Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells.

The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity.

Harnessing type I CRISPR-Cas systems for genome engineering in human cells.

Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea. Target interference relies on a multi-subunit, RNA-guided complex called Cascade, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex.

Mapping the genomic landscape of CRISPR-Cas9 cleavage.

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.

DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks.

The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods.

Activation of Sonic hedgehog signaling in ventricular cardiomyocytes exerts cardioprotection against ischemia reperfusion injuries.

Sonic hedgehog (SHH) is a conserved protein involved in embryonic tissue patterning and development. SHH signaling has been reported as a cardio-protective pathway via muscle repair-associated angiogenesis. The goal of this study was to investigate the role of SHH signaling pathway in the adult myocardium in physiological situation and after ischemia-reperfusion.

TNF-α-mediated caspase-8 activation induces ROS production and TRPM2 activation in adult ventricular myocytes.

Aims: TRPM2 is a Ca(2+)-permeable cationic channel of the transient receptor potential (TRP) superfamily that is linked to apoptotic signalling. Its involvement in cardiac pathophysiology is unknown. The aim of this study was to determine whether the pro-apoptotic cytokine tumour necrosis factor-α (TNF-α) induces a TRPM2-like current in murine ventricular cardiomyocytes.