Oligonucleotide-directed Mutagenesis Research Articles

Biotechnological Innovations in Sustainable Food Production

This review article explores the critical role of biotechnology in addressing the global food security crisis aggravated by the COVID-19 pandemic and Eastern European conflict. It starts by examining the profound impacts of these events on food supply chains and pricing, supported by relevant statistical evidence. The study then discusses agricultural biotechnology, comparing conventional and sustainable agriculture, with an emphasis on genome editing and modification techniques such as Zinc Finger Nucleases (ZFNs), Oligonucleotide-directed mutagenesis (ODM), and CRISPR. The application of RNA interference in agriculture and microbial biotechnology in promoting soil health and sustainable food production is discussed. Furthermore, the review shifts focus to the utilization of microbial biotechnology for soil health enhancement and sustainable food production, encompassing the development of pest-resistant and drought-tolerant crops and biotechnological methods for optimizing water use efficiency. Additionally, it examines biotechnology's applications in food processing, including enhancing nutritional content and improving shelf life and safety. The European Green Deal is analyzed, particularly its influence on agriculture through strategies like Farm to Fork, Biodiversity, and Circular Economy. Finally, the review concludes by addressing policy considerations, ethical challenges, and the necessity of international cooperation in biotechnology research, exemplified by projects like the Golden Rice and Heat-Tolerant Maize for Asia (HTMA), highlighting the multidisciplinary nature and global significance of biotechnological innovations in ensuring sustainable food production.

Overview of sixteen scientific opinions on genetically modified plants obtained by new genomic techniques

RIVM was asked by EFSA in the frame of a procurement (PO/EFSA/GMO/2020/01) to producean overview of 16 scientific opinions published by European competent authorities and national institutions since 2012 ongenetically modified plants obtained by new genomic techniques (NGTs). The 16 opinions were provided by the European Commission in the frame of a mandate asking EFSA to provide an overview on the risk assessment of plants developed through NGTsbased on its own previous and current work and on work carried out at national level. NGTs are defined as techniques capable to change the genetic material of an organism and that have emerged or have been developed since the adoption of the GMO legislation in 2001. Based on this definition for NGTs provided by EC, the following NGTs as described in a report of the Joint Research Council on new plant breeding techniques, published in 2011, were considered: (1) Zinc finger nuclease technology, (defined more broadly as site-directed nuclease technology); (2) oligonucleotide directed mutagenesis; (3) cisgenesis and intragenesis; (4) RNA-dependent DNA methylation; (5) grafting (on genetically modified rootstock); (6) reverse breeding; (7) agro-infiltration and (8) synthetic genomics.The current report presents the information on the defined NGTsmethod descriptionand on the risk assessment of plants developed through these NGTs, as extracted from the 16opinions. In order to extract the relevant information a baseline descriptionfor each NGT was set and inclusion and exclusion criteria for information extraction were defined. Most (14 out of 16) opinions discuss the SDN technology, whereas no opinion contains information on synthetic genomics. A new NGT, base-editing, is described in four opinions and therefore information on this technique is also presented.As specified by the procurement, no critical appraisal onthe information extracted from the scientific opinions was performed.

Implications of the EFSA Scientific Opinion on Site Directed Nucleases 1 and 2 for Risk Assessment of Genome-Edited Plants in the EU

Genome editing is a set of techniques for introducing targeted changes in genomes. It may be achieved by enzymes collectively called site-directed nucleases (SDN). Site-specificity of SDNs is provided either by the DNA binding domain of the protein molecule itself or by RNA molecule(s) that direct SDN to a specific site in the genome. In contrast to transgenesis resulting in the insertion of exogenous DNA, genome editing only affects specific endogenous sequences. Therefore, multiple jurisdictions around the world have exempted certain types of genome-edited organisms from national biosafety regulations completely, or on a case-by-case basis. In the EU, however, the ruling of the Court of Justice on the scope of mutagenesis exemption case C-528/16 indicated that the genome-edited organisms are subject to the GMO Directive, but the practical implications for stakeholders wishing to develop and authorize genome-edited products in the EU remain unclear. European Food Safety Authority in response to a request by European Commission has produced a scientific opinion on plants developed by SDN-1, SDN-2, and oligonucleotide-directed mutagenesis (ODM) genome editing techniques. In this review, I will (1) provide a conceptual background on GMO risk assessment in the EU; (2) will introduce the main conclusions of the EFSA opinion, and (3) will outline the potential impact on the risk assessment of genome-edited plants.

Targeting female flight for genetic control of mosquitoes.

Aedes aegypti Act4 is a paralog of the Drosophila melanogaster indirect flight muscle actin gene Act88F. Act88F has been shown to be haploinsufficient for flight in both males and females (amorphic mutants are dominant). Whereas Act88F is expressed in indirect flight muscles of both males and females, expression of Act4 is substantially female-specific. We therefore used CRISPR/Cas9 and homology directed repair to examine the phenotype of Act4 mutants in two Culicine mosquitoes, Aedes aegypti and Culex quinquefasciatus. A screen for dominant female-flightless mutants in Cx. quinquefasciatus identified one such mutant associated with a six base pair deletion in the CxAct4 coding region. A similar screen in Ae. aegypti identified no dominant mutants. Disruption of the AeAct4 gene by homology-dependent insertion of a fluorescent protein marker cassette gave a recessive female-flightless phenotype in Ae. aegypti. Reproducing the six-base deletion from Cx. quinquefasciatus in Ae. aegypti using oligo-directed mutagenesis generated dominant female-flightless mutants and identified additional dominant female-flightless mutants with other in-frame insertions or deletions. Our data indicate that loss of function mutations in the AeAct4 gene are recessive but that short in-frame deletions produce dominant-negative versions of the AeAct4 protein that interfere with flight muscle function. This makes Act4 an interesting candidate for genetic control methods, particularly population-suppression gene drives targeting female viability/fertility.

Update of Regulatory Options of New Breeding Techniques and Biosafety Approaches among Selected Countries: A Review

A new set of breeding techniques, referred to as New Breeding Techniques developed in the last two decades have potential for enhancing improved productivity in crop and animal breeding globally. These include site directed nucleases based genomic editing procedures-CRISPR and Cas associated proteins, Zinc Finger Nucleases, Meganucleases/Homing Endonucleases and Transcription- Activator Like-Effector Nucleases for genome editing and other technologies including- Oligonucleotide-Directed Mutagenesis, Cisgenesis and intragenesis, RNA-Dependent DNA methylation; Transgrafting, Agroinfiltration, Reverse breeding. There are ongoing global debates on whether the processes of and products emerging from these technologies should be regulated as genetically modified organisms or approved as conventional products. Decisions on whether to regulate as GMOs are based both on understanding of the molecular basis of their development and if the GMO intermediate step was used. For example- cisgenesis, can be developed using Agrobacterium tumefaciens methods of transformation, a process used by GMO but if the selection is properly conducted the intermediate GMO elements will be eliminated and the final product will be identical to the conventionally developed crops. Others like Site Directed Nuclease 3 are regulated as GMOs in countries such as United State of America, Canada, European Union, Argentina, Australia. Progress in genome editing research, testing of genome edited bacterial blight resistant rice, development of Guidelines for regulating new breeding techniques or genome editing in Africa is also covered with special reference to South Africa, Kenya and Nigeria. Science- and evidence-based approach to regulation of new breeding techniques among regulators and policy makers should be strongly supported.

CRISPR/Cas-based precision genome editing via microhomology-mediated end joining.

SummaryGene editing and/or allele introgression with absolute precision and control appear to be the ultimate goals of genetic engineering. Precision genome editing in plants has been developed through various approaches, including oligonucleotide‐directed mutagenesis (ODM), base editing, prime editing and especially homologous recombination (HR)‐based gene targeting. With the advent of CRISPR/Cas for the targeted generation of DNA breaks (single‐stranded breaks (SSBs) or double‐stranded breaks (DSBs)), a substantial advancement in HR‐mediated precise editing frequencies has been achieved. Nonetheless, further research needs to be performed for commercially viable applications of precise genome editing; hence, an alternative innovative method for genome editing may be required. Within this scope, we summarize recent progress regarding precision genome editing mediated by microhomology‐mediated end joining (MMEJ) and discuss their potential applications in crop improvement.

Applicability of the EFSA Opinion on site-directed nucleases type 3 for the safety assessment of plants developed using site-directed nucleases type 1 and 2 and oligonucleotide-directed mutagenesis.

The European Commission requested the EFSA Panel on Genetically Modified Organisms (GMO) to assess whether section 4 (hazard identification) and the conclusions of EFSA's Scientific opinion on the risk assessment of plants developed using zinc finger nuclease type 3 technique (ZFN‐3) and other site‐directed nucleases (SDN) with similar function are valid for plants developed via SDN‐1, SDN‐2 and oligonucleotide‐directed mutagenesis (ODM). In delivering this Opinion, the GMO Panel compared the hazards associated with plants produced via SDN‐1, SDN‐2 and ODM with those associated with plants obtained via both SDN‐3 and conventional breeding. Unlike for SDN‐3 methods, the application of SDN‐1, SDN‐2 and ODM approaches aims to modify genomic sequences in a way which can result in plants not containing any transgene, intragene or cisgene. Consequently, the GMO Panel concludes that those considerations which are specifically related to the presence of a transgene, intragene or cisgene included in section 4 and the conclusions of the Opinion on SDN‐3 are not relevant to plants obtained via SDN‐1, SDN‐2 or ODM as defined in this Opinion. Overall, the GMO Panel did not identify new hazards specifically linked to the genomic modification produced via SDN‐1, SDN‐2 or ODM as compared with both SDN‐3 and conventional breeding. Furthermore, the GMO Panel considers that the existing Guidance for risk assessment of food and feed from genetically modified plants and the Guidance on the environmental risk assessment of genetically modified plants are sufficient but are only partially applicable to plants generated via SDN‐1, SDN‐2 or ODM. Indeed, those guidance documents’ requirements that are linked to the presence of exogenous DNA are not relevant for the risk assessment of plants developed via SDN‐1, SDN‐2 or ODM approaches if the genome of the final product does not contain exogenous DNA.

Outcome of the public consultation on the draft Scientific Opinion on the applicability of the EFSA Opinion on site‐directed nucleases type 3 for the safety assessment of plants developed using site‐directed nucleases type 1 and 2 and oligonucleotide‐directed mutagenesis

The European Food Safety Authority (EFSA) carried out a public consultation to receive input from interested parties on the applicability of the EFSA Opinion on site-directed nucleases type 3 for the safety assessment of plants developed using site-directed nucleases type 1 and 2 and oligonucleotide-directed mutagenesis. This draft scientific opinion was prepared by the GMO Panel, supported by the Working Group on Molecular Characterization. The draft opinion was endorsed by the EFSA GMO Panel for public consultation on 1stApril 2020. The written public consultation was open from 15April 2020 until 5 June 2020. EFSA received comments from 51 different interested parties. EFSA and its GMO Panel wish to thank all stakeholders for their contributions to this work. The present report contains the comments received and details how they have been considered for finalisation of the opinion. The final opinion was adopted at the GMO Panel Plenary meeting on 14October 2020 and will be published in the EFSA Journal.© European Food Safety Authority, 2020

Single-Base Genome Editing in Corynebacterium glutamicum with the Help of Negative Selection by Target-Mismatched CRISPR/Cpf1.

CRISPR/Cpf1 has emerged as a new CRISPR-based genome editing tool because, in comparison with CRIPSR/Cas9, it has a different T-rich PAM sequence to expand the target DNA sequence. Single-base editing in the microbial genome can be facilitated by oligonucleotide-directed mutagenesis (ODM) followed by negative selection with the CRISPR/Cpf1 system. However, single point mutations aided by Cpf1 negative selection have been rarely reported in Corynebacterium glutamicum. This study aimed to introduce an amber stop codon in crtEb encoding lycopene hydratase, through ODM and Cpf1-mediated negative selection; deficiency of this enzyme causes pink coloration due to lycopene accumulation in C. glutamicum. Consequently, on using double-, triple-, and quadruple-basemutagenic oligonucleotides, 91.5-95.3% pink cells were obtained among the total live C. glutamicum cells. However, among the negatively selected live cells, 0.6% pink cells were obtained using single-base-mutagenic oligonucleotides, indicating that very few single-base mutations were introduced, possibly owing to mismatch tolerance. This led to the consideration of various targetmismatched crRNAs to prevent the death of single-base-edited cells. Consequently, we obtained 99.7% pink colonies after CRISPR/Cpf1-mediated negative selection using an appropriate singlemismatched crRNA. Furthermore, Sanger sequencing revealed that single-base mutations were successfully edited in the 99.7% of pink cells, while only two of nine among 0.6% of pink cells were correctly edited. The results indicate that the target-mismatched Cpf1 negative selection can assist in efficient and accurate single-base genome editing methods in C. glutamicum.

CRISPR-Cas9-mediated pinpoint microbial genome editing aided by target-mismatched sgRNAs.

Genome editing has been revolutionized by the CRISPR-Cas9 system. CRISPR-Cas9 is composed of single-molecular guide RNA (sgRNA) and a proteinaceous Cas9 nuclease, which recognizes a specific target sequence and a protospacer adjacent motif (PAM) sequence and, subsequently, cleaves the targeted DNA sequence. This CRISPR-Cas9 system has been used as an efficient negative-selection tool to cleave unedited or unchanged target DNAs during site-specific mutagenesis and, consequently, obtain microbial cells with desired mutations. This study aimed to investigate the genome editing efficiency of the CRISPR-Cas9 system for in vivo oligonucleotide-directed mutagenesis in bacteria. This system successfully introduced two- to four-base mutations in galK in Escherichia coli with high editing efficiencies (81%−86%). However, single-point mutations (T504A or C578A) were rarely introduced with very low editing efficiencies (<3%), probably owing to mismatch tolerance. To resolve this issue, we designed one- or two-base mismatches in the sgRNA sequence to recognize target sequences in galK in E. coli. A single-point nucleotide mutation (T504A or C578A in the galK gene) was successfully introduced in 36%−95% of negatively selected E. coli cells using single-base mismatched sgRNAs. Sixteen targets were randomly selected through genome-wide single-base editing experiments using mismatched sgRNAs. Consequently, out of 48 desired single-base mutations, 25 single bases were successfully edited, using mismatched sgRNAs. Finally, applicable design rules for target-mismatched sgRNAs were provided for single-nucleotide editing in microbial genomes.

Current Status and Future Perspectives of Biofortification in Wheat

Wheat is the main cereal crop worldwide and about 2 billion people suffer from Zn and Fe deficiency because of their dependence just on cereal crops. Three Billion people are malnourished suffering from mild to severe physical and mental disabilities. Vast genetic diversity of Wheat exists in nature that differs in their mineral compositions. Main deficient micronutrients are Provitamin-A, Zn &amp; Fe which deficiencies cause serious physical and mental abnormalities. Different methods of enhancing mineral contents of plants products have been used, out of which biofortification has proved more promising and economical. Bacterial phytoene synthase gene (crtb) and carotene desaturase gene (crti) has been transferred in wheat that has increased carotenoid content but darker colour has less public acceptance.GPC-B1 gene is found to be associated with increase micronutrients but it lowers the overall yield of the plant. Several new methodologies such as oligo-directed mutagenesis, reverse breeding, RNA directed DNA methylation and genome editing have been used for increasing micronutrient composition and their bioavailability. But the combination of Plant Breeding methods with Molecular Techniques will be more useful for advancement in this field.

What is the available evidence for the range of applications of genome-editing as a new tool for plant trait modification and the potential occurrence of associated off-target effects: a systematic map

BackgroundWithin the last decades, genome-editing techniques such as CRISPR/Cas, TALENs, Zinc-Finger Nucleases, Meganucleases, Oligonucleotide-Directed Mutagenesis and base editing have been developed enabling a precise modification of DNA sequences. Such techniques provide options for simple, time-saving and cost-effective applications compared to other breeding techniques and hence genome editing has already been promoted for a wide range of plant species. Although the application of genome-editing induces less unintended modifications (off-targets) in the genome compared to classical mutagenesis techniques, off-target effects are a prominent point of criticism as they are supposed to cause unintended effects, e.g. genomic instability or cell death. To address these aspects, this map aims to answer the following question: What is the available evidence for the range of applications of genome-editing as a new tool for plant trait modification and the potential occurrence of associated off-target effects? This primary question will be considered by two secondary questions: One aims to overview the market-oriented traits being modified by genome-editing in plants and the other explores the occurrence of off-target effects.MethodsA literature search in nine bibliographic databases, Google Scholar, and 47 web pages of companies and governmental agencies was conducted using predefined and tested search strings in English language. Articles were screened on title/abstract and full text level for relevance based on pre-defined inclusion criteria. The relevant information of included studies were mapped using a pre-defined data extraction strategy. Besides a descriptive summary of the relevant literature, a spreadsheet containing all extracted data is provided.ResultsAltogether, 555 relevant articles from journals, company web pages and web pages of governmental agencies were identified containing 1328 studies/applications of genome-editing in model plants and agricultural crops in the period January 1996 to May 2018. Most of the studies were conducted in China followed by the USA. Genome-editing was already applied in 68 different plants. Although most of the studies were basic research, 99 different market-oriented applications were identified in 28 different crops leading to plants with improved food and feed quality, agronomic value like growth characteristics or increased yield, tolerance to biotic and abiotic stress, herbicide tolerance or industrial benefits. 252 studies explored off-target effects. Most of the studies were conducted using CRISPR/Cas. Several studies firstly investigated whether sites in the genome show similarity to the target sequence and secondly analyzed these potential off-target sites by sequencing. In around 3% of the analyzed potential off-target sites, unintended mutations were detected. Only a few studies conducted off-target analyses using unbiased detection methods (e.g. whole genome sequencing). No off-target effects that could be correlated to the genome-editing process were identified in these studies.ConclusionsThe rapid adoption in plant breeding was demonstrated by a considerable number of market oriented applications (crops and traits) described in publications worldwide. Studies investigating off-target effects are very heterogeneous in their structure and design. Therefore, an in-depth assessment regarding their weight of evidence is mandatory.

Solid Phase Assembly of Fully Protected Trinucleotide Building Blocks for Codon-Based Gene Synthesis

The use of pre-formed trinucleotides, representing codons of the 20 canonical amino acids, for oligonucleotide-directed mutagenesis offers the advantage of controlled randomization and generation of “smart libraries”. We here present a method for the preparation of fully protected trinucleotides on solid phase. The key issue of our strategy is the linkage of the starting nucleoside to the solid support via a traceless disulfide linker. Upon trinucleotide assembly, the disulfide bridge is cleaved under reducing conditions, and the fully protected trinucleotide is released with a terminal 3′-OH group.

Oligonucleotide-Directed Mutagenesis by Elimination of a Unique Restriction Site (USE Mutagenesis).

In this method, two oligonucleotide primers are hybridized to the same strand of a denatured double-stranded recombinant plasmid. One primer (the mutagenic primer) introduces the desired mutation into the target sequences, whereas the second primer carries a mutation that destroys a unique restriction site (called "unique site elimination," or USE) in the plasmid. The product of the first part of the method is a heteroduplex plasmid consisting of a wild-type parental strand and a new full-length strand that carries the desired mutation but no longer contains the unique restriction site. In the second phase of the method, the mixed population is incubated with the restriction enzyme that cleaves the unique site. The wild-type molecules are linearized, and the mutated plasmids are resistant to digestion. The mixture of circular heteroduplex DNA and linear wild-type DNA is then used to transform a strain of Escherichia coli that is deficient in repair of mismatched bases. The circular heteroduplex molecules begin to replicate while many of the linear wild-type molecules are unable to reestablish themselves in E. coli cells. Because the mismatched bases are not repaired, the first round of replication generates a wild-type plasmid that carries the original restriction site and a mutated plasmid that does not. DNA from the first set of transformants is recovered, digested once more with the same restriction enzyme to linearize the wild-type molecules, and then used to transform a standard laboratory strain of E. coli.

What is the available evidence for the application of genome editing as a new tool for plant trait modification and the potential occurrence of associated off-target effects: a systematic map protocol

BackgroundPlant breeding is a developing process and breeding methods have continuously evolved over time. In recent years, genome editing techniques such as clustered regularly interspaced short palindromic repeats/CRISPR associated proteins (CRISPR/Cas), transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFN), meganucleases (MN) and oligonucleotide-directed mutagenesis (ODM) enabled a precise modification of DNA sequences in plants. Genome editing has already been applied in a wide range of plant species due to its simplicity, time saving and cost-effective application compared to earlier breeding techniques including classical mutagenesis. Although genome editing techniques induce much less unintended modifications in the genome (off-target effects) compared to classical mutagenesis techniques, off-target effects are a prominent point of criticism as they might cause genomic instability, cytotoxicity and cell death.MethodsThe aim of this systematic map is to address the following primary question: “What is the available evidence for the application of genome editing as a new tool for plant trait modification and the potential occurrence of associated off-target effects”? The primary question will be considered by two secondary questions: One is aimed at the traits being modified by genome editing in plants and the other explores the occurrence of off-target effects. The systematic map will focus on model plants as well as on plants produced for agricultural production that were subjected to genome editing techniques. Academic and grey literature will be searched in English and German language. Inclusion/exclusion criteria were developed for the two secondary questions and will be applied on title/abstract and full text stage. Included studies will be catalogued in a searchable and open access database and study results will be summarized using descriptive statistics. Furthermore, the extracted data will serve as a preparatory step for further in-depth analysis, e.g. by a systematic review.

The Swedish policy approach to directed mutagenesis in a European context

This review describes the Swedish approach to directed mutagenesis in plants and puts it in a comparative European perspective. Directed mutagenesis is accomplished by a number of genome editing techniques; however, the legal status of these techniques and their resulting products is uncertain in the European Union (EU) as there is no political consensus on whether or not these should be regulated as genetically modified organisms (GMOs). A number of cases have developed over the past few years, putting the GMO regulatory framework to the test. These include oilseed rape developed by oligonucleotide-directed mutagenesis, Arabidopsis developed by clustered regularly interspaced short palindromic repeat-Cas9, and the case on mutagenesis for which the French Court requested a preliminary ruling from the Court of Justice of the EU. In this review, the involvement of the Swedish Government and governmental authorities in these cases is described and compared with that of other EU member states and/or EU entity statements and reports. Various approaches to the definition of recombinant nucleic acids are also discussed, as this is crucial for the EU GMO definition thus affecting the legal status of products developed by directed mutagenesis.

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel's method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity.

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel's method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

Prevention of Transcriptional γ-globin Gene Silencing by Inducing The Hereditary Persistence of Fetal Hemoglobin Point Mutation Using Chimeraplast-Mediated Gene Targeting.

Objective Hemoglobin F (HbF) augmentation is considered a clinically beneficial phenomenon in β-hemoglobinopathies. Prevention of γ-globin gene silencing, inspired by the hereditary persistence of fetal hemoglobin, may be a suitable strategy to upregulate HbF expression in these patients. Therefore, our objective was to assess the potential feasibility of induced -117 G→A substitution in HBG promoter in prevention of transcriptional silencing of the γ-globin. Materials and Methods In this experimental study, human peripheral blood-derived hematopoietic stem cells (HSCs) and the K562 cell line were differentiated to erythroid cells. Erythroid maturation was examined using cell morphology parameters and flow cytometry analysis of CD235a expression. A synthesised chimeraplast was transfected to differentiating cells. The efficiency of chimeraplast delivery into target cells was assessed by flow cytometry. Restriction-fragment length polymorphism and DNA sequencing verified oligonucleotide-directed mutagenesis. Gene conversion frequency and globin genes expression was quantified through Allele specific-quantitaive polymerase chain reaction (AS-qPCR) and quantitative-PCR respectively. Results Increase in CD235a-expressing cells along with observations made for different stages of erythroid maturation confirmed erythroid differentiation in HSCs and K562 cells. γ to β-globin gene switching was estimated to be on days 18-21 of HSC differentiation. Flow cytometry analysis showed that more than 70% of erythroid progenitor cells (EPCs) were transfected with the chimeraplast. The highest gene conversion efficiency was 7.2 and 11.1% in EPCs and K562 cells respectively. The induced mutation led to a 1.97-fold decrease in β/γ-globin gene expression in transfected EPCs at the experimental end point (day 28) whereas, due to the absence of β-globin gene expression following K562 differentiation, this rate was not evaluable. ConclusionOur results suggest the effectiveness of chimeraplasty in induction of the mutation of interest in both EPCs and K562 cells. We also demonstrate that the single nucleotide promoter variant was able to significantly inhibit γ-globin gene silencing during erythroid differentiation.

Genome Editing Tools in Plants.

Genome editing tools have the potential to change the genomic architecture of a genome at precise locations, with desired accuracy. These tools have been efficiently used for trait discovery and for the generation of plants with high crop yields and resistance to biotic and abiotic stresses. Due to complex genomic architecture, it is challenging to edit all of the genes/genomes using a particular genome editing tool. Therefore, to overcome this challenging task, several genome editing tools have been developed to facilitate efficient genome editing. Some of the major genome editing tools used to edit plant genomes are: Homologous recombination (HR), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), pentatricopeptide repeat proteins (PPRs), the CRISPR/Cas9 system, RNA interference (RNAi), cisgenesis, and intragenesis. In addition, site-directed sequence editing and oligonucleotide-directed mutagenesis have the potential to edit the genome at the single-nucleotide level. Recently, adenine base editors (ABEs) have been developed to mutate A-T base pairs to G-C base pairs. ABEs use deoxyadeninedeaminase (TadA) with catalytically impaired Cas9 nickase to mutate A-T base pairs to G-C base pairs.