CRISPR/Cas12a collateral cleavage activity for an ultrasensitive assay of RNase H

We herein describe an ultrasensitive RNase H assay by utilizing CRISPR/Cas12a collateral cleavage activity. In this technique, a chimeric duplex probe consisting of activator DNA and its complementary blocker RNA...

A Bioinformatic Workflow for InDel Analysis in the Wheat Multi-Copy α-Gliadin Gene Family Engineered with CRISPR/Cas9

The α-gliadins of wheat, along with other gluten components, are responsible for bread viscoelastic properties. However, they are also related to human pathologies as celiac disease or non-celiac wheat sensitivity. CRISPR/Cas was successfully used to knockout α-gliadin genes in bread and durum wheat, therefore, obtaining low gluten wheat lines. Nevertheless, the mutation analysis of these genes is complex as they present multiple and high homology copies arranged in tandem in A, B, and D subgenomes. In this work, we present a bioinformatic pipeline based on NGS amplicon sequencing for the analysis of insertions and deletions (InDels) in α-gliadin genes targeted with two single guides RNA (sgRNA). This approach allows the identification of mutated amplicons and the analysis of InDels through comparison to the most similar wild type parental sequence. TMM normalization was performed for inter-sample comparisons; being able to study the abundance of each InDel throughout generations and observe the effects of the segregation of Cas9 coding sequence in different lines. The usefulness of the workflow is relevant to identify possible genomic rearrangements such as large deletions due to Cas9 cleavage activity. This pipeline enables a fast characterization of mutations in multiple samples for a multi-copy gene family.

The conserved single-cleavage mechanism of animal DROSHA enzymes

RNase III enzymes typically cleave both strands of double-stranded RNAs (dsRNAs). We recently discovered that a human RNase III, DROSHA, exhibits a single cleavage on the one strand of primary microRNAs (pri-miRNAs). This study revealed that DROSHAs from the other animals, including worms and flies, also show the single cleavage on dsRNAs. Furthermore, we demonstrated that the mechanism of single cleavage is conserved in animal DROSHA enzymes. In addition, the dsRNA-binding domain (dsRBD) and a 3p-strand cleavage-supporting helix (3pCSH) of the DROSHA enzymes foster a weak single cleavage on one strand, which ensures their double cleavages. Disrupting the interaction of dsRBD-RNA and 3pCSH-RNA by an internal loop (IL) and a 3pCSH-loop in the lower stem of pri-miRNAs, respectively, inhibits one of the double cleavages of DROSHAs, and this results in the single cleavage. Our findings expand our understanding of the enzymatic mechanisms of animal DROSHAs. They also indicate that there are currently unknown cellular functions of DROSHA enzymes using their single cleavage activity.

A Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells

CRISPR/Cas9 nucleases hold great potential for gene therapy, but they frequently induce unwanted off-target cleavage. We previously developed a GFP activation assay for detection of DNA cleavage in cells. Here, we demonstrate two novel applications of this assay. First, we use this assay to confirm off-target cleavage that cannot be detected by targeted deep sequencing in cells before. Second, we use this approach to detect multiple alternative PAMs recognized by SpCas9. These noncanonical PAMs are associated with low cleavage activity, but targets associated with these PAMs must be considered as potential off-target sites. Taken together, the GFP activation assay is a powerful platform for DNA cleavage detection in cells.

Spectral analysis of radical metalloenzyme using EPR in Hydrolases

One of the categories of hydrolase is acid phosphatase which is isolated from sweet potato (spAP). It contains metal ions such as Zn, Mn, Fe etc. with molecular weight 110kDa. An aqueous epr spectrum in buffer (pH 5.6) exhibits class II mixed valence nature of Fe+3 together with Fe+2, Mn+2, Zn+2 and Cu+2 metallobiosites. It indicates reduced form of enzyme with gMn(II)= 2.0084 and grad= 2.0041 signals with radical signal. The localized electron interaction with 55Mn+2 nucleus is evidenced by six-hyperfine splitted spin allowed transitions flanked by ten satellite lines. Allowed and forbidden doublets are computed with A (55Mn) coupling constant~96 G and Zfs parameter D=0.029 cm-1. The spin integration of Tyrradical signal is shown in lyophilized sample spectrum (1.029 x 1015 spins /gm). It also shows DNA cleavage activity with pUC 19 plasmid DNA.

The synthesis and evaluation of the antitumor and antibacterial activity of two novel oxovanadium complexes

Two novel oxovanadium(IV) complexes ([VO(hntdtsc)(BPIP)] and [VO(hntdtsc)(MOPIP)] (hntdtsc = 2-hydroxy-1-naphthaldehydethiosemicarbazone, BPIP = 2-(4-bromophenyl)-imidazo[4,5- f]-1,10-phenanthroline, MOPIP = 2-(4-methoxyphenyl)-imidazo[4,5- f]1,10-phenanthroline), are synthesized and characterized. Subsequently, the Methyl Thiazolyl Tetrazolium (MTT) assay is used to investigate the antitumor activity of the ligand and two complexes in vitro.The results indicate that both complexes could significantly inhibit selected tumor cells (SH-SY5Y, MCF-7, and SK-N-SH). In addition, the antibacterial activity of VO(hntdtsc)(BPIP) against Staphylococcus aureus is further investigated. Interestingly, VO(hntdtsc)(BPIP) can efficiently attenuate S. aureus growth and abrogate α-hemolysin secretion and biofilm formation. The plasmid DNA cleavage activity of both complexes is also investigated. The results suggest that supercoiled plasmid DNA is efficiently cleaved after treatment with each complex, which might contribute to the biological activity of these oxovanadium(IV) complexes.

Modeling homing suppression gene drive in haplodiploid organisms

Gene drives have shown great promise for suppression of pest populations. These engineered alleles can function by a variety of mechanisms, but the most common is the CRISPR homing drive, which converts wild-type alleles to drive alleles in the germline of heterozygotes. Some potential target species are haplodiploid, in which males develop from unfertilized eggs and thus have only one copy of each chromosome. This prevents drive conversion, a substantial disadvantage compared to diploids where drive conversion can take place in both sexes. Here, we study the characteristics of homing suppression gene drives in haplodiploids and find that a drive targeting a female fertility gene could still be successful. However, such drives are less powerful than in diploids. They are substantially more vulnerable to high resistance allele formation in the embryo due to maternally deposited Cas9 and gRNA and also to somatic cleavage activity. Examining models of continuous space where organisms move over a landscape, we find that haplodiploid suppression drives surprisingly perform nearly as well as in diploids, possibly due to their ability to spread further before inducing strong suppression. Together, these results indicate that gene drive can potentially be used to effectively suppress haplodiploid populations.

Mechanism of RNA polymerase III termination-associated reinitiation-recycling conferred by the essential function of the N terminal-and-linker domain of the C11 subunit

RNA polymerase III achieves high level tRNA synthesis by termination-associated reinitiation-recycling that involves the essential C11 subunit and heterodimeric C37/53. The C11-CTD (C-terminal domain) promotes Pol III active center-intrinsic RNA 3′-cleavage although deciphering function for this activity has been complicated. We show that the isolated NTD (N-terminal domain) of C11 stimulates Pol III termination by C37/53 but not reinitiation-recycling which requires the NTD-linker (NTD-L). By an approach different from what led to current belief that RNA 3′-cleavage activity is essential, we show that NTD-L can provide the essential function of Saccharomyces cerevisiae C11 whereas classic point mutations that block cleavage, interfere with active site function and are toxic to growth. Biochemical and in vivo analysis including of the C11 invariant central linker led to a model for Pol III termination-associated reinitiation-recycling. The C11 NTD and CTD stimulate termination and RNA 3′-cleavage, respectively, whereas reinitiation-recycling activity unique to Pol III requires only the NTD-linker. RNA 3′-cleavage activity increases growth rate but is nonessential.