Sumoylation of Cas9 at lysine 848 regulates protein stability and DNA binding

CRISPR/Cas9 is a popular genome editing technology. Although widely used, little is known about how this prokaryotic system behaves in humans. An unwanted consequence of eukaryotic Cas9 expression is off-target DNA binding leading to mutagenesis. Safer clinical implementation of CRISPR/Cas9 necessitates a finer understanding of the regulatory mechanisms governing Cas9 behavior in humans. Here, we report our discovery of Cas9 sumoylation and ubiquitylation, the first post-translational modifications to be described on this enzyme. We found that the major SUMO2/3 conjugation site on Cas9 is K848, a key positively charged residue in the HNH nuclease domain that is known to interact with target DNA and contribute to off-target DNA binding. Our results suggest that Cas9 ubiquitylation leads to decreased stability via proteasomal degradation. Preventing Cas9 sumoylation through conversion of K848 into arginine or pharmacologic inhibition of cellular sumoylation enhances the enzyme’s turnover and diminishes guide RNA-directed DNA binding efficacy, suggesting that sumoylation at this site regulates Cas9 stability and DNA binding. More research is needed to fully understand the implications of these modifications for Cas9 specificity.

PCR-free, label-free detection of sequence-specific DNA with single-molecule sensitivity using in vitro N-hybrid system in microfluidic drops

We propose a novel method that can detect DNA with high specificity at the single-molecule level by employing the in vitro N-hybrid strategy realized in sub-picoliter microfluidic drops. It detects target DNA based on the specific interactions of the target-encoded proteins with their partner molecules, and achieves single-molecule sensitivity via signal-transduction and signal-amplification during gene-expression processes in a sub-picoliter droplet, therefore effectively avoiding complicated procedures in labeling-based methods or biases and artifacts in PCR-based methods.

The Rice miR396-GRF-GIF-SWI/SNF Module: A Player in GA Signaling

Rice Growth-Regulating Factors (GRFs) were originally identified to be gibberellin (GA)-induced, but the nature of GA induction has remained unknown because most reports thereafter focused on revealing their roles in growth-promoting activities. GRFs have the WRC (Trp, Arg, Cys) domain to target DNA and contain the QLQ (Gln, Leu, Gln) domain to interact with GRF-Interacting Factor (GIF), which recruits ATP-dependent DNA translocase Switch/Sucrose Non-fermenting (SWI/SNF) for chromatin remodeling. Both GRFs and GIFs exhibit transcriptional activities but GIFs lack a DNA-binding domain. So, GRFs act like a navigator in the GRF-GIF-SWI/SNF complex, determining when and where the complex should work on. The levels of most rice GRFs can be sensitively regulated by miR396, which responds to many developmental and environmental factors. Recent clues from several studies highlight the original question of how GRFs participate in GA signaling. DELLA (contain DELLA motif) protein plays dual roles in controlling the level of GRFs by regulating the level of miR396 and interacting with GRFs. Here we address the question of why this complex plays an essential role in controlling plant growth focusing on the action of GA signaling pivot, DELLA.

Developing Qualitative Plasmid DNA Reference Materials to Detect Mechanisms of Quinolone and Fluoroquinolone Resistance in Foodborne Pathogens

The aim of this study was to develop homogeneous and stable plasmid DNA reference materials for detecting the mechanisms of resistance to quinolones and fluoroquinolones in foodborne pathogens. The DNA fragments of 11 target genes associated with quinolone and fluoroquinolone resistance were artificially synthesized, inserted into plasmid vectors, and transferred into recipient cells. PCR and sequencing of DNA were performed to assess the genetic stability of the target DNA in recombinant Escherichia coli DH5α cells during subculturing for 15 generations. The limit of detection (LOD) of the target DNA was determined using PCR and real-time qualitative PCR (qPCR). The homogeneity and storage stability of plasmid DNA reference materials were evaluated in terms of plasmid DNA quantity, PCR-measured gene expression, and qPCR threshold cycle. All 11 target DNAs were successfully synthesized and inserted into vectors to obtain recombinant plasmids. No nucleotide mutations were identified in the target DNA being stably inherited and detectable in the corresponding plasmids during subculturing of recombinant strains. When the target DNA was assessed using PCR and qPCR, the LOD was ≤1.77 × 105 and 3.26 × 104 copies/μL, respectively. Further, when the reference materials were stored at 37 °C for 13 days, 4 °C for 90 days, and −20 °C for 300 days, each target DNA was detectable by PCR, and no mutations were found. Although the threshold cycle values of qPCR varied with storage time, they were above the LOD, and no significant differences were found in the quantity of each plasmid DNA at different timepoints. Further, the homogeneity and stability of the materials were highly consistent with the requirements of standard reference materials. To summarize, considering that our plasmid DNA reference materials conformed to standard requirements, they can be used to detect the mechanisms of quinolone and fluoroquinolone resistance in foodborne pathogens.

RETINOBLASTOMA RELATED (RBR) interaction with key factors of the RNA-directed DNA methylation (RdDM) pathway

● Transposable elements and other repetitive elements are silenced by the RNA-directed DNA methylation pathway (RdDM). In RdDM, POLIV-derived transcripts are converted into double stranded RNA (dsRNA) by the activity of RDR2 and subsequently processed into 24 nucleotide short interfering RNAs (24-nt siRNAs) by DCL3. 24-nt siRNAs are recruited by AGO4 and serve as guides to direct AGO4-siRNA complexes to chromatin bound POLV-derived transcripts generated from the template/target DNA. The interaction between POLV, AGO4, DMS3, DRD1, RDM1 and DRM2 promotes DRM2-mediated de novo DNA methylation. In silico exploration of Arabidopsis RBR protein partners revealed that several members of the RdDM pathway contain a motif that confers high affinity binding to RBR, including the largest subunits of POLIV and POLV (NRPD1 and NRPE1), the shared second largest subunit of POLIV and POLV (NRPD/E2), RDR1, RDR2, DCL3, DRM2 and SUVR2. We demonstrate that RBR binds to DRM2, DRD1 and SUVR2. We also report that seedlings from loss-of-function mutants in RdDM and in RBR show similar phenotypes in the root apical meristem. Furthermore, we show that RdDM and SUVR2 targets are up-regulated in the 35S::AmiGO-RBR background. Our results suggest a novel mechanism for RBR function in transcriptional gene silencing based on the interaction with key players of the RdDM pathway and opens several new hypotheses, including the convergence of RBR-DRM2 on the transcriptional control of TEs and several cell/tissue and stage-specific target genes.

CRISPR/Cas9 Mediated Genome Editing in Crop Plants

Recently, most genomic research has focused on genome editing methods to develop new technologies that could be easy, reliable, and feasible to edit plant genomes for highly productive agriculture. Genome editing is based on alternating a specific target DNA sequence by adding, replacing, and removing DNA bases. This newest technology called CRISPR/Cas9 seems to be less time-consuming, more effective and used in many research areas of plant genetic research. CRISPR/Cas9 systems have many advantages in comparison with ZFNs and TALENs and has been extensively used for genome editing to many crop plant species. Around 20 crop species are successfully worked out for trait improvements, for example, yield improvement, disease resistance, herbicide tolerance, and biotic and abiotic stress management. This review paper will overview recent advances in CRISPR/Cas genome editing research in detail. The main focus will be on the use of CRISPR/Cas9 technology in plant genome research.

Evolutionary plasticity and functional versatility of CRISPR systems

The principal biological function of bacterial and archaeal CRISPR systems is RNA-guided adaptive immunity against viruses and other mobile genetic elements (MGEs). These systems show remarkable evolutionary plasticity and functional versatility at multiple levels, including both the defense mechanisms that lead to direct, specific elimination of the target DNA or RNA and those that cause programmed cell death (PCD) or induction of dormancy. This flexibility is also evident in the recruitment of CRISPR systems for nondefense functions. Defective CRISPR systems or individual CRISPR components have been recruited by transposons for RNA-guided transposition, by plasmids for interplasmid competition, and by viruses for antidefense and interviral conflicts. Additionally, multiple highly derived CRISPR variants of yet unknown functions have been discovered. A major route of innovation in CRISPR evolution is the repurposing of diverged repeat variants encoded outside CRISPR arrays for various structural and regulatory functions. The evolutionary plasticity and functional versatility of CRISPR systems are striking manifestations of the ubiquitous interplay between defense and “normal” cellular functions.

Amplificação isotérmica mediada por loop para detecção de patógenos de plantas

Disease control is crucial to minimize potential losses in agriculture and thereby maintain high crop yield. However, for its effectiveness, the pathogen must be detected early and correctly in the production fields. Different methods of diagnosis can be used, from those based on symptoms to molecular tests. Loop-mediated isothermal amplification (LAMP) is a molecular technique that has been widely used in several biological fields, due to the ease with which it can be applied. The reaction can be carried out in a single thermal condition, due to the use of Bst DNA polymerase, isolated from the bacterium Bacillus stearothermophilus, which has high displacement activity. LAMP is a highly exponential amplification method that produces the target DNA in amounts 109 -1010 times between 45 and 60 minutes at 60-65°C. Its advantages are the visualization of results directly with the naked eye and the fact that it does not need sophisticated equipment for its application. In phytopathology, the technique has been gaining prominence in the detection of fungi, viruses, bacteria, nematodes and phytoplasmas, as well as in the monitoring of fungicide-resistant fungi. LAMP can benefit agriculture so that early, accurate and sensitive diagnostics can be carried out in the fields of cultivation and minimize losses caused by diseases. In this review, we present and discuss LAMP tests, developed for plant pathogens detection, which can be useful for researchers who wish to use the technique in their research area

Classification of CRISPR/Cas system and its application in tomato breeding

Remarkable diversity in the domain of genome loci architecture, structure of effector complex, array of protein composition, mechanisms of adaptation along with difference in pre-crRNA processing and interference have led to a vast scope of detailed classification in bacterial and archaeal CRISPR/Cas systems, their intrinsic weapon of adaptive immunity. Two classes: Class 1 and Class 2, several types and subtypes have been identified so far. While the evolution of the effector complexes of Class 2 is assigned solely to mobile genetic elements, the origin of Class 1 effector molecules is still in a haze. Majority of the types target DNA except type VI, which have been found to target RNA exclusively. Cas9, the single effector protein, has been the primary focus of CRISPR-mediated genome editing revolution and is an integral part of Class 2 (type II) system. The present review focuses on the different CRISPR types in depth and the application of CRISPR/Cas9 for epigenome modification, targeted base editing and improving traits such as abiotic and biotic stress tolerance, yield and nutritional aspects of tomato breeding.