RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using ...Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
RNA structure is a primary determinant of its function, and methods that merge chemical probing with next generation sequencing have created breakthroughs in the throughput and scale of RNA structure ...characterization. However, little work has been done to examine the effects of library preparation and sequencing on the measured chemical probe reactivities that encode RNA structural information. Here, we present the first analysis and optimization of these effects for selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). We first optimize SHAPE-Seq, and show that it provides highly reproducible reactivity data over a wide range of RNA structural contexts with no apparent biases. As part of this optimization, we present SHAPE-Seq v2.0, a 'universal' method that can obtain reactivity information for every nucleotide of an RNA without having to use or introduce a specific reverse transcriptase priming site within the RNA. We show that SHAPE-Seq v2.0 is highly reproducible, with reactivity data that can be used as constraints in RNA folding algorithms to predict structures on par with those generated using data from other SHAPE methods. We anticipate SHAPE-Seq v2.0 to be broadly applicable to understanding the RNA sequence-structure relationship at the heart of some of life's most fundamental processes.
The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA ...guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery.
Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5'-N
GATT-3'), for NmeCas9 genome editing in human cells.
Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.
Abstract
Introduction
Under the NHS Long Term Plan1, every hospital with a Type 1 Emergency Department (ED) has been asked to provide Acute Frailty Services for at least 70 hours a week. We have ...adopted a liaison model to ensure Comprehensive Geriatric Assessment at the front door. The Frailty Team now includes two Frailty Practitioners, a Specialist Registrar based in ED and the Acute Medical Unit (AMU) with Consultant Physician of the Week support.
Method
The team attend multi-disciplinary meetings in ED and AMU on a daily basis. Frail patients are reviewed and Comprehensive Geriatric Assessments are undertaken together with appropriate signposting. The aim being, to enhance the care of frail patients at the earliest opportunity.
Results
High bed utilisation comes at a cost and a longer length of stay (LOS) is associated with an increased risk of deconditioning. Since introducing incremental changes to our working model, the average LOS of patients on AMU has been reduced from a mean of 1.33 days to 0.82 days. This represents a 38.3% reduction from September 2020 to August 2021. This reduction has been sustained, while 30-day readmission rates have not increased. Feedback has been very positive from all the other teams involved.
Conclusion
Our service has established a more positive working culture to efficiently manage frail patients. The AMU team now feel empowered to make patient-centric decisions regarding patient flow and discharges. Since this data was analysed we have implemented a further change to help enhance the care of frail patients. A list of appropriate patients is emailed to the hospital site team every afternoon to aid getting the right patients into the right beds. The impact of LOS on the frailty wards will then be reviewed following this introduction.
Reference
1. nhs-long-term-plan-june-2019.pdf (longtermplan.nhs.uk).
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