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A quick and solvent-free mechanochemical method was employed for the post-synthetic modification of a Ni-MOF. The Ni-MOF was utilized as an electrocatalyst for oxygen evolution and reduction, and biomass conversion applications.

While CRISPR-based editing most often occurs at DNA sequences with perfect homology to the guide RNA (gRNA), unintended editing can occur at highly homologous regions (i.e., off-target (OT) sites). Due to the pace at which genome editing therapies are approaching clinical applications, there is an emerging need to define effective workflows for investigating OT editing effects. A number of homology-dependent,in silico-basedprediction methods and wet lab-based empirical methods exist to investigate OT editing, but few have been subjected to analytical assessment or head-to-head comparison in human primary cells using anex vivoediting process optimized for high-fidelity gene editing. Therefore, we sought to compare publicly availablein silicotools (COSMID, CCTop, and Cas-OFFinder) as well as empirical methods (CHANGE-Seq, CIRCLE-Seq, DISCOVER-Seq, GUIDE-Seq, and SITE-Seq) in the context ofex vivohematopoietic stem and progenitor cell (HSPC) editing. To do so, we edited CD34+HSPCs using 11 different guide RNAs (gRNAs) complexed with HiFi Cas9, then performed targeted next-generation sequencing of ~200-site panels containing a range of nominated OT sites identified byin silicoand empirical methods. We identified an average of 0.45 OT sites per gRNA at an indel detection limit of 0.5%. This study confirmed the marked improvement in specificity with HiFi Cas9 compared to wild-type Cas9 without compromising on-target activity when delivered as an RNP. Additionally, all HiFi Cas9 OT sites using a standard 20nt gRNA were identified by all OT detection methods with one exception (SITE-seq did not identify an OT generated by an AAVS1 gRNA). This resulted in high sensitivity for the majority of OT nomination tools, however due to the large number of false positives called by most methods,in silico-based COSMID and empirical methods DISCOVER-Seq and GUIDE-Seq attained the highest positive predictive value. We did not find the empirical methods identified off-target sites that were not also identified by bioinformatic methods when delivered as an RNP complex. Finally, this study supports that refined bioinformatic algorithms could be developed that maintain both high sensitivity as well as positive predictive value which would enable more efficient identification of potential off-target sites without compromising a thorough examination for any given gRNA.

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR–Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.

BACKGROUND There are many parameters that are considered to affect function and the extrusion time of the ventilation tubes (Grommets) inserted for patients with secretory otitis media (glue ear/otitis media with effusion). Our objective is to study such parameters and their significance in order to better understand the disease process. METHODS A total of 84 patients who underwent grommet insertion were retrospectively evaluated for the relationship between various parameters such as extrusion time, age, gender, adenoid size, status of tympanic membrane at the time of insertion, the nature of the fluid aspirated during procedure, pre-operative impedance audiogram and combination of adenoidectomy procedure. RESULTS The mean time of extrusion of grommet in our study was found to be 254 days. Although rest of the parameters did not have much significance in the process of extrusion, we found that history of previous ventilation tube insertion took shorter time to extrude (mean, 203 days) and serous effusion (mean, 188 days) took shorter time to extrude than mucoid effusion (mean, 271 days). CONCLUSIONS The mean time for extrusion of ventilation tube (grommet) was 254 days in our study. The characteristics of the middle ear effusion and a history of previous ventilation tube insertion had significant impact on the extrusion time. The quality of effusion can give us a clinical clue to ventilation tube extrusion time, which in turn explains about the return of middle ear function

Background This study aims at comparing the pure tone audiogram and tympanogram results of a measured thickness (1 mm) conchal cartilage graft material (1.0-mm CC) with full-thickness conchal cartilage (FTCC) used for inactive chronic suppurative otitis media (CSOM) mucosal disease with moderate size perforation. Results A total of 40 successful cartilage tympanoplasty procedures were included in the prospective quasi-randomised study. Patients aged between 15 and 55 years with inactive CSOM—moderate size perforation in the tympanic membrane were included. The patients were assessed at the end of 12 weeks with a pure tone audiogram and a tympanogram. The results were compared to know the compliance of the tympanic membrane (TM) between the two groups and the difference in audiometric gain (air-bone gap closure). The air-bone gap gain was better in the 1.0-mm CC group of patients (12) compared to the FTCC group of patients (7). The difference was statistically significant (p = 0.04). The 1.0-mm CC group of patients showed better compliance (0.43) compared to FTCC group of patients (0.73). The air volume and compliance were significantly better in the 1-mm CC group compared to the FTCC group (p = 0.031 and 0.01 respectively). Conclusion The use of sliced cartilage seems to have a better role in reconstructing the TM when compared to full-thickness cartilage as graft material. It is indeed beneficial to have a compliant TM. Though the results cannot be compared with temporalis fascia grafts, the use of 1.0-mm CC has its advantage in producing an audio logically acceptable outcome in tympanoplasty surgery.

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The most commonly used Cas9 variant, Streptococcus pyogenes Cas9 (SpCas9), is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific than wild-type (WT) SpCas9. However, systematic comparisons of the cleavage activities of these Cas9 variants have not been reported. In this study, we employed our high-throughput in vitro cleavage assay to compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9, and HiFi Cas9). We observed that all Cas9s tested were able to cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on the target sequence and Cas9 variant. For targets with multiple mismatches, SaCas9 and engineered SpCas9 variants are more prone to nicking, while WT SpCas9 creates double-strand breaks (DSB). These differences in cleavage rates and DSB formation may account for the varied specificities observed in genome editing studies. Our analysis reveals mismatch position-dependent, off-target nicking activity of Cas9 variants which have been underreported in previous in vivo studies.

Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.

An important advantage of delivering CRISPR reagents into cells as a ribonucleoprotein (RNP) complex is the ability to edit genes without reagents being integrated into the genome. Transient presence of RNP molecules in cells can reduce undesirable off-target effects. One method for RNP delivery into plant cells is the use of a biolistic gun. To facilitate selection of transformed cells during RNP delivery, a plasmid carrying a selectable marker gene can be co-delivered with the RNP to enrich for transformed/edited cells. In this work, we compare targeted mutagenesis in rice using three different delivery platforms: biolistic RNP/DNA co-delivery; biolistic DNA delivery; and Agrobacterium-mediated delivery. All three platforms were successful in generating desired mutations at the target sites. However, we observed a high frequency (over 14%) of random plasmid or chromosomal DNA fragment insertion at the target sites in transgenic events generated from both biolistic delivery platforms. In contrast, integration of random DNA fragments was not observed in transgenic events generated from the Agrobacterium-mediated method. These data reveal important insights that must be considered when selecting the method for genome-editing reagent delivery in plants, and emphasize the importance of employing appropriate molecular screening methods to detect unintended alterations following genome engineering.

CRISPR–Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR–Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR–Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR–Cas systems. This diversity has enabled further expansion of CRISPR–Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR–Cas systems.

Cas12a (formerly Cpf1) is an RNA-guided endonuclease in the CRISPR-Cas immune system that can be easily programmed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for precise genome editing applications. This specificity is contradictory to the natural role of Cas12a as an immune effector against rapidly evolving phages. However, the native cleavage specificity and activity remains to be fully understood. We employed high-throughput in vitro cleavage assays to determine and compare the native specificities of three Cas12a orthologs. Surprisingly, we observed pervasive nicking of randomized target libraries, with strong nicking activity observed against targets with up to four mismatches. Nicking and cleavage activities are dependent on mismatch type and position, and vary depending on the Cas12a ortholog and crRNA sequence. Our high-throughput and biochemical analysis further reveal that Cas12a has robust activated non-specific nicking and weak non-specific dsDNA degradation activity in trans. Together, our findings reveal Cas12a cleavage activities that could be beneficial in the context of bacterial CRISPR-Cas immunity but may be detrimental for genome editing technology.

Bacteriophage-encoded anti-CRISPR (Acr) proteins were previously thought to inhibit CRISPR-mediated immunity by acting as physical barriers against the binding or cleavage of DNA. Two new studies report that recently discovered type V Acr proteins use enzymatic activities to shut down the Cas12a endonuclease, providing a multi-turnover ‘off switch’ for CRISPR-based immunity and technology.

Hashimoto’s encephalopathy (HE) is a rare autoimmune disorder with neurological and neuropsychiatric manifestations and elevated titres of anti-thyroid antibodies. Here we are reporting a case of HE in a 19-year-old girl who presented with seizure-like episodes, confusion, and behavioural disturbances with catatonic symptoms such as posturing, echopraxia, echolalia, and ambivalence. Patient did not respond to antipsychotics and anticonvulsants. On further investigation, patient was found to have high serum anti-TPO antibodies of about 1261 U/mL with euthyroid status, which supported a suspicion of HE. Our consultant neurologist confirmed the diagnosis and she was started on injection of methylprednisolone 750 mg OD. Since patient started showing clinical improvement, her antipsychotic medications were tapered off. On follow-up, patient has recovered and is functioning well. Since HE is a diagnosis of exclusion, very high anti-TPO antibodies and good response to steroids supported the diagnosis of HE in this patient after excluding other etiological possibilities. This case has been reported because the clinical presentation was predominantly neurobehavioural manifestations which is uncommon with HE.