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Type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one strand of a double-helical DNA substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we detect intrinsic instability in DNA flanking the RNA-3' side of R-loops, which Cas12a can exploit to expose second-strand DNA for cutting. Interestingly, DNA flanking the RNA-5' side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

Aedes aegypti is the principal mosquito vector for many arboviruses that increasingly infect millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has sparked significant enthusiasm for genetic control of mosquitoes; however, no such system has been developed in Ae. aegypti. To fill this void, here we develop several CRISPR-based split gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission.

Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies1-5. Here we report two improvements that increase the efficiency of CRISPR-Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3+) T cells, CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived6 hematopoietic stem progenitor cells (HSPCs).

Gene-drive systems in diploid organisms bias the inheritance of one allele over another. CRISPR-based gene-drive expresses a guide RNA (gRNA) into the genome at the site where the gRNA directs Cas9-mediated cleavage. In the presence of Cas9, the gRNA cassette and any linked cargo sequences are copied via homology-directed repair (HDR) onto the homologous chromosome. Here, we develop an analogous CRISPR-based gene-drive system for the bacterium Escherichia coli that efficiently copies a gRNA cassette and adjacent cargo flanked with sequences homologous to the targeted gRNA/Cas9 cleavage site. This "pro-active" genetic system (Pro-AG) functionally inactivates an antibiotic resistance marker on a high copy number plasmid with ~ 100-fold greater efficiency than control CRISPR-based methods, suggesting an amplifying positive feedback loop due to increasing gRNA dosage. Pro-AG can likewise effectively edit large plasmids or single-copy genomic targets or introduce functional genes, foreshadowing potential applications to biotechnology or biomedicine.

The CRISPR/Cas system is a highly specific genome editing tool capable of distinguishing alleles differing by even a single base pair. Target sites might carry genetic variations that are not distinguishable by sgRNA designing tools based on one reference genome. AlleleAnalyzer is an open-source software that incorporates single-nucleotide variants and short insertions and deletions to design sgRNAs for precisely editing 1 or multiple haplotypes of a sequenced genome, currently supporting 11 Cas proteins. It also leverages patterns of shared genetic variation to optimize sgRNA design for different human populations. AlleleAnalyzer is available at https://github.com/keoughkath/AlleleAnalyzer .

BackgroundRewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve in a predictable manner using targeted systems.ResultsHere, we report that persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). We demonstrate that the histone methyltransferase requirement can be locus specific. Co-targeting Ezh2-dCas9, but not KRAB-dCas9, with DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50 days (approximately 57 cell divisions) and triggered an epigenetic switch to a heterochromatic environment. An increase in H3K27 trimethylation and DNA methylation was stably maintained and accompanied by a sustained loss of H3K27 acetylation. Interestingly, substitution of Ezh2-dCas9 with KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the off-target loci tested. Conversely, robust hypermethylation was observed at HER2. We further demonstrated that Ezh2-dCas9 required full-length DNMT3L for maximal activity and that co-targeting DNMT3L was sufficient for persistent repression by Ezh2-dCas9 or KRAB-dCas9.ConclusionsThese data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci. Fine-tuning of targeting tools is a necessity to engineer epigenetic memory at any given locus in any given cell type.

Base editors are a new family of programmable genome editing tools that fuse ssDNA (single stranded DNA) modifying enzymes to catalytically inactive CRISPR-associated (Cas) endonucleases to induce highly efficient single base changes. With dozens of base editors now reported, it is apparent that these tools are highly modular; many combinations of ssDNA modifying enzymes and Cas proteins have resulted in a variety of base editors, each with its own unique properties and potential uses. In this perspective, we describe currently available base editors, highlighting their modular nature and describing the various options available for each component. Furthermore, we briefly discuss applications in synthetic biology and genome engineering where base editors have presented unique advantages over alternative techniques.

Base editors are a new family of programmable genome editing tools that fuse ssDNA (single stranded DNA) modifying enzymes to catalytically inactive CRISPR-associated (Cas) endonucleases to induce highly efficient single base changes. With dozens of base editors now reported, it is apparent that these tools are highly modular; many combinations of ssDNA modifying enzymes and Cas proteins have resulted in a variety of base editors, each with its own unique properties and potential uses. In this perspective, we describe currently available base editors, highlighting their modular nature and describing the various options available for each component. Furthermore, we briefly discuss applications in synthetic biology and genome engineering where base editors have presented unique advantages over alternative techniques.

Bullying behavior is currently very widespread in the school environment. The school as a place for students to study and pursue education becomes an environment that is no longer conducive to supporting the learning process. Therefore, this study aims to see the importance of the teacher's role as a guide in dealing with bullying behavior that occurs in the classroom. This research is qualitative descriptive research. The instruments used are observation sheets, interviews, and reflection. The conclusion obtained from this study is that the teacher as a mentor is very important in overcoming bullying behavior because the teacher acts as a role model, as well as a person who has authority in the classroom. As a supervisor the teacher pays attention to things that can be done, namely giving advice, instilling character education, as well as role models in the form of active action, namely giving motivation and exemplifying friendly and polite interaction patterns. The result is the creation of a classroom atmosphere that has good communication quality which has an impact on reducing bullying behavior in class. BAHASA INDONESIA ABSTRACT: Perilaku perundungan saat ini sangat marak terjadi di lingkungan sekolah. Oleh karena itu, sekolah sebagai wadah bagi para siswa untuk belajar dan menempuh pendidikan menjadi lingkungan yang tidak lagi kondusif dalam mendukung proses pembelajaran. Oleh karena itu, penelitian ini bertujuan untuk melihat pentingnya peran guru sebagai pembimbing dalam mengatasi perilaku perundungan yang terjadi di kelas. Penelitian ini adalah penelitian deskriptif kualitatif. Instrumen yang digunakan adalah lembar observasi, wawancara, dan refleksi. Kesimpulan yang diperoleh dari penelitian ini adalah guru sebagai pembimbing sangat penting dalam mengatasi perilaku perundungan dikarenakan guru berperan sebagai teladan sekaligus pribadi yang memiliki otoritas di dalam kelas. Sebagai pembimbing guru menaruh perhatian terhadap hal-hal yang bisa dilakukan yakni memberi nasihat, menanamkan pendidikan karakter, juga teladan dalam bentuk tindakan aktif yaitu memberi motivasi dan mencontohkan pola interaksi yang ramah dan santun. Hasilnya berupa terciptanya suasana kelas yang memiliki kualitas komunikasi yang baik yang berdampak pada menurunnya perilaku perundungan di kelas.

As a result of persistent weakening or loss of function of one of the senses or organs, which are stated in articles 87, 89 and 95 of the Turkish Penal Code, penalty increases are applied at varying rates depending on whether the act was committed by negligence, willful or torture. The need for a separate guide has become more evident today, as there are deficiencies in the guide used for the evaluation of these situations, and the existing debates about the charts used to determine the financial compensation recommended to be used to support the guide. The existing problems in the evaluation of the function of the senses and organs also bring the problem of fair distribution of justice. Considering the problems in practice, a separate guide is needed for the evaluation of sensory and organ function, which can be considered as a second-line forensic medical evaluation. Since the definition of organ is evaluated differently in the Turkish Penal Code and in the medical literature, first of all, it will be important to ensure a common language unity in practice, to define all the body parts that will be included in the concept of organ in forensic medicine practices. Including studies in the literature in the guide will provide a more reassuring approach in updating the guide. What are the concepts of organs and senses from a forensic medicine perspective? What should be considered in the forensic medical examination in the evaluation of their functions? In our study, the concepts of permanent weakening/loss of the function of one of the senses or organs will be discussed in terms of legal and forensic medicine and solutions will be discussed by revealing the problems experienced in practice.

Abstract Background and objective The Cortical Bone Trajectory (CBT) technique provides an alternative method for fixation in the lumbar spine in patients with osteoporosis. An accuracy CBT screw placement could improve mechanical stability and reduce complication rates. Purpose The purpose of this study is to explore the accuracy of cortical screw placement with the application of implanted spinous process clip (SPC) guide. Methods and materials Four lumbar specimens with T12-S1 were used to access the accuracy of the cortical screw. The SPC-guided planning screws were compared to the actual inserted screws by superimposing the vertebrae and screws preoperative and postoperative CT scans. According to preoperative planning, the SPC guide was adjusted to the appropriate posture to allow the K-wire drilling along the planned trajectory. Pre and postoperative 3D-CT reconstructions was used to evaluate the screw accuracy according to Gertzbein and Robbins classification. Intraclass correlation coefficients (ICCs) and Bland–Altman plots were used to examine SPC-guided agreements for CBT screw placement. Results A total of 48 screws were documented in the study. Clinically acceptable trajectory (grades A and B) was accessed in 100% of 48 screws in the planning screws group, and 93.8% of 48 screws in the inserted screws group (p = 0.242). The incidence of proximal facet joint violation (FJV) in the planning screws group (2.1%) was comparable to the inserted screws group (6.3%) (p = 0.617). The lateral angle and cranial angle of the planned screws (9.2 ± 1.8° and 22.8 ± 5.6°) were similar to inserted screws (9.1 ± 1.7° and 23.0 ± 5.1°, p = 0.662 and p = 0.760). Reliability evaluated by intraclass correlation coefficients and Bland–Altman showed good consistency in cranial angle and excellent results in lateral angle and distance of screw tip. Conclusions Compared with preoperative planning screws and the actually inserted screws, the SPC guide could achieve reliable execution for cortical screw placement.

Copyright of Automobile Technology & Material is the property of Automobile Technology & Material Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of West China Journal of Stomatology is the property of Sichuan University, West China College of Stomatology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Higher education has begun to shift from a teacher-centred instructional approach to a student-centred learning approach; many instructors have embraced this change by developing flipped classrooms. In the flipped classroom, students complete pre-work prior to attending class; this pre-work is often in the form of a video recording, lecture recording or reading. Class time is then repurposed to focus on knowledge application and synthesis rather than delivery. As such, it is suggested that instructors become less sage- and more guide-like in their teaching approach. The impact of the flipped classroom has been mostly positive in research; however, it is unclear whether students actually note and/or value the change in the role of the instructor in the flipped classroom. This study sought to evaluate the perception of the ideal instructor according to students following their experience in a flipped classroom course. Data were collected on students' perceptions of the instructor prior to, during, and following a flipped classroom experience. Overall, students valued the instructor's role as a moderator rather than information-deliverer; however, the most important aspect of the flipped classroom, according to the students in this study, was the ability of the flipped classroom to foster greater interaction and learning between students. [ABSTRACT FROM AUTHOR]

Introduction: Fixation of femoral neck fractures with cannulated screws is an accepted method. This study aimed to compare a novel guide device and conventional technique with regard to the accuracy, operation time, fluoroscopy numbers, and drilling attempts during the insertion of cannulated screws to internal fixation of femoral neck fractures. Methods: This retrospective study included 60 patients with intracapsular femoral neck fractures who were treated with cannulated screws from January 2020 to June 2021. Three screws were inserted into the femoral neck by conventional technique or using the novel guide device. The operative time, total drilling attempts, and fluoroscopy numbers were evaluated. The precision of implant placement was evaluated by screw parallelism and spread. The patients were followed for 12–24 months. Results: The fluoroscopy numbers and operation time of the guide-device-assisted group were shorter than for the conventional group (p < 0.05). The total drilling attempts were significantly lower with the guide device than in the conventional group (p < 0.05). The guide-device-assisted group had better screw parallelism and greater spread than the conventional group (p < 0.05). Conclusions: The novel guide device may be an effective assistant instrument for internal fixation of femoral neck fracture with cannulated screws. [ABSTRACT FROM AUTHOR]

BACKGROUND: Complications such as infection, arrhythmia, pulmonary embolism, heart failure, and deep vein thrombosis exist after total knee arthroplasty, and venous thromboembolism is an important risk factor for surgical failure. Therefore, effective postoperative thrombosis monitoring is particularly important. OBJECTIVE: To discuss the early warning value of thrombus molecular markers in the patients after total knee arthroplasty complicated with venous thromboembolism. METHODS: A retrospective analysis of the data of 80 patients, who received total knee arthroplasty in Hebei Hospital of Integrated Traditional Chinese and Western Medicine between April and July 2021, was performed. The patients were divided into thrombus group (n=39) and non-thrombus group (n=41) based on thrombus formation 3 days after operation. Totally 40 people who had a health checkup during the same period were chosen as the control group. Thrombus molecular markers were tested in each group: thrombin-antithrombin complex, plasmin-antiplasmin complex, thrombomodulin, and tissue plasminogen activator inhibitory complex. The markers were examined in total knee arthroplasty patients on the day before surgery and 8 hours after surgery. The markers were tested once in the control group. RESULTS AND CONCLUSION: (1) One day before surgery: thrombin-antithrombin complex and plasmin-antiplasmin complex levels in the non-thrombus group were higher than those in the control group (P < 0.05). Thrombin-antithrombin complex and thrombomodulin levels in the thrombus group were higher than those in the non-thrombus group (P < 0.05). The thrombus molecular marker levels of the thrombus group were higher than those in the control group (P < 0.05). (2) At 8 hours postoperatively, the thrombus molecular marker levels of the thrombus group were higher than those in the non-thrombus group (P < 0.05). (3) Logistics regression analysis showed that the single index thrombomodulin had the best prediction efficiency (ACU=0.814, sensitivity 76.70%, specificity 83.80%, P=0.000). Combined detection of thrombin-antithrombin complex and thrombomodulin could improve prediction efficiency (ACU=0.822, sensitivity 83.30%, specificity 76.80%, P=0.000). The single index thrombin-antithrombin complex had the best prediction efficiency (ACU=0.898, sensitivity 83.30%, specificity 84.80%, P=0.000). Combined detection of thrombin-antithrombin complex and plasmin-antiplasmin complex could improve prediction efficiency (ACU=0.897, sensitivity 88.30%, P=0.000). (4) Spearman’s linear correlation analysis showed that tissue plasminogen activator inhibitory complex and plasminantiplasmin complex had a good positive correlation (r 2 =0.209, P < 0.05). (5) It is concluded that thrombomodulin can be used as an early warning indicator before total knee arthroplasty. Thrombin-antithrombin complex is a specific indicator of venous thromboembolism occurrence. Combined detection of thrombin-antithrombin complex, plasmin-antiplasmin complex, thrombomodulin, and tissue plasminogen activator inhibitory complex can improve prediction efficiency for venous thromboembolism. [ABSTRACT FROM AUTHOR]

BACKGROUND: Percutaneous pedicle screw internal fixation is a common minimally invasive operation for clinical treatment of vertebral fractures. The key steps of this operation are puncture positioning and accurate screw placement, which are of great importance for postoperative recovery of patients. OBJECTIVE: To design a coplanar screw placement guide for minimally invasive percutaneous pedicle screw internal fixation, and to observe its clinical value in the treatment of thoracolumbar fractures. METHODS: Totally 60 patients with thoracolumbar fracture treated by minimally invasive percutaneous pedicle screw internal fixation were randomly divided into two groups, with 30 cases in each group. The control group was treated with traditional screw placement technique, and the study group was treated with coplanar screw placement guide assisted screw placement. The screw placement and operation, recent orthopedical and reduction effects, long-term functional recovery and quality of life were compared between the two groups.RESULTS AND CONCLUSION: (1) There were no significant differences in postoperative drainage volume, intraoperative blood loss, hospital stay, fracture healing time, or correction rate between the two groups (P > 0.05). The study group got shorter operation time, fewer intraoperative fluoroscopy times, and higher coplanar rate compared with the control group (all P < 0.05). (2) Visual analogue scale scores in both groups were lower than those before surgery, and the pain scores in the study group at 1, 3 and 6 months after surgery were lower than those in the control group (P < 0.05). (3) At the last follow-up, the anterior edge height ratio, Cobb angle and ODI score of the study group were higher than those of the control group (P < 0.05), while the loss rate of the study group was lower than that of the control group (P < 0.05). (4) It is concluded that the self-made coplanar screw placement guide used in minimally invasive percutaneous pedicle screw technique in the treatment of thoracolumbar fracture has advantages in shortening the operation time, improving the accuracy of screw implantation, reducing radiation leakage and relieving pain, which is benefit for improving long-term quality of life. [ABSTRACT FROM AUTHOR]

The use of exploratory network analysis has increased in psychopathology research over the past decade. A benefit of exploratory network analysis is the wealth of information it can provide; however, a single analysis may generate more inferences than what can be discussed in one manuscript (e.g., centrality indices of each node). This necessitates that authors choose which results to discuss in further detail and which to omit. Without a guide for this process, the likelihood of a biased interpretation is high. We propose that the integration of theory throughout the research process makes the interpretation of exploratory networks more manageable for the researcher and more likely to result in an interpretation that advances science. The goals of this paper are to differentiate between exploratory and confirmatory network analyses, discuss the utility of exploratory work, and provide a practical framework that uses theory as a guide to interpret exploratory network analyses. [ABSTRACT FROM AUTHOR]

Martin Buber was untrained in psychology, yet his teaching provides helpful guidance for a psychological science of suffering. His ideas deserve attention at three distinct levels. For each of these, his ideas align with research findings, but also push beyond them. At the individual level, Buber’s radical approach to relationships disrupts typical social cognitive cycles of suffering and can thereby build a defense against suffering. At the community level, he provides guidance that can help create a society that cares for people who suffer. At the dyadic level, Buber’s guidance also matters. His ideas point toward a therapeutic dyad that can help address suffering when the individual and community responses are not sufficient. Specifically, he guides us toward a holistic view of the person that transcends labels and also toward ineffable human relations. Here again, his ideas align with empirical research, but push beyond. Buber’s unique take on relationships has much to offer scholars seeking to understand and alleviate suffering. Some might perceive Buber as ignoring evil. That possible criticism and others deserve consideration. Nonetheless, readiness to adjust theory in response to Buber and other psychological outsiders may be valuable when developing a psychology of suffering.

Digital micromirror devices have gained popularity in wavefront shaping, offering a high frame rate alternative to liquid crystal spatial light modulators. They are relatively inexpensive, offer high resolution, are easy to operate, and a single device can be used in a broad optical bandwidth. However, some technical drawbacks must be considered to achieve optimal performance. These issues, often undocumented by manufacturers, mostly stem from the device’s original design for video projection applications. Herein, we present a guide to characterize and mitigate these effects. Our focus is on providing simple and practical solutions that can be easily incorporated into a typical wavefront shaping setup.

Internal climate variability (ICV) often violates the assumptions of statistical methods, and the climate research community does not have an established approach for addressing resulting biases. Here we argue for a technique we call climate model Large-Ensemble Monte-Carlo (LENS-MC) to inform the selection of statistical methods for real-world application. Until now, scientists have often made best efforts to select methods based on assumptions about the mathematical properties of ICV. LENS-MC relaxes these assumptions and justifies method selection, potentially for a wide range of statistical analyses. We demonstrate LENS-MC using a case study of statistical errors in 20 year trends in global temperature and top-of-atmosphere flux series, comparing results with standard ordinary least squares (OLS). OLS commonly underestimates trend uncertainties, resulting in a higher likelihood of falsely reporting statistically significant trends or changes in trends, for example reporting p < 0.05 in 20 year temperature trends when the statistics are actually equivalent to p < 0.56. LENS-MC tests result in the selection of methods that almost eliminate the low bias in OLS trend standard errors. Using the suggested methods, researchers are less likely to mistakenly report significant trends, and LENS-MC could be widely applied to statistical climate analysis for which model output is available, provided that model ICV displays similar statistical structure, such as in autocorrelation, to observed ICV.

In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of premature-aging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications.

Base editing is a genome editing strategy that induces specific single-nucleotide changes within genomic DNA. Two major DNA base editors, cytosine base editors and adenine base editors, that consist of a Cas9 protein linked to a deaminase enzyme that catalyzes targeted base conversion directed by a single-guide RNA have been developed. This strategy has been used widely for precise genome editing because, unlike CRISPR-Cas nuclease-based genome editing systems, this strategy does not create double-strand DNA breaks that often result in high levels of undesirable indels. However, recent papers have reported that DNA base editors can cause substantial off-target editing in both genomic DNA and RNA. The off-target editing described in these studies is primarily independent of guide RNA and arises from the promiscuous reactivity of the deaminase enzymes used in DNA base editors. In this Perspective, we discuss the development of DNA base editors, the guide RNA-independent off-target activity reported in recent studies, and strategies that improve the selectivity of DNA base editors.

Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.

Understanding of repair outcomes after Cas9-induced DNA cleavage is still limited, especially in primary human cells. We sequence repair outcomes at 1,656 on-target genomic sites in primary human T cells and use these data to train a machine learning model, which we have called CRISPR Repair Outcome (SPROUT). SPROUT accurately predicts the length, probability and sequence of nucleotide insertions and deletions, and will facilitate design of SpCas9 guide RNAs in therapeutically important primary human cells.

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.

Most methods for the detection of nucleic acids require many reagents and expensive and bulky instrumentation. Here, we report the development and testing of a graphene-based field-effect transistor that uses clustered regularly interspaced short palindromic repeats (CRISPR) technology to enable the digital detection of a target sequence within intact genomic material. Termed CRISPR-Chip, the biosensor uses the gene-targeting capacity of catalytically deactivated CRISPR-associated protein 9 (Cas9) complexed with a specific single-guide RNA and immobilized on the transistor to yield a label-free nucleic-acid-testing device whose output signal can be measured with a simple handheld reader. We used CRISPR-Chip to analyse DNA samples collected from HEK293T cell lines expressing blue fluorescent protein, and clinical samples of DNA with two distinct mutations at exons commonly deleted in individuals with Duchenne muscular dystrophy. In the presence of genomic DNA containing the target gene, CRISPR-Chip generates, within 15 min, with a sensitivity of 1.7 fM and without the need for amplification, a significant enhancement in output signal relative to samples lacking the target sequence. CRISPR-Chip expands the applications of CRISPR-Cas9 technology to the on-chip electrical detection of nucleic acids.

The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes.

BackgroundRewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve in a predictable manner using targeted systems.ResultsHere, we report that persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). We demonstrate that the histone methyltransferase requirement can be locus specific. Co-targeting Ezh2-dCas9, but not KRAB-dCas9, with DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50 days (approximately 57 cell divisions) and triggered an epigenetic switch to a heterochromatic environment. An increase in H3K27 trimethylation and DNA methylation was stably maintained and accompanied by a sustained loss of H3K27 acetylation. Interestingly, substitution of Ezh2-dCas9 with KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the off-target loci tested. Conversely, robust hypermethylation was observed at HER2. We further demonstrated that Ezh2-dCas9 required full-length DNMT3L for maximal activity and that co-targeting DNMT3L was sufficient for persistent repression by Ezh2-dCas9 or KRAB-dCas9.ConclusionsThese data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci. Fine-tuning of targeting tools is a necessity to engineer epigenetic memory at any given locus in any given cell type.

The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it is difficult to determine the function of single transcription factor binding sites within larger transcription networks. Here, we use deactivated Cas9 (dCas9) to disrupt binding to specific sites, a method we term CRISPRd. Since CRISPR guide RNAs are longer than transcription factor binding sites, flanking sequence can be used to target specific sites. Targeting dCas9 to an Oct4 site in the Nanog promoter displaced Oct4 from this site, reduced Nanog expression, and slowed division. In contrast, disrupting the Oct4 binding site adjacent to Pax6 upregulated Pax6 transcription and disrupting Nanog binding its own promoter upregulated its transcription. Thus, we can easily distinguish between activating and repressing binding sites and examine autoregulation. Finally, multiple guide RNA expression allows simultaneous inhibition of multiple binding sites, and conditionally destabilized dCas9 allows rapid reversibility.

To accelerate the development of an inclusive culture in biomedical engineering (BME), we must accept complexity, seek to understand our own privilege, speak out about diversity, learn the difference between intent and impact, accept our mistakes, and learn how to engage in difficult conversations. In turn, we will be rewarded by the ideas, designs, devices and discoveries of a new generation of problem solvers and thought leaders who bring diverse experiences and perspectives.

Citrus sudden death-associated virus (CSDaV) is a member of the genus Marafivirus in the family Tymoviridae, and has been associated with citrus sudden death (CSD) disease in Brazil. Difficulties in the purification of CSDaV from infected citrus plants have prevented progress in the investigation of the role of this virus in CSD and an understanding of its molecular biology. In this work, we have constructed a full-length cDNA clone of CSDaV driven by the 35S promoter (35SRbz-CSDaV). Agrobacterium tumefaciens-mediated inoculation of 35SRbz-CSDaV in Nicotiana benthamiana plants enabled a fast recovery of large amounts of virions from the agroinfiltrated leaves, which allowed a better molecular characterization of CSDaV. In vivo analyses of mutant versions of 35SRbz-CSDaV revealed the expression strategies used by CSDaV for production of the capsid proteins (CPs). We showed that CSDaV virions contain three forms of CP, each of which is generated from the same coding sequence, but by different mechanisms. The major CPp21 is a product of direct translation by leaky scanning from the second start codon in the subgenomic RNA (sgRNA), whereas the minor CPs, p25 and p23, are produced by direct translation from the first start codon in the sgRNA and by trans-proteolytic cleavage processing derived from the p25 precursor, respectively. Together, these findings contribute to advance our understanding of CSDaV genome expression strategies. In addition, the construction and characterization of the CSDaV infectious clone represent important steps towards the investigation of the role of this virus in CSD and of its use as a tool for citrus biotechnology.

External guide sequences (EGSs) signify the short RNAs that induce ribonuclease P (RNase P), an enzyme responsible for processing the 5' termini of tRNA, to specifically cleave a target mRNA by forming a precursor tRNA-like complex. Hence, the EGS technology may serve as a potential strategy for gene-targeting therapy. Our previous studies have revealed that engineered EGS variants induced RNase P to efficiently hydrolyze target mRNAs. In the present research, an EGS variant was designed to be complementary to the mRNA coding for human cytomegalovirus (HCMV) major capsid protein (MCP), which is vital to form the viral capsid. In vitro, the EGS variant was about 80-fold more efficient in inducing human RNase P-mediated cleavage of the target mRNA than a natural tRNA-derived EGS. Moreover, the expressed variant and natural tRNA-originated EGSs led to a decrease of MCP expression by 98% and 73%-74% and a decrease of viral growth by about 10,000- and 200-fold in cells infected with HCMV, respectively. These results reveal direct evidence that the engineered EGS variant has higher efficiency in blocking the expression of HCMV genes and viral growth than the natural tRNA-originated EGS. Therefore, our findings imply that the EGS variant can be a potent candidate agent for the treatment of infections caused by HCMV.

CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.

Gene-drive systems developed in several organisms result in super-Mendelian inheritance of transgenic insertions. Here, we generalize this "active genetic" approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. We test two configurations for allelic-drive: one, copy-cutting, in which a non-preferred allele is selectively targeted for Cas9/guide RNA (gRNA) cleavage, and a more general approach, copy-grafting, that permits selective inheritance of a desired allele located in close proximity to the gRNA cut site. We also characterize a phenomenon we refer to as lethal-mosaicism that dominantly eliminates NHEJ-induced mutations and favors inheritance of functional cleavage-resistant alleles. These two efficient allelic-drive methods, enhanced by lethal mosaicism and a trans-generational drive process we refer to as "shadow-drive", have broad practical applications in improving health and agriculture and greatly extend the active genetics toolbox.

Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.

We present in vivo sequence-specific RNA base editing via adenosine deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). To achieve this, we systematically engineered adRNAs to harness ADARs, and comprehensively evaluated the specificity and activity of the toolsets in vitro and in vivo via two mouse models of human disease. We anticipate that this platform will enable tunable and reversible engineering of cellular RNAs for diverse applications.

A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR-Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity1-4. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR-Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR-Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.

The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR-CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coli and human genome modification. Eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and a domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.

CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory "anti-CRISPR" (Acr) proteins, including six inhibitors (AcrIIA1-AcrIIA6) that can block DNA cutting and genome editing by type II-A CRISPR-Cas9 enzymes. We show here that AcrIIA2 and its more potent homolog, AcrIIA2b, prevent Cas9 binding to DNA by occluding protein residues required for DNA binding. Cryo-EM-determined structures of AcrIIA2 or AcrIIA2b bound to S. pyogenes Cas9 reveal a mode of competitive inhibition of DNA binding that is distinct from other known Acrs. Differences in the temperature dependence of Cas9 inhibition by AcrIIA2 and AcrIIA2b arise from differences in both inhibitor structure and the local inhibitor-binding environment on Cas9. These findings expand the natural toolbox for regulating CRISPR-Cas9 genome editing temporally, spatially, and conditionally.

Molecules capable of directing changes to nucleic acid sequences are powerful tools for molecular biology and promising candidates for the therapeutic correction of disease-causing mutations. However, unwanted reactions at off-target sites complicate their use. Here we report selective combinations of mutant editing enzyme and directing oligonucleotide. Mutations in human ADAR2 (adenosine deaminase acting on RNA 2) that introduce aromatic amino acids at position 488 reduce background RNA editing. This residue is juxtaposed to the nucleobase that pairs with the editing site adenine, suggesting a steric clash for the bulky mutants. Replacing this nucleobase with a hydrogen atom removes the clash and restores editing activity. A crystal structure of the E488Y mutant bound to abasic site-containing RNA shows the accommodation of the tyrosine side chain. Finally, we demonstrate directed RNA editing in vitro and in human cells using mutant ADAR2 proteins and modified guide RNAs with reduced off-target activity.

The sterile insect technique (SIT) is an environmentally safe and proven technology to suppress wild populations. To further advance its utility, a novel CRISPR-based technology termed precision guided SIT (pgSIT) is described. PgSIT mechanistically relies on a dominant genetic technology that enables simultaneous sexing and sterilization, facilitating the release of eggs into the environment ensuring only sterile adult males emerge. Importantly, for field applications, the release of eggs will eliminate burdens of manually sexing and sterilizing males, thereby reducing overall effort and increasing scalability. Here, to demonstrate efficacy, we systematically engineer multiple pgSIT systems in Drosophila which consistently give rise to 100% sterile males. Importantly, we demonstrate that pgSIT-generated sterile males are fit and competitive. Using mathematical models, we predict pgSIT will induce substantially greater population suppression than can be achieved by currently-available self-limiting suppression technologies. Taken together, pgSIT offers to potentially transform our ability to control insect agricultural pests and disease vectors.

CRISPR-Cas9 gene-editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology for use in primary human CD4+ T cells to create a high-throughput platform for analyzing the role of host factors in HIV infection and pathogenesis. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated CD4+ T cells by nucleofection. These cells are then assayed for editing efficiency and expanded for use in downstream cellular, genetic, or protein-based assays. This platform supports the rapid, arrayed generation of multiple gene manipulations and is widely adaptable across culture conditions, infection protocols, and downstream applications. Here, we present detailed protocols for crRNP synthesis, primary T-cell culture, 96-well nucleofection, molecular validation, and HIV infection, and discuss additional considerations for guide and screen design, as well as crRNP multiplexing. Taken together, this procedure allows high-throughput identification and mechanistic interrogation of HIV host factors in primary CD4+ T cells by gene knockout, validation, and HIV spreading infection in as little as 2-3 weeks.

Presence of the integrated endogenous banana streak virus (eBSV) in the B genome of plantain (AAB) is a major challenge for breeding and dissemination of hybrids. As the eBSV activates into infectious viral particles under stress, the progenitor Musa balbisiana and its derivants, having at least one B genome, cannot be used as parents for crop improvement. Here, we report a strategy to inactivate the eBSV by editing the virus sequences. The regenerated genome-edited events of Gonja Manjaya showed mutations in the targeted sites with the potential to prevent proper transcription or/and translational into functional viral proteins. Seventy-five percent of the edited events remained asymptomatic in comparison to the non-edited control plants under water stress conditions, confirming inactivation of eBSV into infectious viral particles. This study paves the way for the improvement of B genome germplasm and its use in breeding programs to produce hybrids that can be globally disseminated.

RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas proteins have recently emerged as versatile tools to investigate and engineer the genome. The programmability of CRISPR-Cas has proven especially useful for probing genomic function in high-throughput. Facile single-guide RNA library synthesis allows CRISPR-Cas screening to rapidly investigate the functional consequences of genomic, transcriptomic, and epigenomic perturbations. Furthermore, by combining CRISPR-Cas perturbations with downstream single-cell analyses (flow cytometry, expression profiling, etc.), forward screens can generate robust data sets linking genotypes to complex cellular phenotypes. In the following review, we highlight recent advances in CRISPR-Cas genomic screening while outlining protocols and pitfalls associated with screen implementation. Finally, we describe current challenges limiting the utility of CRISPR-Cas screening as well as future research needed to resolve these impediments. As CRISPR-Cas technologies develop, so too will their clinical applications. Looking ahead, patient centric functional screening in primary cells will likely play a greater role in disease management and therapeutic development.