CRISPR/Cas9 systems are a versatile tool for genome editing due to the highly efficient targeting of DNA sequences complementary to their RNA guide strands. However, it has been shown that RNA-guided Cas9 nuclease cleaves genomic DNA sequences containing mismatches to the guide strand. A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large mammalian genomes. Here we show that genomic sites could be cleaved by CRISPR/Cas9 systems when DNA sequences contain insertions (‘DNA bulge’) or deletions (‘RNA bulge’) compared to the RNA guide strand, and Cas9 nickases used for paired nicking can also tolerate bulges in one of the guide strands. Variants of single-guide RNAs (sgRNAs) for four endogenous loci were used as model systems, and their cleavage activities were quantified at different positions with 1- to 5-bp bulges. We further investigated 114 putative genomic off-target loci of 27 different sgRNAs and confirmed 15 off-target sites, each harboring a single-base bulge and one to three mismatches to the guide strand. Our results strongly indicate the need to perform comprehensive off-target analysis related to DNA and sgRNA bulges in addition to base mismatches, and suggest specific guidelines for reducing potential off-target cleavage.

Genome editing with engineered nucleases has recently emerged as an approach to correct genetic mutations by enhancing homologous recombination with a DNA repair template. However, many genetic diseases, such as Duchenne muscular dystrophy (DMD), can be treated simply by correcting a disrupted reading frame. We show that genome editing with transcription activator-like effector nucleases (TALENs), without a repair template, can efficiently correct the reading frame and restore the expression of a functional dystrophin protein that is mutated in DMD. TALENs were engineered to mediate highly efficient gene editing at exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from Duchenne patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. Finally, exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein-coding regions of the genome, as predicted by in silico target site analysis. This strategy integrates the rapid and robust assembly of active TALENs with an efficient gene-editing method for the correction of genetic diseases caused by mutations in non-essential coding regions that cause frameshifts or premature stop codons.

Targeted gene addition to mammalian genomes is central to biotechnology, basic research and gene therapy. For example, gene targeting to the ROSA26 locus by homologous recombination in embryonic stem cells is commonly used for mouse transgenesis to achieve ubiquitous and persistent transgene expression. However, conventional methods are not readily adaptable to gene targeting in other cell types. The emerging zinc finger nuclease (ZFN) technology facilitates gene targeting in diverse species and cell types, but an optimal strategy for engineering highly active ZFNs is still unclear. We used a modular assembly approach to build ZFNs that target the ROSA26 locus. ZFN activity was dependent on the number of modules in each zinc finger array. The ZFNs were active in a variety of cell types in a time- and dose-dependent manner. The ZFNs directed gene addition to the ROSA26 locus, which enhanced the level of sustained gene expression, the uniformity of gene expression within clonal cell populations and the reproducibility of gene expression between clones. These ZFNs are a promising resource for cell engineering, mouse transgenesis and pre-clinical gene therapy studies. Furthermore, this characterization of the modular assembly method provides general insights into the implementation of the ZFN technology.

BackgroundWhile dissemination and implementation (D&I) science has grown rapidly, there is an ongoing need to understand how to build and sustain capacity in individuals and institutions conducting research. There are three inter-related domains for capacity building: people, settings, and activities. Since 2008, Washington University in St. Louis has dedicated significant attention and resources toward building D&I research capacity. This paper describes our process, challenges, and lessons with the goal of informing others who may have similar aims at their own institution.ActivitiesAn informal collaborative, the Washington University Network for Dissemination and Implementation Research (WUNDIR), began with a small group and now has 49 regular members. Attendees represent a wide variety of settings and content areas and meet every 6 weeks for half-day sessions. A logic model organizes WUNDIR inputs, activities, and outcomes. A mixed-methods evaluation showed that the network has led to new professional connections and enhanced skills (e.g., grant and publication development). As one of four, ongoing, formal programs, the Dissemination and Implementation Research Core (DIRC) was our first major component of D&I infrastructure. DIRC’s mission is to accelerate the public health impact of clinical and health services research by increasing the engagement of investigators in later stages of translational research. The aims of DIRC are to advance D&I science and to develop and equip researchers with tools for D&I research. As a second formal component, the Washington University Institute for Public Health has provided significant support for D&I research through pilot projects and a small grants program. In a third set of formal programs, two R25 training grants (one in mental health and one in cancer) support post-doctoral scholars for intensive training and mentoring in D&I science. Finally, our team coordinates closely with D&I functions within research centers across the university. We share a series of challenges and potential solutions.ConclusionOur experience in developing D&I research at Washington University in St. Louis shows how significant capacity can be built in a relatively short period of time. Many of our ideas and ingredients for success can be replicated, tailored, and improved upon by others.Electronic supplementary materialThe online version of this article (doi:10.1186/s13012-017-0634-4) contains supplementary material, which is available to authorized users.