Search

Abstract Clinical implementation of therapeutic genome editing relies on efficient in vivo delivery and the safety of CRISPR-Cas tools. Previously, we identified PsCas9 as a Type II-B family enzyme capable of editing mouse liver genome upon adenoviral delivery without detectable off-targets and reduced chromosomal translocations. Yet, its efficacy remains insufficient with non-viral delivery, a common challenge for many Cas9 orthologues. Here, we sought to redesign PsCas9 for in vivo editing using lipid nanoparticles. We solve the PsCas9 ribonucleoprotein structure with cryo-EM and characterize it biochemically, providing a basis for its rational engineering. Screening over numerous guide RNA and protein variants lead us to develop engineered PsCas9 (ePsCas9) with up to 20-fold increased activity across various targets and preserved safety advantages. We apply the same design principles to boost the activity of FnCas9, an enzyme phylogenetically relevant to PsCas9. Remarkably, a single administration of mRNA encoding ePsCas9 and its guide formulated with lipid nanoparticles results in high levels of editing in the Pcsk9 gene in mouse liver, a clinically relevant target for hypercholesterolemia treatment. Collectively, our findings introduce ePsCas9 as a highly efficient, and precise tool for therapeutic genome editing, in addition to the engineering strategy applicable to other Cas9 orthologues.

Summary: CRISPR genome editing is a promising tool for translational research but can cause undesired editing outcomes, both on target at the edited locus and off target at other genomic loci. Here, we investigate the occurrence of deleterious on-target effects (OnTEs) in human stem cells after insertion of disease-related mutations by homology-directed repair (HDR) and gene editing using non-homologous end joining (NHEJ). We identify large, mono-allelic genomic deletions and loss-of-heterozygosity escaping standard quality controls in up to 40% of edited clones. To reliably detect such events, we describe simple, low-cost, and broadly applicable quantitative genotyping PCR (qgPCR) and single-nucleotide polymorphism (SNP) genotyping-based tools and suggest their usage as additional quality controls after editing. This will help to ensure the integrity of edited loci and increase the reliability of CRISPR editing.

Introduction The current study examined associations between affective and smoking-related factors, and dispositional mindfulness among smokers seeking enrollment in a cessation program in Brazil. Methods Participants were first-time treatment seeking adult smokers (N=90) on a waiting list for a government-sponsored cessation program. Pearson’s bivariate correlations assessed relationships between the primary outcome variable (dispositional mindfulness) and each explanatory variable (nicotine dependence, depression, anxiety, and state positive/negative affect). Linear regression analyses evaluated the unique contribution of each explanatory variable when controlling for the others. Results The sample (N=90) was predominantly female (n=71) and most (79%) had 11 or fewer years of education. In the final regression model, a total of 36.2% of the variance in dispositional mindfulness was accounted for by positive affect (B=0.81, p