Genotyping strategies for detecting CRISPR mutations in polyploid species: a case study-based approach in hexaploid wheat

As a versatile tool for genome engineering, CRISPR-Cas9 has been revolutionizing the field of molecular biology, biotechnology, and crop improvement. By precisely targeting pre-selected genomic sites, CRISPR-Cas9 primarily induces insertions or deletions (indels) of variable size. Despite the significant advance in the technology per se, detecting these indels is the major and difficult part of the CRISPR program in polyploid species, like wheat, with relatively low mutation rates. A plethora of methods are available for detecting mutations, but no method is perfect for all mutation types. In this case study, we demonstrated a new, protocol for capturing length polymorphism from small indels using a nested PCR approach. This new method is tractable, efficient, and cost-effective in detecting and genotyping indels >3-bp. We also discussed the major genotyping platforms used in our wheat CRISPR projects, such as mismatch cleavage assay, restriction enzyme assay, ribonucleoprotein assay, and Sanger sequencing, for their advantages and pitfalls in wheat CRISPR mutation detection.

Azoxystrobin Impairs Neuronal Migration and Induces ROS Dependent Apoptosis in Cortical Neurons

Fungicides often cause genotoxic stress and neurodevelopmental disorders such as autism (ASD). Fungicide-azoxystrobin (AZOX) showed acute and chronic toxicity to various organisms, and remained a concern for ill effects in developing neurons. We evaluated the neurotoxicity of AZOX in developing mouse brains, and observed prenatal exposure to AZOX reduced neuronal viability, neurite outgrowth, and cortical migration process in developing brains. The 50% inhibitory concentration (IC50) of AZOX for acute (24 h) and chronic (7 days) exposures were 30 and 10 μM, respectively. Loss in viability was due to the accumulation of reactive oxygen species (ROS), and inhibited neurite outgrowth was due to the deactivation of mTORC1 kinase activity. Pretreatment with ROS scavenger- N-acetylcysteine (NAC) reserved the viability loss and forced activation of mTORC1 kinase revived the neurite outgrowth in AZOX treated neurons. Intra-amniotic injection of AZOX coupled with in utero electroporation of GFP-labelled plasmid in E15.5 mouse was performed and 20 mg/kg AZOX inhibited radial neuronal migration. Moreover, the accumulation of mitochondria was significantly reduced in AZOX treated primary neurons, indicative of mitochondrial deactivation and induction of apoptosis, which was quantified by Bcl2/Bax ratio and caspase 3 cleavage assay. This study elucidated the neurotoxicity of AZOX and explained the possible cure from it.

piCRISPR: Physically Informed Features Improve Deep Learning Models for CRISPR/Cas9 Off-Target Cleavage Prediction

CRISPR/Cas programmable nuclease systems have become ubiquitous in the field of gene editing. With progressing development, applications in in vivo therapeutic gene editing are increasingly within reach, yet limited by possible adverse side effects from unwanted edits. Recent years have thus seen continuous development of off-target prediction algorithms trained on in vitro cleavage assay data gained from immortalised cell lines. Here, we implement novel deep learning algorithms and feature encodings for off-target prediction and systematically sample the resulting model space in order to find optimal models and inform future modelling efforts. We lay emphasis on physically informed features, hence terming our approach piCRISPR, which we gain on the large, diverse crisprSQL off-target cleavage dataset. We find that our best-performing model highlights the importance of sequence context and chromatin accessibility for cleavage prediction and outperforms state-of-the-art prediction algorithms in terms of area under precision-recall curve.

A Quantitative Arabidopsis IRE1a Ribonuclease-Dependent in vitro mRNA Cleavage Assay for Functional Studies of Substrate Splicing and Decay Activities

The unfolded protein response (UPR) is an adaptive eukaryotic reaction that controls the protein folding capacities of the endoplasmic reticulum (ER). The most ancient and well-conserved component of the UPR is Inositol-Requiring Enzyme 1 (IRE1). Arabidopsis IRE1a (AtIRE1) is a transmembrane sensor of ER stress equipped with dual protein kinase and ribonuclease (RNase) activities, encoded by its C-terminal domain. In response to both physiological stresses and pathological perturbations, AtIRE1a directly cleaves bZIP60 (basic leucine zipper 60) mRNA. Here, we developed a quantitative in vitro cleavage assay that combines recombinant AtIRE1a protein that is expressed in Nicotiana benthamiana and total RNA isolated from Arabidopsis leaves. Wild-type AtIRE1a as well as its variants containing point mutations in the kinase or RNase domains that modify its cleavage activity were employed to demonstrate their contributions to cleavage activity levels. We show that, when exposed to total RNA in vitro, the AtIRE1a protein cleaves bZIP60 mRNA. Depletion of the bZIP60 transcript in the reaction mixture can be precisely quantified by a qRT-PCR-mediated assay. This method facilitates the functional studies of novel plant IRE1 variants by allowing to quickly and precisely assess the effects of protein mutations on the substrate mRNA cleavage activity before advancing to more laborious, stable transgenic approaches in planta. Moreover, this method is readily adaptable to other plant IRE1 paralogs and orthologs, and can also be employed to test additional novel mRNA substrates of plant IRE1, such as transcripts undergoing degradation through the process of regulated IRE1-dependent decay (RIDD). Finally, this method can also be modified and expanded to functional testing of IRE1 interactors and inhibitors, as well as for studies on the molecular evolution of IRE1 and its substrates, providing additional insights into the mechanistic underpinnings of IRE1-mediated ER stress homeostasis in plant tissues.

Functional Validation of cas9/guideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8′-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.

Spc1 regulates the signal peptidase-mediated processing of membrane proteins

Signal peptidase (SPase) cleaves the signal sequences (SSs) of secretory precursors. It contains an evolutionarily conserved membrane protein subunit, Spc1 that is dispensable for the catalytic activity of SPase, and its role remains unknown. In the present study, we investigated the function of yeast Spc1. First, we set up an in vivo SPase cleavage assay using secretory protein CPY variants with SSs modified in the n and h regions. When comparing the SS cleavage efficiencies of these variants in cells with or without Spc1, we found that signal-anchored sequences became more susceptible to cleavage by SPase without Spc1. Further, SPase-mediated processing of model membrane proteins was enhanced in the absence of but reduced upon overexpression of Spc1. Spc1 was co-immunoprecipitated with proteins carrying uncleaved signal-anchored or transmembrane (TM) segments. Taken together, these results suggest that Spc1 protects TM segments from SPase action, thereby sharpening substrate selection for SPase and acting as a negative regulator for the SPase-mediated processing of membrane proteins.

Functional Validation of cas9/guideRNA Constructs for Site-directed Mutagenesis of Triticale ABA8’OH1 loci

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like Triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in Triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the Triticale ABA 8’-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in Triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in Triticale genes.

Ferulic Acid Inhibits Topoisomerase I of Mycobacterium Tuberculosis

IntroductionVarious drugs are first line anti-TB drugs for more than thirty years. Since ferulic acid have shown promising inhibition growth of M. tuberculosis and M. smegmatis cell.ObjectiveIn this study, we have explored the inhibitory effect of ferulic acid against Mycobacterium tuberculosis topoisomerase I. We further investigated the mechanism of ferulic acid by various experiments.MethodsDNA relaxation assays was done for supercoiled pUC18 DNA, oligonucleotide cleavage assay was performed for 5ʹ-end-labeled 32-mer harboring the STS (Strong Topoisomerase Site) annealed to a complimentary sequence. Growth inhibition (MIC) values were calculated by performing resazurin reduction microplate assay (REMA).ResultsIn this study, we describe the growth inhibition of M. tuberculosis and M. smegmatis through Mycobacterium tuberculosis topoisomerase I (MttopoI) by ferulic acid. We have further investigated the mechanism of ferulic acid by analysing the two step of topoisomerase 1 (topoI) reaction cycle. Ferulic acid was able to stimulate cleavage which further leads to cleavage-relegation equilibrium. Moreover, it was shown that ferulic acid inhibited topoI overexpressing cells at low minimum inhibitory concentration (MIC). Ferulic acid affects the activity of DNA relexation in the mutations in DxDxE motif of metal binding mutants which contributes to the reaction of enzyme with the drug.ConclusionIn conclusion, our results indicate that ferulic acid leads to combat microbial infection and act as toxins to MstopoI and MttopoI. Moreover, targeting metal coordination to topoisomerases might be a general strategy to develop new lead molecules.