Genome Modification Research Articles

Versatile Cloning Strategy for Efficient Multigene Editing in Arabidopsis.

CRISPR-Cas9 technology has become an essential tool for plant genome editing. Recent advancements have significantly improved the ability to target multiple genes simultaneously within the same genetic background through various strategies. Additionally, there has been significant progress in developing methods for inducible or tissue-specific editing. These advancements offer numerous possibilities for tailored genome modifications. Building upon existing research, we have developed an optimized and modular strategy allowing the targeting of several genes simultaneously in combination with the synchronized expression of the Cas9 endonuclease in the egg cell. This system allows significant editing efficiency while avoiding mosaicism. In addition, the versatile system we propose allows adaptation to inducible and/or tissue-specific edition according to the promoter chosen to drive the expression of the Cas9 gene. Here, we describe a step-by-step protocol for generating the binary vector necessary for establishing Arabidopsis edited lines using a versatile cloning strategy that combines Gateway® and Golden Gate technologies. We describe a versatile system that allows the cloning of as many guides as needed to target DNA, which can be multiplexed into a polycistronic gene and combined in the same construct with sequences for the expression of the Cas9 endonuclease. The expression of Cas9 is controlled by selecting from among a collection of promoters, including constitutive, inducible, ubiquitous, or tissue-specific promoters. Only one vector containing the polycistronic gene (tRNA-sgRNA) needs to be constructed. For that, sgRNA (composed of protospacers chosen to target the gene of interest and sgRNA scaffold) is cloned in tandem with the pre-tRNA sequence. Then, a single recombination reaction is required to assemble the promoter, the zCas9 coding sequence, and the tRNA-gRNA polycistronic gene. Each element is cloned in an entry vector and finally assembled according to the Multisite Gateway® Technology. Here, we detail the process to express zCas9 under the control of egg cell promoter fused to enhancer sequence (EC1.2en-EC1.1p) and to simultaneously target two multiple C2 domains and transmembrane region protein genes (MCTP3 and MCTP4, respectively at3g57880 and at1g51570), using one or two sgRNA per gene. Key features • A simple method for Arabidopsis edited lines establishment using CRISPR-Cas9 technology • Versatile cloning strategy combining various technologies for convenient cloning (Gateway®, Golden Gate) • Multigene targeting with high efficiency.

Callus Induction Followed by Regeneration and Hairy Root Induction in Common Buckwheat.

This section describes a set of methods for callus induction followed by the successful regeneration of whole plants and obtaining a culture of transgenic hairy roots from buckwheat plants (Fagopyrum esculentum Moench.). Callus induction and regeneration are key steps for many biotechnological, genetic, and breeding approaches, such as genetic modification, production of biologically active compounds, and propagation of valuable germplasm. Induction of hairy roots using Agrobacterium rhizogenes is also an important tool for functional gene research and plant genome modification. While many efforts were invested into the development of the corresponding protocols, they are not equally efficient for different cultivars. Here, we have tested and optimized the protocols of callus induction, regeneration, and transformation using A. rhizogenes for a set of cultivars of F. esculentum, including wild ancestor of cultivated buckwheat F. esculentum ssp. ancestrale and a self-pollinated accession KK8. The optimal medium for callus induction is Murashige-Skoog basal medium with 3% sucrose which includes hormones 2,4-dichlorophenoxyacetic acid 2mg/L and kinetin 2mg/L; for shoot initiation 6-benzylaminopurine 2mg/L, kinetin 0.2mg/L, and indole-3-acetic acid 0.2mg/L; for shoot multiplication 6-benzylaminopurine 3mg/L and indole-3-acetic acid 0.2mg/L; and for root initiation half-strength Murashige-Skoog medium with 1.5% sucrose and indole-3-butyric acid 1mg/L. A. rhizogenes R1000 strain proved to be the most efficient in inducing hairy roots in buckwheat and T-DNA transfer from binary vectors. Seedling explants cut at the root area and immersed in agrobacterium suspension, as well as prickling the cotyledonary area with agrobacteria dipped syringe needle, are the most labor-effective methods of infection, allowing to initiate hairy root growth in 100% of explants.

CRISPR-Cas9-Mediated Genome Editing in Paenibacillus polymyxa.

In recent years, the clustered regularly interspaced palindromic repeats-Cas (CRISPR-Cas) technology has become the method of choice for precision genome editing in many organisms due to its simplicity and efficacy. Multiplex genome editing, point mutations, and large genomic modifications are attractive features of the CRISPR-Cas9 system. These applications facilitate both the ease and velocity of genetic manipulations and the discovery of novel functions. In this protocol chapter, we describe the use of a CRISPR-Cas9 system for multiplex integration and deletion modifications, and deletions of large genomic regions by the use of a single guide RNA (sgRNA), and, finally, targeted point mutation modifications in Paenibacillus polymyxa.

ARTIFICIAL INTELLIGENCE, GENOM, CYBERSPACE AND SPACE – CONTEMPORARY THREATS TO THE SECURITY OF THE STATE AND NATIONS

Among many contemporary threats, which include threats in the military, economic or social sphere, issues related to threats in cyberspace, artificial intelligence (AI), genetic modifications of the human genome and threats related to with space exploration. This work, based on a qualitative analysis of the literature, aims to identify selected, often overlooked and not functioning in the common awareness of contemporary threats to state security. The analysis undertaken allows us to conclude that an important factor contributing to the increase in the level of threat to the security of the state and nation today are the contemporary "black swans" in the security sphere, the weakness of political leadership, unknown directions of AI development and changes in the human genome, threats related to the outer space exploration and threats in cyberspace.

CRISPR-based genomic modification method in the treatment of hematological diseases

Mutations in hematopoietic stem cells are the main cause of most blood diseases. Hematopoietic stem cell transplantation, which is the main treatment for hematopoietic stem cell disorders ,brings many negative effects according to the clinical cases, such as high toxic side effects, limited donor source, high recurrence rate, and high treatment cost .CRISPR/Cas acts as an immune system in prokaryote that grants them an ability to resist invasion by exogenous mobile genetic elements (MGE), which is a new generation of gene editing technology after the appearance of TALEN and ZFN. The immune system functions in three steps: adaptation, expression, and interference. It enables genomic modification by unwinding the double strands of DNA and inducing DNA repair processes such as homologydirected repair (HDR) and/or non-homologous end joining (NHEJ). So, this paper discusses the utilization of Crispr system in the treatment of -hemoglobinopathy, Chronic myeloid leukemia (CML) , Hemophilia and summarizes the current limitations of this technology, such as the editing efficiency, potential immunogenicity of editing tools and off-target effects , while it still remains promising as to its future prospects for research. This technology is very likely to replace traditional therapies and achieve a complete cure for the blood disease.

Principle and applications of CRISPR/Cas system

With the continuous in-depth research and exploration of the CRISPR/Cas9 system by researchers, people's ability to edit biological genes is also constantly improving. In recent years, gene editing technology as a hot technology has been paid more and more attention, it has played an important role in crop breeding, disease treatment, molecular diagnosis and other aspects. Among the gene editing technologies that have been developed, the emerging CRISPR/Cas technology gene editing system has become the most widely used gene editing technology at present. Compared with the first two generations of gene-editing technologies: ZFN and TALEN, CRISPR/Cas system has the advantages of short cycle, high efficiency, low cost, and has great potential for the development of animal and plant breeding. The development of CRISPR/Cas9 technology has promoted the progress of modern life sciences. With these tools for efficient, simple, and precise genome modification, transcriptional regulation, and epigenetic editing, CRISPR systems have the potential to revolutionize genetics, medicine, and agriculture. In this review, the basic principle of CRISPR/Cas technology and its application will be introduced and illustrated.

Preliminary characterization of biomolecular processes related to plasticity in Acyrthosiphon pisum

Global warming strongly impacts many organisms' development, distribution and population structure. This problem has attracted the attention of many scientists to understand and study its actual effects, especially on insects influenced by environmental temperatures. Aphids are a model for studies of the genetics and physiology of stress. Aphids are characterized by parthenogenetic reproduction, which limits the effects of recombination on evolutionary processes, and have shown resistance to various biotic and abiotic stresses. This study was based on the hypothesis that aphids have optimized, over time, genetic mechanisms capable to give them plasticity through genome modifications mediated by transposition. To understand and evaluate the effects of heat stress, the expression levels of transposases and methylases were analyzed in mothers and daughters. Our results show that after four days from the thermal shock, methylation decreases in both mothers and daughters, while transposition significantly increases in daughters, thus generating gene variability, essential for adaptation.

Islamic Perspectives on Polygenic Testing and Selection of IVF Embryos (PGT-P) for Optimal Intelligence and Other Non-Disease-Related Socially Desirable Traits.

In recent years, the genetic testing and selection of IVF embryos, known as preimplantation genetic testing (PGT), has gained much traction in clinical assisted reproduction for preventing transmission of genetic defects. However, a more recent ethically and morally controversial development in PGT is its possible use in selecting IVF embryos for optimal intelligence quotient (IQ) and other non-disease-related socially desirable traits, such as tallness, fair complexion, athletic ability, and eye and hair colour, based on polygenic risk scores (PRS), in what is referred to as PGT-P. Artificial intelligence (AI) and machine learning-based analysis of big data sets collated from genome sequencing of specific human ethnic populations can be used to estimate an individual embryo's likelihood of developing such multifactorial traits by analysing the combination of specific genetic variants within its genome. Superficially, this technique appears compliant with Islamic principles and ethics. Because there is no modification of the human genome, there is no tampering with Allah's creation (taghyīr khalq Allah). Nevertheless, a more critical analysis based on the five maxims of Islamic jurisprudence (qawa'id fiqhiyyah) that are often utilized in discourses on Islamic bioethics, namely qaṣd (intention), yaqın̄ (certainty), ḍarar (injury), ḍarūra (necessity), and `urf (custom), would instead reveal some major ethical and moral flaws of this new medical technology in the selection of non-disease-related socially desirable traits, and its non-compliance with the spirit and essence of Islamic law (shariah). Muslim scholars, jurists, doctors, and biomedical scientists should debate this further and issue a fatwa on this new medical technology platform.

Modifying Our Genes: Theology, Science and “Playing God”

Modifying Our Genes: Theology, Science and “Playing God”

Codonopsis pilosula polysaccharide alleviates rotenone-induced murine brain organoids death through downregulation of gene body DNA methylation modification in the ZIC4/PGM5/CAMTA1 axis

Here, the protective mechanism of Codonopsis pilosula polysaccharide (CpP) against mouse brain organoids (mBO) damage was analyzed, and the rotenone affected the genomic epigenetic modifications and physiological activity of mouse brain organoids was examined. Pathological experiments have shown that rotenone significantly damaged the subcellular organelles of mouse brain organoids. According to RRBS-Seq, rotenone significantly promoted gene body hypermethylation modifications in mouse brain organoids. Molecular biology experiments have confirmed that rotenone significantly promoted the hypermethylation modification of Zic4, Pgm5, and Camta1 gene bodies in mouse brain organoids, and their expression levels were significantly lower than those of the control group. Bioinformatic analysis suggested that multiple binding motif of transcription factors ZIC4 (Zinc finger protein of the cerebellum 4) were present at the promoters of both the Pgm5 (Phosphoglucomutase 5) and Camta1 (Calmodulin binding transcription activator 1) genes. When the expression of Zic4 was silenced, the proliferation of mouse brain organoids was significantly reduced and the expression level of PGM5 was also significantly decreased. In addition, Codonopsis pilosula polysaccharide treatment of mouse brain organoids significantly reduced the cytotoxicity of rotenone, promoted cell cycle progression, increased intracellular glutathione activity, significantly induced the demethylation modification of the Zic4, Pgm5, and Camta1 gene bodies, and promoted the high expression of ZIC4 and PGM5. Therefore, the study confirmed that Codonopsis pilosula polysaccharide alleviated rotenone-induced mouse brain organoids death by downregulating DNA gene bodies methylation modification of the Zic4/Pgm5/Camta1 axis.

Improved production of Taxol® precursors in S. cerevisiae using combinatorial in silico design and metabolic engineering

BackgroundIntegrated metabolic engineering approaches that combine system and synthetic biology tools enable the efficient design of microbial cell factories for synthesizing high-value products. In this study, we utilized in silico design algorithms on the yeast genome-scale model to predict genomic modifications that could enhance the production of early-step Taxol® in engineered Saccharomyces cerevisiae cells.ResultsUsing constraint-based reconstruction and analysis (COBRA) methods, we narrowed down the solution set of genomic modification candidates. We screened 17 genomic modifications, including nine gene deletions and eight gene overexpressions, through wet-lab studies to determine their impact on taxadiene production, the first metabolite in the Taxol® biosynthetic pathway. Under different cultivation conditions, most single genomic modifications resulted in increased taxadiene production. The strain named KM32, which contained four overexpressed genes (ILV2, TRR1, ADE13, and ECM31) involved in branched-chain amino acid biosynthesis, the thioredoxin system, de novo purine synthesis, and the pantothenate pathway, respectively, exhibited the best performance. KM32 achieved a 50% increase in taxadiene production, reaching 215 mg/L. Furthermore, KM32 produced the highest reported yields of taxa-4(20),11-dien-5α-ol (T5α-ol) at 43.65 mg/L and taxa-4(20),11-dien-5-α-yl acetate (T5αAc) at 26.2 mg/L among early-step Taxol® metabolites in S. cerevisiae.ConclusionsThis study highlights the effectiveness of computational and integrated approaches in identifying promising genomic modifications that can enhance the performance of yeast cell factories. By employing in silico design algorithms and wet-lab screening, we successfully improved taxadiene production in engineered S. cerevisiae strains. The best-performing strain, KM32, achieved substantial increases in taxadiene as well as production of T5α-ol and T5αAc. These findings emphasize the importance of using systematic and integrated strategies to develop efficient yeast cell factories, providing potential implications for the industrial production of high-value isoprenoids like Taxol®.

Cisgenics and intragenics: boon or bane for crop improvement

Recent advances in molecular biology and genetic engineering have made it possible to increase agricultural yields when compared to conventional methods. However, lots of challenges are to be addressed due to changing climatic conditions. Although genetically modified organisms (GMOs) have proven their potential in a few crops, but needs to be explored in majority of the field/vegetable crops to overcome food and nutritional security in view of alarming population explosion. In spite of advantages from GMO crops due to the presence of foreign DNA, queries regarding their safety, environmental dangers and health effects needs to be addressed. One of the major environmental issues concerning transgenic crops is the mixing of genetic components across species that cannot hybridize naturally. Due to these limitations, new revolutionary technologies have been developed, such as intragenesis and cisgenesis for the transformation and development of superior plants. While cisgenesis entails genetic modification employing a complete copy of natural genes with their native regulatory components that only belong to sexually compatible species, intragenesis refers to the transfer of unique combinations of genes and regulatory sequence inside the same species. In cisgenesis, the donor genes are the same genes employed in conventional breeding. The two benefits of cisgenics are avoiding linkage drag and making greater use of existing gene alleles. This method significantly shortens the time it takes to breed plants by combining conventional methods with cutting-edge biotechnological tools. Because of this, plant genomes can be altered without causing drastic changes to the whole plant population and the environmental effects of cisgenic plants cannot be compared to those of transgenics. Transgenesis and cisgenesis share the same transformation methods; hence, cisgenic, intragenic and transgenic plants produced through random insertion do not pose any distinct risks with regard to host genome modifications. In contrast, using new genome techniques lessens the dangers related to potential unintentional changes to the host DNA. The use of cisgenesis and intragenesis as alternatives to transgenesis has been restricted to a small number of species due to incomplete understanding of the required regulatory sequences.

Exosomes for CRISPR-Cas9 Delivery: The Cutting Edge in Genome Editing.

Gene mutation correction was challenging until the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas). CRISPR is a new era for genome modification, and this technology has bypassed the limitations of previous methods such as zinc-finger nuclease and transcription activator-like effector nuclease. Currently, this method is becoming the method of choice for gene-editing purposes, especially therapeutic gene editing in diseases such as cardiovascular, neurological, renal, genetic, optical, and stem cell, as well as blood disorders and muscular degeneration. However, finding the optimum delivery system capable of carrying this large complex persists as the main challenge of this technology. Therefore, it would be ideal if the delivery vehicle could direct the introduction of editing functions to specific cells in a multicellular organism. Exosomes are membrane-bound vesicles with high biocompatibility and low immunogenicity; they offer the best and most reliable way to fill the CRISPR/Cas9 system delivery gap. This review presents the current evidence on the molecular mechanisms and challenges of CRISPR/Cas9-mediated genome modification. Also, the role of CRISPR/Cas9 in the development of treatment and diagnosis of numerous disorders, from malignancies to viral infections, has been discussed. Lastly, the focus is on new advances in exosome-delivery technologies that may play a role in CRISPR/Cas9 delivery for future clinical settings.

Survey of attitude to human genome modification in Nigeria.

Gene editing and mitochondrial replacement therapy (MRT) are biotechnologies used to modify the host nuclear and mitochondrial DNA, respectively. Gene editing is the modification of a region of the host genome using site-specific nucleases, in particular the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. Heritable and somatic genome editing (HGE and SGE) are used in gene therapy. MRT is a technique used to substitute the defective mitochondria in the recipient embryo with a female donor healthy mitochondrion in order to prevent the inheritance of mothers' defective mitochondria resulting in the change of mitochondria of the entire generation to come. To evaluate the perception of the Nigerian citizens on human genome modification, two survey forms were created and distributed in-person and majorly online. There was a total of 268 responses, 188 from the public and 80 from health workers and bio-scientists. The results showed poor knowledge about gene editing and MRT by the Nigerian public, but its use to prevent and cure inherited diseases was supported. Morality and religion have great influence on the attitude of Nigerians towards genome modification, but the influence of religion and morality is not unequivocal. Multiple regression analysis of Nigerian public responses shows that gender (females), age (19-30years), monthly income (NGN 0 to 30,000), and level of education (tertiary) are significantly associated with approval of human genome editing, but the survey of health workers and bio-scientists shows no significant association except for females who approve and Muslims who disapprove of human genome editing.

Modular cytosine base editing promotes epigenomic and genomic modifications.

Prokaryotic and eukaryotic adaptive immunity differ considerably. Yet, their fundamental mechanisms of gene editing via Cas9 and activation-induced deaminase (AID), respectively, can be conveniently complimentary. Cas9 is an RNA targeted dual nuclease expressed in several bacterial species. AID is a cytosine deaminase expressed in germinal centre B cells to mediate genomic antibody diversification. AID can also mediate epigenomic reprogramming via active DNA demethylation. It is known that sequence motifs, nucleic acid structures, and associated co-factors affect AID activity. But despite repeated attempts, deciphering AID's intrinsic catalytic activities and harnessing its targeted recruitment to DNA is still intractable. Even recent cytosine base editors are unable to fully recapitulate AID's genomic and epigenomic editing properties. Here, we describe the first instance of a modular AID-based editor that recapitulates the full spectrum of genomic and epigenomic editing activity. Our 'Swiss army knife' toolbox will help better understand AID biology per se as well as improve targeted genomic and epigenomic editing.

CRISPR-Cas9 technology: As an efficient genome modification tool in the cancer diagnosis and treatment.

Cancer is the second most common cause of death globally and is a major public health concern. Managing this disease is difficult due to its multiple stages and numerous genetic and epigenetic changes. Traditional cancer diagnosis and treatment methods have limitations, making it crucial to develop new modalities to combat the increasing burden of cancer. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has transformed genetic engineering due to its simplicity, specificity, low cytotoxicity, and cost-effectiveness. It has been proposed as an effective technology to enhance cancer diagnosis and treatment strategies. This article presents the most recent discoveries regarding the structure, mechanism, and delivery methods of the highly powerful genome editing tool, CRISPR-Cas9. In terms of diagnosis, the article examines the role of CRISPR-Cas9 in detecting microRNAs and DNA methylation, and discusses two popular gene detection techniques that utilize the CRISPR-Cas system: DNA endonuclease-targeted CRISPR trans reporterand specific high sensitivity enzymatic reporter unlocking. Regarding treatment, the article explores several genes that have been identified and modified by CRISPR-Cas9 for effective tumorigenesis of common cancers such as breast, lung, and colorectal cancer. The present review also addresses the challenges and ethical issues associated with using CRISPR-Cas9 as a diagnostic and therapeutic tool. Despite some limitations, CRISPR-Cas9-based cancer diagnosis has the potential to become the next generation of cancer diagnostic tools, and the continuous progress of CRISPR-Cas9 can greatly aid in cancer treatment.

Noodles, the all-in-one system for on-target efficiency analysis of CRISPR guide RNAs

The efficiency of clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA (gRNA) targeting is critical for CRISPR associated protein 9 (Cas9)-dependent genomic modifications. Here, we developed Noodles, an all-in-one system to test the on-target activity of gRNAs easily and efficiently. Single-strand annealing repair mechanism of the split luciferase gene allows a positive selection of gRNAs efficiently driving nuclease activity of Cas9 from Streptococcus pyogenes (SpCas9). Our system can reliably validate in silico-predicted gRNAs before implementing them for in vitro and in vivo applications. Altogether, Noodles might be an asset for researchers and bioengineers, saving their time and efforts, while keeping the screening efficient and sensitive.•All-in-one dual-luciferase system to easily probe on-target activity of gRNAs based on homology-directed repair mechanism.•Easy-to-subclone spCas9-based 2-plasmid system comprising Renilla luciferase for transfection efficiency control.

Enhanced osteoblastic differentiation of parietal bone in a novel murine model of mucopolysaccharidosis type II

Mucopolysaccharidosis type II (MPS II, OMIM 309900) is an X-linked disorder caused by a deficiency of lysosomal enzyme iduronate-2-sulfatase (IDS). The clinical manifestations of MPS II involve cognitive decline, bone deformity, and visceral disorders. These manifestations are closely associated with IDS enzyme activity, which catalyzes the stepwise degradation of heparan sulfate and dermatan sulfate. In this study, we established a novel Ids-deficient mice and further assessed the enzyme's physiological role. Using DNA sequencing, we found a genomic modification of the Ids genome, which involved the deletion of a 138-bp fragment spanning from intron 2 to exon 3, along with the insertion of an adenine at the 5′ end of exon 3 in the mutated allele. Consistent with previous data, our Ids-deficient mice showed an attenuated enzyme activity and an enhanced accumulation of glycosaminoglycans. Interestingly, we noticed a distinct enlargement of the calvarial bone in both neonatal and young adult mice. Our examination revealed that Ids deficiency led to an enhanced osteoblastogenesis in the parietal bone, a posterior part of the calvarial bone originating from the paraxial mesoderm and associated with an enhanced expression of osteoblastic makers, such as Col1a and Runx2. In sharp contrast, cell proliferation of the parietal bone in these mice appeared similar to that of wild-type controls. These results suggest that the deficiency of Ids could be involved in an augmented differentiation of calvarial bone, which is often noticed as an enlarged head circumference in MPS II-affected individuals.

Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia.

Clostridium species have gained attention in industrial and medical applications, and the development of genetic tools has enabled the advancement of the CRISPR-Cas systems for these bacteria. Compared to the primarily used Cas9 from Streptococcus pyogenes, the utilization of Cas12a (previously known as Cpf1) proteins remains incomplete in clostridia, although they exhibit potential advantages, including T-rich recognition for Clostridium genomes and lower toxicity. In this study, we expanded the CRISPR-Cas tools in clostridia by establishing a CRISPR-Cas12a system with two different cas12a genes (Ascas12a from Acidaminococcus and Fncas12a from Francisella novicida). The optimized tetracycline-inducible systems were initially determined by the glucuronidase reporter and were used to drive expression of the cas12a genes and crRNAs. Our results demonstrate that the CRISPR-FnCas12a system offers flexible target selection in clostridia, and a specific folding pattern of the precursor crRNA is important to enable high mutation generation efficiency. By using sacB (encoding levansucrase) as a negative marker for plasmid curing and determining the optimal size of the donor DNA template for gene integration in the CRISPR-FnCas12a system, we achieved highly efficient and rapid genome modification, exemplified by the successful engineering of clostridia (Clostridium butyricum and Clostridium sporogenes) to produce near-infrared fluorescence from biliverdin and hemin.IMPORTANCEContinued efforts in developing the CRISPR-Cas systems will further enhance our understanding and utilization of Clostridium species. This study demonstrates the development and application of a genome-engineering tool in two Clostridium strains, Clostridium butyricum and Clostridium sporogenes, which have promising potential as probiotics and oncolytic agents. Particular attention was given to the folding of precursor crRNA and the role of this process in off-target DNA cleavage by Cas12a. The results provide the guidelines necessary for efficient genome engineering using this system in clostridia. Our findings not only expand our fundamental understanding of genome-engineering tools in clostridia but also improve this technology to allow use of its full potential in a plethora of biotechnological applications.

Endothelial cell-expressed long non-coding RNAs in cardiac regeneration

Abstract Endothelial cells (ECs) are the most abundant non-myocyte cell types in the heart. Upon cardiac injury, revascularization can occur through proliferation and migration of ECs from existing blood vessels in the surrounding myocardium. In animal models, revascularization is crucial to stimulate cardiomyocyte survival and regeneration. In the human heart, establishing blood flow could improve outcomes of tissue engineering or cell based therapy to regenerate the heart after injury. In addition to establishing blood flow, EC also secrete factors in a paracrine manner which can act on neighbouring cardiomyocytes. We investigate a role for non-coding RNA in the regulation of endothelial cell function and their potential role in neovascularization and cardiac regeneration. A large portion of the genome is transcribed into non-coding RNA, which does not encode protein. Many long non-coding RNAs (lncRNAs) have been shown to be involved in important regulatory processes such as genomic imprinting and chromatin modification. To identify lncRNAs which could be involved in cardiac regeneration, we isolated ECs from neonatal mice at postnatal day 1 and 5.5. The potential for cardiac regeneration was observed in mice at postnatal day 1, but this capacity is lost by day 5.5. Differential expression of lncRNAs in ECs was assessed by RNA sequencing. Three potential target lncRNAs, which also have a human homologue, were selected. NR2F2-AS1 and HAND2-AS1 were upregulated at postnatal day 1, whereas FOXD2-AS1 was upregulated at day 5.5. These genes were selected for further validation in human cardiac microvascular endothelial cells (CMEC). Knockdown of NR2F2-AS1 resulted in a decrease in proliferation but did not affect angiogenic sprouting. Loss of HAND2-AS1 resulted in a trend to impaired sprouting but did not affect proliferation. FOXD2-AS1 knockdown resulted in a significant decrease in angiogenic sprouting. We observed no change in the rate of migration after knockdown of any of the three genes. Knockdown of the lncRNAs did not affect expression of the antisense protein coding transcripts, therefore the effects we observe are likely mediated by the lncRNAs. In conclusion, lncRNAs NR2F2-AS1, HAND2-AS1 and FOXD2-AS1 regulate different aspects of EC function such as proliferation and sprouting. Modulation of expression of these transcripts therefore may be considered as a therapeutic tool to promote cardiac regeneration.