Maximizing the Efficacy of CRISPR/Cas Homology-Directed Repair Gene Targeting

Clustered regularly interspaced short palindromic repeats/CRISPR-associated system (CRISPR/Cas) is a powerful gene editing tool that can introduce double-strand breaks (DSBs) at precise target sites in genomic DNA. In mammalian cells, the CRISPR/Cas-generated DSBs can be repaired by either template-free error-prone end joining (e.g., non-homologous end joining/microhomology-mediated end joining [NHEJ]/[MMEJ]) or templated error-free homology-directed repair (HDR) pathways. CRISPR/Cas with NHEJ/MMEJ DNA repair results in various length insertions/deletion mutations (indels), which can cause frameshift mutations leading to a stop codon and subsequent gene-specific knockout (i.e., loss of function). In contrast, CRISPR/Cas with HDR DNA repair, utilizing an exogenous repair template harboring specific nucleotide (nt) changes, can be employed to intentionally edit out or introduce mutations or insertions at specific genomic sites (i.e., targeted gene knock-in). This review provides an overview of HDR-based gene-targeting strategies to facilitate the knock-in process, including improving gRNA cleavage efficiency, optimizing HDR efficacy, decreasing off-target effects, suppressing NHEJ/MMEJ activity, and thus expediting the screening of CRISPR/Cas-edited clonal cells

The mediating role of entrepreneurial orientation in the task-environment-performance relationship: a meta-analysis of 30 years of research

Pre-print version. Final version published by Sage; available online at http://online.sagepub.com/Despite the importance of the external task environment for firm performance little is known about the mechanisms that enable firms to benefit from a specific environmental setting. We argue that firms adjust their entrepreneurial orientation (EO) to the external environment and use it as a mechanism to transform the advantages provided by the environment into above-average performance levels. Specifically, we use meta-analysis supplemented by structural equation modeling to explore the links between environmental munificence, hostility, dynamism and complexity, EO, and firm performance in a mediation model. Our results suggest that environmental munificence, dynamism and complexity affect EO and, in turn, firm performance. We discuss our meta-analytical findings with respect to their theoretical contribution and their practical implications.This research was partially supported by a research grant by the Volkswagen Foundation (II/82 408)