations and among great ape species.ResultsWe report a stronger correlation between GC frequency and recombination for transitions than for transversions. Notably, CpG transitions are most strongly affected by gBGC. We show that the strength of gBGC differs for transitions and transversions but that its overall strength is positively correlated with effective population sizes of human populations and great ape species, with some notable exceptions, such as a stronger effect of gBGC on non-CpG transitions in populations of European descent. We study the dependence of gBGC dynamics on flanking nucleotides and show that some mutation types evolve in opposition to the gBGC expectation, likely due to hypermutability of specific nucleotide contexts.ConclusionsDifferences in GC-biased gene conversion are evident between different mutation types, and dependent on sex-specific recombination, population size and flanking nucleotide context. Our results therefore highlight the importance of different gBGC dynamics experienced by GC-changing mutations and their impact on nucleotide composition evolution.

Prime editing is achievable in the tetraploid potato, but needs improvement

Since its discovery and first applications for genome editing in plants, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology has revolutionized plant research and precision crop breeding. Although the classical CRISPR-Cas9 system is highly useful for the introduction of targeted small mutations for knock-out applications, this system is mostly inefficient for the introduction of precise and predictable nucleotide substitutions. Recently, the prime editing (PE) technology has been developed in human cells, allowing the introduction of all kinds of mutations, including the simultaneous generation of nucleotide transitions and transversions. Therefore, this system holds great promises for the production of gain-of-function mutants and for the improvement of precision breeding in crops. In this study, we report on the successful use of prime editing in the tetraploid and highly heterozygous potato (Solanum tuberosum) with the introduction of simultaneous nucleotide transitions and transversions in the StALS1 gene.

Energy mapping of the genetic code and genomic domains: implications for code evolution and molecular Darwinism

When the iconic DNA genetic code is expressed in terms of energy differentials, one observes that information embedded in chemical sequences, including some biological outcomes, correlate with distinctive free energy profiles. Specifically, we find correlations between codon usage and codon free energy, suggestive of a thermodynamic selection for codon usage. We also find correlations between what are considered ancient amino acids and high codon free energy values. Such correlations may be reflective of the sequence-based genetic code fundamentally mapping as an energy code. In such a perspective, one can envision the genetic code as composed of interlocking thermodynamic cycles that allow codons to ‘evolve’ from each other through a series of sequential transitions and transversions, which are influenced by an energy landscape modulated by both thermodynamic and kinetic factors. As such, early evolution of the genetic code may have been driven, in part, by differential energetics, as opposed exclusively by the functionality of any gene product. In such a scenario, evolutionary pressures can, in part, derive from the optimization of biophysical properties (e.g. relative stabilities and relative rates), in addition to the classic perspective of being driven by a phenotypical adaptive advantage (natural selection). Such differential energy mapping of the genetic code, as well as larger genomic domains, may reflect an energetically resolved and evolved genomic landscape, consistent with a type of differential, energy-driven ‘molecular Darwinism’. It should not be surprising that evolution of the code was influenced by differential energetics, as thermodynamics is the most general and universal branch of science that operates over all time and length scales.

Shed skin as a source of DNA for genotyping-by-sequencing (GBS) in reptiles

Association and genetic mapping studies aimed at linking genotype to phenotype are powerful tools that require large numbers of samples, complicating their use in long-lived species with low fecundity. Shed skins of snakes and other reptiles contain DNA; are a safe and ethical way of non-invasively sampling large numbers of individuals; and provide a simple mechanism by which to involve the public in scientific research. Here we test whether the DNA in dried shed skins mailed to us from citizen scientists is suitable for reduced representation sequencing approaches, specifically genotyping-by-sequencing (GBS). We find that shed skin samples provide DNA of sufficient quality and quantity for GBS, although libraries from shed skin resulted in fewer sequenced reads than libraries from snap-frozen muscle, and contained slightly fewer variants (70,685 SNPs versus 97,724). This issue is a direct result of lower read counts of the shed skin samples, and can be rectified quite simply with deeper sequencing. Skin-derived libraries also have a very slight (but significantly different) profile of transitions and transversions, suggesting that DNA damage occurs but is minimal. We conclude that shed skin-derived DNA is a good source of genomic DNA for a variety of genetic studies, and use it to identify sex-linked scaffolds in the corn snake genome.

A QM/QTAIM research under the magnifying glass of the DPT tautomerisation of the wobble mispairs involving 2-aminopurine

In this study, a comprehensive survey of the changes of the physico-chemical parameters at each point of the IRC for the biologically important T·2AP*(w) ↔ T*·2AP(w) and G·2AP*(w) ↔ G*·2AP(w) DPT tautomerisation reactions involved in the point mutations (transitions and transversions) induced by 2-aminopurine (2AP) in DNA is provided.

Evaluation of genotoxicity of acetamiprid using PCR technique on mosquito genome

The present studies deal with the evaluation of the genotoxic potential of acetamiprid at LD40 on a mosquito Culex quinquefasciatus by adopting polymerase chain reaction technique (PCR). This technique was used for detecting DNA damage by amplifying ribosomal DNA internal transcribed spacer 2 (ITS 2) regions. The amplified products were sequenced and the results of treated and non-treated controls were compared using Clustal W software programme. The results were studied in the form of deletions, additions, transitions and transversions of the bases. The DNA band amplified from control stocks consisted of 444 bases while those from LD40 treated individuals were comprised of 448 bases. The total number of mutations caused in the treated stock was 230 out of which 84 were transitions, 117 transversions, 13 deletions and 16 additions. Thus, it was evident that acetamiprid has a potential to promote gene mutations in the individuals exposed to its semilethal doses.