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A method for CRISPR-based genome editing that harnesses cellular non-homologous end joining activity to achieve targeted DNA knock-in in non-dividing tissues. A novel method for knock-in gene integration A current challenge in genome editing is achieving efficient targeted integration of transgenes in post-mitotic cells. These authors develop a method for CRISPR-based genome editing that harnesses the non-homologous-end-joining double-strand-break repair pathway to achieve targeted knock-in in dividing and non-dividing tissues. Although further development is needed to increase efficacy, the authors show the potential application of this method for targeted knock-in in post-mitotic neurons and other non-dividing tissues, and provide initial exploratory data on its potential application for disease correction in retinal pigment epithelium models. Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient 1 , especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders 2 . Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) 3 , 4 technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.

Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing. In-depth characterization of adeno-associated virus (AAV)-mediated CRISPR delivery is still lacking. Here, the authors show high levels of integration into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in vivo.

Clustered regularly interspaced short palindromic repeat-CRISPR-associated protein (CRISPR-Cas) systems, found in nature as microbial adaptive immune systems, have been repurposed into an important tool in biological engineering and genome editing, providing a programmable platform for precision gene targeting. These tools have immense promise as therapeutics that could potentially correct disease-causing mutations. However, CRISPR-Cas gene editing components must be transported directly to the nucleus of targeted cells to exert a therapeutic effect. Thus, efficient methods of delivery will be critical to the success of therapeutic genome editing applications. Here, we review current strategies available for in vivo delivery of CRISPR-Cas gene editing components and outline challenges that need to be addressed before this powerful tool can be deployed in the clinic. CRISPR is a novel gene editing tool that has the potential for multiple in vivo applications. Cas9 can target virtually any gene through complementarity to a synthetically produced gRNA. A major obstacle to in vivo implementation of CRISPR-mediated genome editing is an efficient, targeted delivery vehicle. Cas9 may be delivered to cells in DNA, mRNA, or protein format, and each mode has unique strengths, weaknesses, and delivery requirements. A variety of physical and chemical delivery vectors are available for Cas9 delivery.

Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed.

Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that a number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo and in the human retina in vitro. We demonstrate applications for recording and stimulation, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.

Early STOP codons created with CRISPR base editors leads to gene knockout with high efficiency and does not stress cells with double-strand DNA breaks. CRISPR-STOP can target the majority of human genes and is useful for genetic screens. CRISPR–Cas9-induced DNA damage may have deleterious effects at high-copy-number genomic regions. Here, we use CRISPR base editors to knock out genes by changing single nucleotides to create stop codons. We show that the CRISPR-STOP method is an efficient and less deleterious alternative to wild-type Cas9 for gene-knockout studies. Early stop codons can be introduced in ∼17,000 human genes. CRISPR-STOP-mediated targeted screening demonstrates comparable efficiency to WT Cas9, which indicates the suitability of our approach for genome-wide functional screenings.

The number of fully sequenced fungal genomes is rapidly increasing. Since genetic tools are poorly developed for most filamentous fungi, it is currently difficult to employ genetic engineering for understanding the biology of these fungi and to fully exploit them industrially. For that reason there is a demand for developing versatile methods that can be used to genetically manipulate non-model filamentous fungi. To facilitate this, we have developed a CRISPR-Cas9 based system adapted for use in filamentous fungi. The system is simple and versatile, as RNA guided mutagenesis can be achieved by transforming a target fungus with a single plasmid. The system currently contains four CRISPR-Cas9 vectors, which are equipped with commonly used fungal markers allowing for selection in a broad range of fungi. Moreover, we have developed a script that allows identification of protospacers that target gene homologs in multiple species to facilitate introduction of common mutations in different filamentous fungi. With these tools we have performed RNA-guided mutagenesis in six species of which one has not previously been genetically engineered. Moreover, for a wild-type Aspergillus aculeatus strain, we have used our CRISPR Cas9 system to generate a strain that contains an AACU_pyrG marker and demonstrated that the resulting strain can be used for iterative gene targeting.

Astrocytes are a type of glial cell that tile the CNS. They interact with multiple cell types, including neurons, glial cells and blood vessels, and are involved or implicated in brain disorders. Progress has been made in understanding astrocytes, but the field lacks detailed information concerning how they perform their multifarious functions, and how and when they influence the operations of the neural circuits with which they interact. One recognized bottleneck to progress has been the paucity of reliable tools with which to explore astrocytes within the adult vertebrate CNS in vivo. However, improved tools for molecular, genetic, morphological and physiological assessments have been developed recently or have been adapted from their original purposes to study neurons and are now being used to systematically document and interrogate astrocyte biology in vivo. These tools, their uses and limitations, and the insights that they afford are summarized in this Review.Much progress has been made in understanding astrocytes, but details on their functions and interactions remain difficult to determine. Yu, Nagai and Khakh give an overview of recent advances in the toolbox for molecular, genetic, morphological and physiological investigations into astrocytes.

High-throughput CRISPR screens have shown great promise in functional genomics. We present MAGeCK-VISPR, a comprehensive quality control (QC), analysis, and visualization workflow for CRISPR screens. MAGeCK-VISPR defines a set of QC measures to assess the quality of an experiment, and includes a maximum-likelihood algorithm to call essential genes simultaneously under multiple conditions. The algorithm uses a generalized linear model to deconvolute different effects, and employs expectation-maximization to iteratively estimate sgRNA knockout efficiency and gene essentiality. MAGeCK-VISPR also includes VISPR, a framework for the interactive visualization and exploration of QC and analysis results. MAGeCK-VISPR is freely available at http://bitbucket.org/liulab/mageck-vispr .