CRISPR-Cas9 knockin mice for genome editing and cancer modeling.

CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.

Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing.

Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here, we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.

Multiple Input Sensing and Signal Integration Using a Split Cas12a System.

The ability to integrate biological signals and execute a functional response when appropriate is critical for sophisticated cell engineering using synthetic biology. Although the CRISPR-Cas system has been harnessed for synthetic manipulation of the genome, it has not been fully utilized for complex environmental signal sensing, integration, and actuation. Here, we develop a split dCas12a platform and show that it allows for the construction of multi-input, multi-output logic circuits in mammalian cells. The system is highly programmable and can generate expandable AND gates with two, three, and four inputs. It can also incorporate NOT logic by using anti-CRISPR proteins as an OFF switch. By coupling the split dCas12a design to multiple tumor-relevant promoters, we provide a proof of concept that the system can implement logic gating to specifically detect breast cancer cells and execute therapeutic immunomodulatory responses.

Scalable biological signal recording in mammalian cells using Cas12a base editors.

Biological signal recording enables the study of molecular inputs experienced throughout cellular history. However, current methods are limited in their ability to scale up beyond a single signal in mammalian contexts. Here, we develop an approach using a hyper-efficient dCas12a base editor for multi-signal parallel recording in human cells. We link signals of interest to expression of guide RNAs to catalyze specific nucleotide conversions as a permanent record, enabled by Cas12's guide-processing abilities. We show this approach is plug-and-play with diverse biologically relevant inputs and extend it for more sophisticated applications, including recording of time-delimited events and history of chimeric antigen receptor T cells' antigen exposure. We also demonstrate efficient recording of up to four signals in parallel on an endogenous safe-harbor locus. This work provides a versatile platform for scalable recording of signals of interest for a variety of biological applications.

Frailty and transcatheter valve intervention: A narrative review.

Frailty is a common clinical syndrome that portends poor peri-procedural outcomes and increased mortality following transcatheter valve interventions. We reviewed frailty assessment tools in transcatheter intervention cohorts to recommend a pathway for preprocedural frailty assessment in patients referred for transcatheter valve procedures, and evaluated current evidence for frailty interventions and their efficacy in transcatheter intervention. We recommend the use of a frailty screening instrument to identify patients as frail, with subsequent referral for comprehensive geriatric assessment in these patients, to assist in selecting appropriate patients and then optimizing them for transcatheter valve interventions. Interventions to reduce preprocedural frailty are not well defined, however, data from limited cohort studies support exercise-based interventions to increase functional capacity and reduce frailty in parallel with preprocedural medical optimization.

Macrotroponin in the COVID-19 Era: An Under-Recognised Cause of Persistent Troponin Elevation.

Troponin is an important diagnostic tool, however, as the assay sensitivity and frequency of testing has increased in the COVID-19 era, a new cohort of patients with persistently elevated troponin has emerged. Interfering antibodies should be considered in patients with persistent and stable troponin elevation, where there is no ongoing cause.

Using Intravascular Lithotripsy to Facilitate Transfemoral Arterial Access for Transcatheter Aortic Valve Implantation.

Peripheral vascular assessment is important in pre-procedural planning for transcatheter aortic valve implantation (TAVI). While alternative vascular access sites have been used in patients with hostile iliofemoral anatomy, femoral access has been established as the superior access method for procedural outcomes. Intravascular lithotripsy (IVL) can facilitate transfemoral access for TAVI in patients with calcific stenoses of the iliofemoral arteries. This How-To-Do-It article describes the procedural planning and methods for performing IVL in these patients.

Using Coronary Artery Calcium Scoring as Preventative Health Tool to Reduce the High Burden of Cardiovascular Disease in Indigenous Australians.

BACKGROUND: Indigenous Australians suffer higher rates of ischaemic heart disease resulting in premature mortality. Despite this, Indigenous Australians undergo less cardiovascular investigation and intervention than their non-Indigenous counterparts. Recent evidence suggests that computed tomography coronary angiography (CTCA) is not only able to accurately predict cardiovascular risk, but also results in reduced rates of myocardial infarction and cardiovascular death. METHODS: This is a prospective longitudinal study of patients in regional Australia referred for CTCA at a regional centre from 2012 to 2017. Patients were identified as Indigenous at registration. Results were recorded from formal radiology reports. Logistic regression was used to compare calcium score, as a measure of coronary artery disease burden in Indigenous and non-Indigenous patients. RESULTS: Indigenous patients are 2.8 times more likely to have a higher burden of coronary artery disease than non-Indigenous patients, even after accounting for the higher rate of cardiovascular risk factors in the Indigenous population (OR 2.77; p = 0.008). In the study population, Indigenous patients were well represented as compared to the background population. CONCLUSION: This is the first study of CTCA in an Indigenous Australian population, and one of the first using CTCA for an Indigenous population worldwide. It demonstrates a higher burden of cardiovascular disease for Indigenous Australians, independent of the higher rate of cardiovascular risk factors. Access to CTCA presents an opportunity to reduce the rate of myocardial infarction and early mortality in the Indigenous Australian population.

Standard dose epinephrine versus placebo in out of hospital cardiac arrest: A systematic review and meta-analysis.

INTRODUCTION: Out of hospital cardiac arrest (OHCA) is a time critical and heterogeneous presentation. The most appropriate management strategies remain an issue for debate. The aim of this systematic review and meta-analysis was to determine the association of epinephrine versus placebo with return of spontaneous circulation, survival to hospital admission, survival to hospital discharge and neurological outcomes in out of hospital cardiac arrest. METHODS: A systematic review of five databases was performed from inception to August 2018. Only randomised controlled trials were considered eligible for inclusion. The primary outcome was survival to hospital discharge. Secondary outcomes were ROSC, survival to hospital admission, neurological function on discharge and three-month survival. All studies were assessed for level of evidence and risk of bias. RESULTS: Five randomised controlled trials with 17,635 patients were identified for inclusion. Use of epinephrine was associated with increased ROSC (OR = 3.10; 95% CI = 2.16 to 4.45; I2 = 74%; p < 0.0001) and increased survival to hospital admission OR = 2.52; 95% CI = 1.63 to 3.88; I2 = 94%; p < 0.0001). However, epinephrine was not associated with increased survival to discharge (OR = 1.09; 95% CI = 0.48 to 2.47; I2 = 77%; p = 0.84) or differences in neurological outcomes (OR = 0.81; 95% CI = 0.34 to 1.96). DISCUSSION: This study was a systematic review and meta-analysis of epinephrine versus placebo in OHCA. The use of epinephrine was associated with improved ROSC and survival to hospital admission. However, use of epinephrine was not associated with a significant difference in survival to hospital discharge, neurological outcomes or survival to 3 months. Further research is required to control for the confounders during inpatient management.

Advanced airway management in out of hospital cardiac arrest: A systematic review and meta-analysis.

OBJECTIVES: To assess the difference in survival and neurological outcomes between endotracheal tube (ETT) intubation and supraglottic airway (SGA) devices used during out-of-hospital cardiac arrest (OHCA). METHODS: A systematic search of five databases was performed by two independent reviewers until September 2018. Included studies reported on (1) OHCA or cardiopulmonary resuscitation, and (2) endotracheal intubation versus supraglottic airway device intubation. Exclusion criteria (1) stimulation studies, (2) selectively included/excluded patients, (3) in-hospital cardiac arrest. Odds Ratios (OR) with random effect modelling was used. Primary outcomes: (1) return of spontaneous circulation (ROSC), (2) survival to hospital admission, (3) survival to hospital discharge, (4) discharge with a neurologically intact state. RESULTS: Twenty-nine studies (n = 539,146) showed that overall, ETT use resulted in a heterogeneous, but significant increase in ROSC (OR = 1.44; 95%CI = 1.27 to 1.63; I2 = 91%; p < 0.00001) and survival to admission (OR = 1.36; 95%CI = 1.12 to 1.66; I2 = 91%; p = 0.002). There was no significant difference in survival to discharge or neurological outcome (p > 0.0125). On sensitivity analysis of RCTs, there was no significant difference in ROSC, survival to admission, survival to discharge or neurological outcome (p > 0.0125). On analysis of automated chest compression, without heterogeneity, ETT provided a significant increase in ROSC (OR = 1.55; 95%CI = 1.20 to 2.00; I2 = 0%; p = 0.0009) and survival to admission (OR = 2.16; 95%CI = 1.54 to 3.02; I2 = 0%; p < 0.00001). CONCLUSIONS: The overall heterogeneous benefit in survival with ETT was not replicated in the low risk RCTs, with no significant difference in survival or neurological outcome. In the presence of automated chest compressions, ETT intubation may result in survival benefits.

Pulmonary arterial compliance as a measure of right ventricular loading in mitral regurgitation.

Pulmonary hypertension (PH) is a common and prognostically important complication of mitral regurgitation (MR). Mean pulmonary arterial pressure (mPAP) and pulmonary vascular resistance (PVR) are traditionally used to diagnose PH, however these indices measure static rather than pulsatile load, leading to an incomplete representation of pulmonary vascular load on the right ventricle (RV). Pulmonary arterial compliance (PAC) is one method for quantifying pulsatile load, and is both a stronger predictor of prognosis in left heart failure, as well as a more sensitive measure of early pulmonary vascular dysfunction than PVR. With the expansion of transcatheter mitral and tricuspid valve therapies, there is renewed interest to more accurately define the load imposed by the pulmonary vasculature on the RV, especially in the early phase, prior to the onset of chronic PH. This review discusses the pathophysiology of pH in left heart failure and MR, the utility of PAC as a measure of RV afterload, and its calculation for clinical use and interpretation, underlining the utility of PAC as an adjunct for assessing pulmonary vascular haemodynamics.

Multiplexed genome regulation in vivo with hyper-efficient Cas12a.

Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR-Cas12a systems enable versatile multiple-genomic-loci targeting by processing numerous CRISPR RNAs (crRNAs) from a single transcript; however, their low efficiency has hindered in vivo applications. Through structure-guided protein engineering, we developed a hyper-efficient Lachnospiraceae bacterium Cas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low concentrations of crRNA. We demonstrate that hyperdCas12a has comparable off-target effects compared with the wild-type system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a activator and a single crRNA array simultaneously activating the endogenous Oct4, Sox2 and Klf4 genes in the retina of post-natal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene-modulation and gene-therapy applications.

Chd8 Mutation Leads to Autistic-like Behaviors and Impaired Striatal Circuits.

Autism spectrum disorder (ASD) is a heterogeneous disease, but genetically defined models can provide an entry point to studying the molecular underpinnings of this disorder. We generated germline mutant mice with loss-of-function mutations in Chd8, a de novo mutation strongly associated with ASD, and demonstrate that these mice display hallmark ASD behaviors, macrocephaly, and craniofacial abnormalities similar to patient phenotypes. Chd8+/- mice display a broad, brain-region-specific dysregulation of major regulatory and cellular processes, most notably histone and chromatin modification, mRNA and protein processing, Wnt signaling, and cell-cycle regulation. We also find altered synaptic physiology in medium spiny neurons of the nucleus accumbens. Perturbation of Chd8 in adult mice recapitulates improved acquired motor learning behavior found in Chd8+/- animals, suggesting a role for CHD8 in adult striatal circuits. These results support a mechanism linking chromatin modification to striatal dysfunction and the molecular pathology of ASD.

Recovery of Chinese hamster ovary host cell proteins for proteomic analysis.

Identification and characterization of Chinese hamster ovary (CHO) host cell protein (HCP) impurities by proteomic techniques can aid bioprocess design and lead to more efficient development and improved biopharmaceutical manufacturing operations. Recovery of extracellular CHO HCP for proteomic analysis is particularly challenging due to the relatively low protein concentration and complex composition of media. In this article, we report the development of optimized protocols that improve proteome capture for CHO HCP. Eleven precipitation protocols were screened for protein recovery and optimized for a subset of precipitants by a design of experiments (DOE) approach. Because total protein recovery does not fully replicate a proteomics experiment, or detect non-protein agents that may interfere with proteomic methods, a subset of precipitation conditions were compared by two-dimensional electrophoresis and liquid chromatography coupled with mass spectrometry, with optimized recovery shown to differ between the two proteomic methods. This work demonstrates broadly applicable methods that can be applied as initial steps to optimize sample preparation of any sample type for proteomic analysis, and presents optimized precipitation protocols for extracellular CHO HCP recovery, which can vary appreciably between gel-based and shotgun proteomic methods.