An enhanced CRISPR repressor for targeted mammalian gene regulation.

The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB-MeCP2, to nuclease-dead Cas9. We demonstrate the system's superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits

Cas9 gRNA engineering for genome editing, activation and repression

We demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.National Human Genome Research Institute (U.S.) (P50 HG005550)United States. Department of Energy (DE-FG02-02ER63445)Wyss Institute for Biologically Inspired EngineeringUnited States. Army Research Office (DARPA W911NF-11-2-0054)National Science Foundation (U.S.)United States. National Institutes of Health (5R01CA155320-04)United States. National Institutes of Health (P50 GM098792)National Cancer Institute (U.S.) (5T32CA009216-34)Massachusetts Institute of Technology. Department of Biological EngineeringHarvard Medical School. Department of GeneticsDefense Threat Reduction Agency (DTRA) (HDTRA1-14-1-0006

Comparative Analysis of Cas9 Activators Across Multiple Species

Several groups have generated programmable transcription factors based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems and assess the role of cooperativity in maximizing gene expression

Highly-efficient Cas9-mediated transcriptional programming

The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).National Human Genome Research Institute (U.S.) (Grant P50 HG005550)United States. Dept. of Energy (Grant DE-FG02-02ER63445)Wyss Institute for Biologically Inspired EngineeringNational Science Foundation (U.S.). Graduate Research FellowshipMassachusetts Institute of Technology. Department of Biological EngineeringHarvard Medical School. Department of Genetic

Peak Tricuspid Regurgitation Jet Velocity and Kidney Outcomes in Patients With Heart Failure With Preserved Ejection Fraction

Introduction: Although venous congestion secondary to elevated pulmonary artery pressure (PAP) has been hypothesized to worsen kidney function, the association of peak tricuspid regurgitation jet velocity (pTRV), a surrogate of PAP, with kidney outcomes remains uncertain in heart failure (HF) with preserved ejection fraction (HFpEF). Methods: This post hoc analysis of the Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist (TOPCAT) trial analyzed participants with a left ventricular ejection fraction (LVEF) of ≥45% who had pTRV measured by echocardiography at baseline. For the cross-sectional analysis, the association of baseline pTRV with baseline estimated glomerular filtration rate (eGFR) was assessed using linear regression. For the longitudinal analysis, the association of baseline pTRV with decline in eGFR of ≥30% and doubling of serum creatinine was assessed using Cox proportional hazards models. Results: Among 450 participants, the mean (SD) baseline age, LVEF, pTRV, and eGFR were 72.3 (9.6) years, 58.2% (7.4%), 2.8 (0.5) m/s, and 62.1 (18.7) ml/min per 1.73 m2, respectively. Each 1 SD higher pTRV was associated with a lower baseline eGFR (coefficient, −1.79; 95% confidence interval [CI], −3.48 to −0.10 ml/min per 1.73 m2). Over a median (interquartile range) follow-up of 3.0 (2.0–4.4) years, 203 (45%) patients experienced ≥30% eGFR decline, and 48 (11%) experienced creatinine doubling. Each 1 SD higher pTRV was associated with a 20% higher risk of ≥30% eGFR decline (hazard ratio [HR], 1.20; 95% CI, 1.04–1.39) and a 45% higher risk of creatinine doubling (HR, 1.45; 95% CI, 1.09–1.94). Conclusions: Higher pTRV was associated with lower eGFR at baseline, and higher risk of ≥30% eGFR decline and creatinine doubling among patients with HFpEF

Precise Cas9 targeting enables genomic mutation prevention

Here, we present a generalized method of guide RNA “tuning” that enables Cas9 to discriminate between two target sites that differ by a single-nucleotide polymorphism. We employ our methodology to generate an in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.National Human Genome Research Institute (U.S.) ( Grant P50 HG005550)Wyss Institute for Biologically Inspired EngineeringUnited States. Defense Threat Reduction Agency (Grant HDTRA1-15-1-0051)Paul G. Allen Frontiers Grou

Precise Cas9 targeting enables genomic mutation prevention

ABSTRACTHere we present a generalized method of guide RNA “tuning” that enables Cas9 to discriminate between two target sites that differ by a single nucleotide polymorphism. We employ our methodology to generate a novelin vivomutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the designed mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.</jats:p

Cas9 gRNA Engineering for Genome Editing, Activation and Repression

We demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.Accepted Manuscrip