H. Bart van der Worp and colleagues discuss the controversies and possibilities of translating the results of animal experiments into human clinical trials.

Nigrostriatal dopaminergic neurons undergo sprouting around the margins of a striatal wound. The mechanism of this periwound sprouting has been unclear. In this study, we have examined the role played by the macrophage and microglial response that follows striatal injury. Macrophages and activated microglia quickly accumulate after injury and reach their greatest numbers in the first week. Subsequently, the number of both cell types declines rapidly in the first month and thereafter more slowly. Macrophage numbers eventually cease to decline, and a sizable group of these cells remains at the wound site and forms a long-term, highly activated resident population. This population of macrophages expresses increasing amounts of glial cell line-derived neurotrophic factor mRNA with time. Brain-derived neurotrophic factor mRNA is also expressed in and around the wound site. Production of this factor is by both activated microglia and, to a lesser extent, macrophages. The production of these potent dopaminergic neurotrophic factors occurs in a similar spatial distribution to sprouting dopaminergic fibers. Moreover, dopamine transporter-positive dopaminergic neurites can be seen growing toward and embracing hemosiderin-filled wound macrophages. The dopaminergic sprouting that accompanies striatal injury thus appears to result from neurotrophic factor secretion by activated macrophages and microglia at the wound site.

No abstract

No single animal model is able to encompass all of the variables known to affect human ischemic stroke. This review highlights the major strengths and weaknesses of the most commonly used animal models of acute ischemic stroke in the context of matching model and experimental aim. Particular emphasis is placed on the relationships between outcome and underlying vascular variability, physiologic control, and use of models of comorbidity. The aim is to provide, for novice and expert alike, an overview of the key controllable determinants of experimental stroke outcome to help ensure the most effective application of animal models to translational research.

CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications but identifying unwanted off-target mutations is important for clinical translation. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.

21Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a 22 target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay 23 for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach 24 takes advantage of the recruitment of endogenous DNA repair factors for genome-wide 25 identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely 26 to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base 27 resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene 28 editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene 29 editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides 30 an unprecedented view of events that precede repair of the affected sites. 31 strengths, they also have certain weaknesses. Naïve prediction algorithms are for the most part 41 based on sequence similarity and currently have limited predictive power with very high false-42 positive rates (10). Assays that induce DSBs in vitro, such as Digenome-Seq (5), CIRCLE-Seq (6) 43 and SITE-Seq (7), have high sensitivity but dramatically under-or overestimate the number of 44 target sites that are actually modified in cellular models or in vivo (11). Nuclease concentration 45 within the cell (7), delivery method (ribonucleoprotein (RNP) vs. plasmid) (7, 12, 13) as well as 46 more complex cellular properties such as chromatin accessibility (14, 15) have been shown to 47

Lewy bodies are made from insoluble, phosphorylated α-synuclein, but the earliest changes that precipitate such pathology still remain conjecture. In this study, we quantify and identify relationships between the levels of the main pathologic form of phosphorylated α-synuclein over the course of Parkinson's disease in regions affected early through to end-stage disease. Brain tissue samples from 33 cases at different disease stages and 13 controls were collected through the Australian Network of Brain Banks. 500 mg of frozen putamen (affected preclinically) and frontal cortex (affected late) was homogenized, fractionated and α-synuclein levels evaluated using specific antibodies (syn-1, BD Transduction Laboratories; S129P phospho-α-synuclein, Elan Pharmaceuticals) and quantitative western blotting. Statistical analyses assessed the relationship between the different forms of α-synuclein, compared levels between groups, and determined any changes over the disease course. Soluble S129P was detected in controls with higher levels in putamen compared with frontal cortex. In contrast, insoluble α-synuclein occurred in Parkinson's disease with a significant increase in soluble and lipid-associated S129P, and a decrease in soluble frontal α-synuclein over the disease course. Increasing soluble S129P in the putamen correlated with increasing S129P in other fractions and regions. These data show that soluble non-phosphorylated α-synuclein decreases over the course of Parkinson's disease, becoming increasingly phosphorylated and insoluble. The finding that S129P α-synuclein normally occurs in vulnerable brain regions, and in Parkinson's disease has the strongest relationships to the pathogenic forms of α-synuclein in other brain regions, suggests a propagating role for putamenal phospho-α-synuclein in disease pathogenesis.

Spotting off-targets from gene editing Unintended genomic modifications limit the potential therapeutic use of gene-editing tools. Available methods to find off-targets generally do not work in vivo or detect single-nucleotide changes. Three papers in this issue report new methods for monitoring gene-editing tools in vivo (see the Perspective by Kempton and Qi). Wienert et al. followed the recruitment of a DNA repair protein to DNA breaks induced by CRISPR-Cas9, enabling unbiased detection of off-target editing in cellular and animal models. Zuo et al. identified off-targets without the interference of natural genetic heterogeneity by injecting base editors into one blastomere of a two-cell mouse embryo and leaving the other genetically identical blastomere unedited. Jin et al. performed whole-genome sequencing on individual, genome-edited rice plants to identify unintended mutations. Cytosine, but not adenine, base editors induced numerous single-nucleotide variants in both mouse and rice. Science , this issue p. 286 , p. 289 , p. 292 ; see also p. 234