Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing.

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

Effective coping strategies utilised by medical students for mental health disorders during undergraduate medical education-a scoping review.

BACKGROUND: Coping denotes cognitive, emotional and behavioural struggles to tackle a troubled person-environment association. Therefore, coping strategies (CSs) are vital for mental well-being. Widespread research studies have explored this domain, targeting caregivers, nurses, physicians and medical teachers, but limited research has been done to explore the common CSs utilised by medical students at the undergraduate medical education level. Therefore, we aimed to identify the frequently occurring CSs and their effects on mental health disorders (MHDs) through the evidence available in the existing literature. METHODS: For this scoping review, we searched the available literature (articles published from January 1, 1986, to March 31, 2021) on CSs at Google Scholar, PubMed and Scopus using the terms coping, medical students and undergraduate medical education. We included in our search all peer-reviewed journal articles whose central topics were the CSs employed by undergraduate medical students of any age, nationality, race and gender. RESULTS: From among the 2,134 articles that were found, 24 were ultimately included in the study. The articles were authored in 14 countries, allowing us to gather broader data to answer our research question. The first identified theme (MHDs) had four subthemes: stress (55% of the articles), depression (30%), anxiety (25%) and burnout (15%). The second theme (CSs), on the other hand, had eight subthemes: support seeking (60%), active coping (40%), acceptance (40%), avoidance/denial (40%), substance abuse (35%), faith/religion (25%), sports (25%) and miscellaneous (40%). CONCLUSIONS: Themes and subthemes were identified about the most common CSs utilised by undergraduate medical students to tackle common MHDs in the context of medical education. Among the most used CSs was support (social and emotional) seeking. Teaching medical students how to cope with challenging times is essential.

Scale development and validation for usage of social networking sites during COVID-19.

This research aims to explore the usage of social networking sites in the midst of the COVID-19 pandemic. To achieve the objective, this study develops and validates a scale to measure the usage of social networking sites. Data was collected via a google form link through social networking sites in India viz. Facebook, Instagram, Twitter, Telegram and WhatsApp. A total of 204 responses were found accurate and useable for the study. The population of the study includes people residing in India. Data was analyzed applying SPSS 20 and AMOS 22.0. To design and validate the scale, both Exploratory Factor Analysis (EFA) and Confirmatory Factor Analysis (CFA) were used in conjunction with each other. The EFA results indicate a six-factor structure, explaining 73.43% of the cumulative variance, with all the items loading above 0.70. Thus, all the 20 items of six constructs meet this threshold limit. Therefore, this 20-items questionnaire emerges as an effective tool for assessing social networking usage. This study developed and validated the scale of usage of social networking sites to measure the six constructs informativeness, education, entertainment, shopping, socialization and social cause. Social media developers and analysts can use this scale from a technological standpoint to redesign and refine their websites for a better user interface. Since the research is primarily focused on India, extrapolating the results to other countries is difficult.

The roles of emotional intelligence, neuroticism, and academic stress on the relationship between psychological distress and burnout in medical students.

BACKGROUND: Stress and burnout commonly threaten the mental health of medical students in Malaysia and elsewhere. This study aimed to explore the interrelations of psychological distress, emotional intelligence, personality traits, academic stress, and burnout among medical students. METHODS: A cross-sectional study was conducted with 241 medical students. Validated questionnaires were administered to measure burnout, psychological distress, emotional intelligence, personality traits, and academic stress, respectively. A structural equation modelling analysis was performed by AMOS. RESULTS: The results suggested a structural model with good fit indices, in which psychological distress and academic stress were noted to have direct and indirect effects on burnout. The burnout levels significantly increased with the rise of psychological distress and academic stress. Neuroticism was only found to have significant indirect effects on burnout, whereby burnout increased when neuroticism increased. Emotional intelligence had a significant direct effect on lowering burnout with the incremental increase of emotional intelligence, but it was significantly reduced by psychological distress and neuroticism. CONCLUSION: This study showed significant effects that psychological distress, emotional intelligence, academic stress, and neuroticism have on burnout. Academic stress and neuroticism significantly increased psychological distress, leading to an increased burnout level, while emotional intelligence had a significant direct effect on reducing burnout; however, this relationship was compromised by psychological distress and neuroticism, leading to increased burnout. Several practical recommendations for medical educators, medical students, and medical schools are discussed.

Design and optimization of a probiotic tablet for gastrointestinal tolerance by a simplex-centroid mixture.

In this study, a simplex-centroid mixture design using design of experiment (DOE) software was implemented to evaluate the effect of biopolymers as excipients, which are hydroxypropyl methylcellulose, and alginate, on the gastrointestinal tolerance of probiotic tablet containing Saccharomyces boulardii. Microbial viability and dissolution time were used to evaluate the ideal formulation made using 39.01% carboxymethylcellulose and 60.99% alginate as excipients, which protected the probiotics from the acidic condition in the stomach with good dissolution time. The formulated probiotic tablet is more stable in terms of viability when stored at 4 °C compared to room temperature. However, the viability remains above 106 CFU/tablet after six months of storage at room temperature. This study shows that the simplex-centroid mixture design is valid and can be used to formulate probiotic tablets that possess gastrointestinal tolerance. This study can lead to the development of commercial production of probiotic yeast tablets with gastrointestinal tolerance.

Scoping Review of frequently highlighted attributes of Medical Professionalism in an Undergraduate Medical Education Context.

BACKGROUND AND OBJECTIVES: Medical Professionalism (MP) establishes the trust between society and doctors. We aimed at finding frequently highlighted qualities of MP in the literature. METHODS: We searched PubMed and Scopus for attributes of MP, using terms, "Professionalism," "Medical Students," and "Undergraduate Medical Education". We included English language, original research articles with MP attributes from the perspective of undergraduate medical education, any nationality, race, gender, and age range, as the central topic of the article. Papers published from January 1st 1986 to 29th February 2020 were included. RESULTS: From 1349 identified articles, finally, 18 were included, authored in 10 countries, collectively contributing to answering the scoping review question. Two themes were identified: (1) Nurturing of MP, 11 (61.11%) out of 18 included articles, highlighted "respect" as the most dominant attribute as it appeared in 6 (54.55%) out of 11 reviews, "communication" 5 (45.45 %) studies and "honesty" and "integrity" 4 (36.36%). (2) Assessment of MP, 7 (38.89%) studies, and majority, 4 (57.14 %) assessed MP using American Board of Internal Medicine's elements of MP, viz, "altruism, accountability, excellence, duty, honor and integrity, respect for others." CONCLUSIONS: Themes exemplified MP's most discoursed issues. The attributes are frequently used worldwide. MP deliberates as a commitment toward the individual patient, society, and necessitates transforming from its present generic form to more explicit details.

Terminal Uridylyl Transferase Mediated Site-Directed Access to Clickable Chromatin Employing CRISPR-dCas9.

Locus-specific interrogation of target genes employing functional probes such as proteins and small molecules is paramount in decoding the molecular basis of gene function and designing tools to modulate its downstream effects. In this context, CRISPR-based gene editing and targeting technologies have proved tremendously useful, as they can be programmed to target any gene of interest by simply changing the sequence of the single guide RNA (sgRNA). Although these technologies are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using CRISPR system is not well developed due to the lack of adequate techniques. Here, we have devised an innovative technology called sgRNA-Click (sgR-CLK) that harnesses the power of bioorthogonal click chemistry for remodeling guide RNA to display synthetic molecules on target genes. sgR-CLK employs a novel posttranscriptional chemoenzymatic labeling platform wherein a terminal uridylyl transferase (TUTase) was repurposed to generate clickable sgRNA of choice by site-specific tailoring of multiple azide-modified nucleotide analogues at the 3' end. The presence of a minimally invasive azide handle assured that the sgRNAs are indeed functional. Notably, an azide-tailed sgRNA targeting the telomeric repeat served as a Trojan horse on the CRISPR-dCas9 system to guide synthetic tags (biotin) site-specifically on chromatin employing copper-catalyzed or strain-promoted click reactions. Taken together, sgR-CLK presents a significant advancement on the utility of bioorthogonal chemistry, TUTase, and the CRISPR toolbox, which could offer a simplified solution for site-directed display of small molecule probes and diagnostic tools on target genes.

Epilepsy Surgery in Hospital Universiti Sains Malaysia: Our Experiences since 2004.

Epilepsy surgery has been performed by a few centres in Malaysia, including Hospital Universiti Sains Malaysia (HUSM). To date, a total of 15 patients have undergone epilepsy surgery in HUSM. The epilepsy surgery included anterior temporal lobectomy (ATL) with amygdalohippocampectomy (AH) and Vagal nerve stimulation (VNS). The surgical outcomes of the patients were assessed using the International League Against Epilepsy (ILAE) outcome scale. The ILAE scores for patients who underwent ATL with AH were comparatively better than those who underwent VNS. One of the patient who underwent ATL with AH and frontal lesionectomy was found to have psychosis during follow up. Epilepsy surgery has proven to be an important treatment for medically resistant epilepsy. Thus it is important to raise public awareness regarding epilepsy and its treatment.

Influence of high-pressure homogenization, ultrasonication, and supercritical fluid on free astaxanthin extraction from ß-glucanase-treated Phaffia rhodozyma cells.

In this study astaxanthin production by Phaffia rhodozyma was enhanced by chemical mutation using ethyl methane sulfonate. The mutant produces a higher amount of astaxanthin than the wild yeast strain. In comparison to supercritical fluid technique, high-pressure homogenization is better for extracting astaxanthin from yeast cells. Ultrasonication of dimethyl sulfoxide, hexane, and acetone-treated cells yielded less astaxanthin than ß-glucanase enzyme-treated cells. The combination of ultrasonication with ß-glucanase enzyme is found to be the most efficient method of extraction among all the tested physical and chemical extraction methods. It gives a maximum yield of 435.71 ± 6.55 µg free astaxanthin per gram of yeast cell mass.

CRISPR/Cas9 Genome Editing for Tissue-Specific In Vivo Targeting: Nanomaterials and Translational Perspective.

Clustered randomly interspaced short palindromic repeats (CRISPRs) and its associated endonuclease protein, i.e., Cas9, have been discovered as an immune system in bacteria and archaea; nevertheless, they are now being adopted as mainstream biotechnological/molecular scissors that can modulate ample genetic and nongenetic diseases via insertion/deletion, epigenome editing, messenger RNA editing, CRISPR interference, etc. Many Food and Drug Administration-approved and ongoing clinical trials on CRISPR adopt ex vivo strategies, wherein the gene editing is performed ex vivo, followed by reimplantation to the patients. However, the in vivo delivery of the CRISPR components is still under preclinical surveillance. This review has summarized the nonviral nanodelivery strategies for gene editing using CRISPR/Cas9 and its recent advancements, strategic points of view, challenges, and future aspects for tissue-specific in vivo delivery of CRISPR/Cas9 components using nanomaterials.

Effectiveness of peer-led education on knowledge, attitude and risk behavior practices related to HIV among students at a Malaysian public university--a randomized controlled trial.

OBJECTIVE: Develop, implement and evaluate the effectiveness of a peer-led education program related to HIV/AIDS among university students. DESIGN: randomized controlled trial with 276 university students at Faculty of Medicine and Health Sciences University Putra Malaysia (UPM), Serdang in 2011. INTERVENTION: A peer-led education program on HIV prevention by university students. OUTCOME: differences in knowledge, attitude and risk behavior practices related to HIV between baselines, immediate follow-up after intervention and after three months. RESULTS: Significant improvement in sound knowledge in the intervention group as compared to the control group (Odds ratio, 1.75; 95% CI 1.01, 3.00; p=0.04) and improvement in good attitude related to HIV (Odds ratio 2.22; 95% CI 1.37, 3.61; p=0.01). The odds of high substance risk behavior was significantly reduced in the intervention group as compared to the control group (Odds ratio 0.07; 95% CI 0.02, 0.34; p=0.01). The association between good knowledge and intervention was modified by the different time points (baseline, immediately after intervention and 3 months after intervention), ethnicity and gender. CONCLUSION: Peer-led education program in HIV prevention improves knowledge, attitude and substance risk behavior. Changes in sexual risk behavior may require a longer follow-up.

The cognitive underpinnings of emerging mathematical skills: executive functioning, patterns, numeracy, and arithmetic.

BACKGROUND: Exposure to mathematical pattern tasks is often deemed important for developing children's algebraic thinking skills. Yet, there is a dearth of evidence on the cognitive underpinnings of pattern tasks and how early competencies on these tasks are related to later development. AIMS: We examined the domain-specific and domain-general determinants of performances on pattern tasks by using (a) a standardized test of numerical and arithmetic proficiency and (b) measures of executive functioning, respectively. SAMPLE: Participants were 163 6-year-olds enrolled in primary schools that typically serve families from low to middle socioeconomic backgrounds. METHOD: Children were administered a battery of executive functioning (inhibitory, switching, updating), numerical and arithmetic proficiency (the Numerical Operations task from the Wechsler Individual Achievement Test-II), and three types of pattern tasks. RESULTS: Contrary to findings from the adult literature, we found all the executive functioning measures coalesced into two factors: updating and an inhibition/switch factor. Only the updating factor predicted performances on the pattern tasks. Although performance on the pattern tasks were correlated with numerical and arithmetic proficiency, findings from structural equation modelling showed that there were no direct or independent relationships between them. CONCLUSIONS: The findings suggest that the bivariate relationships between pattern, numeracy, and arithmetic tasks are likely due to their shared demands on updating resources. Unlike older children, these findings suggest that for 6-year-olds, better numerical and arithmetic proficiency, without accompanying advantages in updating capacities, will no more likely lead to better performance on the pattern tasks.

Green extraction of lutein from marigold flower petals, process optimization and its potential to improve the oxidative stability of sunflower oil.

Marigold flower petals are considered the richest source of lutein which possesses immense applications in the food and health sector. The study was undertaken to improve the stability of sunflower oil by enriching it with lutein extracted from marigold flower petals using safe and green technology. The extraction of lutein was optimized using Box-Behnken design by ultrasound-assisted extraction (UAE) employing sunflower oil as a solvent. The impact of three independent variables i.e., ultrasonic intensity, solid to solvent ratio, and extraction time were evaluated on the amount of lutein extracted and its antioxidant activity. Highest amount of lutein (21.23 mg/g) was extracted by employing ultrasonic intensity of 70 W/m2, extraction time of 12.5 min, and solid to solvent ratio of 15.75%. FT-IR spectra of lutein extracted by ultrasound and conventional extraction show similar peaks depicting that ultrasound does not have any impact on the functionality of lutein. Sunflower oil incorporated with lutein at 1000 PPM and the synthetic antioxidant (TBHQ) showed good oxidative stability than oil with 500 PPM lutein and no lutein during accelerated storage for a month. The oxidative stability was shown by different oil samples in the following order: TBHQ = 1000PPM lutein˃500PPM lutein ˃control oil. It was concluded that the ultrasound technique extracts lutein efficiently from marigold flowers and this lutein was effective in improving the oxidative stability of sunflower oil under accelerated storage conditions.

Cationic lipopolymeric nanoplexes containing the CRISPR/Cas9 ribonucleoprotein for genome surgery.

sgRNA/Cas9 ribonucleoproteins (RNPs) provide a site-specific robust gene-editing approach avoiding the mutagenesis and unwanted off-target effects. However, the high molecular weight (∼165 kDa), hydrophilicity and net supranegative charge (∼-20 mV) hinder the intracellular delivery of these RNPs. In the present study, we have prepared cationic RNPs lipopolymeric nanoplexes that showed a size of 117.3 ± 7.64 nm with +6.17 ± 1.04 mV zeta potential and >90% entrapment efficiency of RNPs. Further, these RNPs lipopolymeric nanoplexes showed good complexation efficiency and were found to be stable for 12 h with fetal bovine serum. These RNPs lipopolymeric nanoplexes did not induce any significant cytotoxicity in HEK293T cells, and were efficiently uptaken via a clathrin-mediated pathway with optimal transfection efficiency and nuclear localization after 48 h. Further, HEK293T cells having the mGFP insert were used as a cell line model for gene editing, wherein the loss of the mGFP signal was observed as a function of gene editing after transfection with mGFP targeting RNPs lipopolymeric nanoplexes. Further, the T7 endonuclease and TIDE assay data showed a decent gene editing efficiency. Additionally, the lipopolymeric nanoplexes were able to transfect muscle cells in vivo, when injected intra-muscularly. Collectively, this study explored the potential of cationic lipopolymeric nanoplexes for delivering gene-editing endonucleases.

The role of open innovation, hotel service quality and marketing strategy in hotel business performance.

These days, instead of developing and marketing new products in the market, organizations are shifting towards commercialization. Therefore, open innovation is of much importance in this era of industrialization. In this study, open innovation is used as a strategic tool to improve the service innovation and organizational performance of the Malaysian hospitality industry. Therefore, the objective of this research study is to examine the role of open innovation in business performance of Malaysian hospitality industry. A survey is conducted with the management of the organizations of the hospitality industry to collect data. Partial Least Square-Structural equation modeling (PLS-SEM) tool is used to test the hypothesis and get results. The findings of the study highlighted that open innovation is the main source of boosting organizational performance in the hospitality industry of Malaysia. The increase in open innovation practices among hotels can increase the business performance by increasing return on investment, return on assets, return on sales and return on equity. The term open innovation incorporates ICT, innovation in the organization, and internal and external knowledge management. Accumulation of these elements forms a mechanism that results in an increase in organizational performance by incorporating service innovation. Hence, this study is most suitable to develop strategies for service innovation using open innovation that improves organizational performance.

FnCas9-based CRISPR diagnostic for rapid and accurate detection of major SARS-CoV-2 variants on a paper strip.

The COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacted global health, causing increased mortality among elderly patients and individuals with comorbid conditions. During the passage of the virus through affected populations, it has undergone mutations, some of which have recently been linked with increased viral load and prognostic complexities. Several of these variants are point mutations that are difficult to diagnose using the gold standard quantitative real-time PCR (qRT-PCR) method and necessitates widespread sequencing which is expensive, has long turn-around times, and requires high viral load for calling mutations accurately. Here, we repurpose the high specificity of Francisella novicida Cas9 (FnCas9) to identify mismatches in the target for developing a lateral flow assay that can be successfully adapted for the simultaneous detection of SARS-CoV-2 infection as well as for detecting point mutations in the sequence of the virus obtained from patient samples. We report the detection of the S gene mutation N501Y (present across multiple variant lineages of SARS-CoV-2) within an hour using lateral flow paper strip chemistry. The results were corroborated using deep sequencing on multiple wild-type (n = 37) and mutant (n = 22) virus infected patient samples with a sensitivity of 87% and specificity of 97%. The design principle can be rapidly adapted for other mutations (as shown also for E484K and T716I) highlighting the advantages of quick optimization and roll-out of CRISPR diagnostics (CRISPRDx) for disease surveillance even beyond COVID-19. This study was funded by Council for Scientific and Industrial Research, India.

SARS-CoV-2, the virus responsible for COVID-19, has a genome made of RNA (a nucleic acid similar to DNA) that can mutate, potentially making the disease more transmissible, and more lethal. Most countries have monitored the rise of mutated strains using a technique called next generation sequencing (NGS), which is time-consuming, expensive and requires skilled personnel. Sometimes the mutations to the virus are so small that they can only be detected using NGS. Finding cheaper, simpler and faster SARS-CoV-2 tests that can reliably detect mutated forms of the virus is crucial for public health authorities to monitor and manage the spread of the virus. Lateral flow tests (the same technology used in many pregnancy tests) are typically cheap, fast and simple to use. Typically, lateral flow assay strips have a band of immobilised antibodies that bind to a specific protein (or antigen). If a sample contains antigen molecules, these will bind to the immobilised antibodies, causing a chemical reaction that changes the colour of the strip and giving a positive result. However, lateral flow tests that use antibodies cannot easily detect nucleic acids, such as DNA or RNA, let alone mutations in them. To overcome this limitation, lateral flow assays can be used to detect a protein called Cas9, which, in turn, is able to bind to nucleic acids with specific sequences. Small changes in the target sequence change how well Cas9 binds to it, meaning that, in theory, this approach could be used to detect small mutations in the SARS-CoV-2 virus. Kumar et al. made a lateral flow test that could detect a Cas9 protein that binds to a nucleic acid sequence found in a specific mutant strain of SARS-CoV-2. This Cas9 was highly sensitive to changes in its target sequence, so a small mutation in the target nucleic acid led to the protein binding less strongly, and the signal from the lateral flow test being lost. This meant that the lateral flow test designed by Kumar et al. could detect mutations in the SARS-CoV-2 virus at a fraction of the price of NGS approaches if used only for diagnosis. The lateral flow test was capable of detecting mutant viruses in patient samples too, generating a colour signal within an hour of a positive sample being run through the assay. The test developed by Kumar et al. could offer public health authorities a quick and cheap method to monitor the spread of mutant SARS-CoV-2 strains; as well as a way to determine vaccine efficacy against new strains.

Rapid and accurate nucleobase detection using FnCas9 and its application in COVID-19 diagnosis.

Rapid detection of DNA/RNA pathogenic sequences or variants through point-of-care diagnostics is valuable for accelerated clinical prognosis, as witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are tough to implement with limited resources, necessitating the development of accurate and robust alternative strategies. Here, we report FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a direct Cas9 based enzymatic readout for detecting nucleobase and nucleotide sequences without trans-cleavage of reporter molecules. We also demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs), including heterozygous carriers, and present a simple web-tool JATAYU to aid end-users. FELUDA is semi-quantitative, can adapt to multiple signal detection platforms, and deploy for versatile applications such as molecular diagnosis during infectious disease outbreaks like COVID-19. Employing a lateral flow readout, FELUDA shows 100% sensitivity and 97% specificity across all ranges of viral loads in clinical samples within 1hr. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection closer to home.

Complementary treatment of psychotic and epileptic patients in malaysia.

The objective of this article is to describe and compare the use of traditional/complementary medicine (T/CM) among psychotic (schizophrenia and schizophreniform disorder) and epileptic Malay patients in peninsular Malaysia. There were 60 patients in each group. T/CM consultation was uniformly spread across all levels of education and social status. We could not find a single over-riding factor that influenced the decision to seek T/CM treatment because the decision to seek such treatment was complex and the majority of decisions were made by others. Fifty-three patients (44.2%), consisting of 37 (61.7%) psychotic and 16 (26.7%) epileptic patients had consulted Malay traditional healers (bomoh) and/or homeopathic practitioners in addition to modern treatment; of these, only three had consulted bomoh and homeopathic practitioners at the same time. The use of T/CM was significantly higher in psychotic than in epileptic Malay patients.

A G-quadruplex motif at the 3' end of sgRNAs improves CRISPR-Cas9 based genome editing efficiency.

Originating as a component of prokaryotic adaptive immunity, the type II CRISPR/Cas9 system has been repurposed for targeted genome editing in various organisms. Although Cas9 can bind and cleave DNA efficiently under in vitro conditions, its activity inside a cell can vary dramatically between targets owing to the differences between genomic loci and the availability of enough Cas9/sgRNA (single guide RNA) complex molecules for cleavage. Most methods have so far relied on Cas9 protein engineering or base modifications in the sgRNA sequence to improve CRISPR/Cas9 activity. Here we demonstrate that a structure based rational design of sgRNAs can enhance the efficiency of Cas9 cleavage in vivo. By appending a naturally forming RNA G-quadruplex motif to the 3' end of sgRNAs we can improve its stability and target cleavage efficiency in zebrafish embryos without inducing off-target activity, thereby underscoring its value in the design of better and optimized genome editing triggers.