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CRISPOR.org is a web tool for genome editing experiments with the CRISPR-Cas9 system. It finds guide RNAs in an input sequence and ranks them according to different scores that evaluate potential off-targets in the genome of interest and predict on-target activity. The list of genomes is continuously expanded, with more 150 genomes added in the last two years. CRISPOR tries to provide a comprehensive solution from selection, cloning and expression of guide RNA as well as providing primers needed for testing guide activity and potential off-targets. Recent developments include batch design for genome-wide CRISPR and saturation screens, creating custom oligonucleotides for guide cloning and the design of next generation sequencing primers to test for off-target mutations. CRISPOR is available from http://crispor.org, including the full source code of the website and a stand-alone, command-line version.

CRISPR (clustered regularly interspaced short palindromic repeats) genome-editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep-sequencing data resulting from genome-editing experiments. However, these tools are typically developed in isolation and oftentimes are not readily translatable into laboratory-based experiments. Here, we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome-editing experiment. This protocol provides instructions for sgRNA design with CRISPOR (computational tool for the design, evaluation, and cloning of sgRNA sequences), experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso (computational tool for analysis of genome-editing outcomes from deep-sequencing data). This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts, as well as computational data analysis that can be performed in 1-2 d by both computational and noncomputational biologists alike using web-based and/or command-line versions.

BackgroundThe success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements.ResultsWe conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening.ConclusionsTo make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website ( http://crispor.org ) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here.

The CRISPR-Cas revolution is taking place in virtually all fields of life sciences. Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient, relying only on the design of a synthetic single guide RNA (sgRNA) and its co-expression with Cas9. Here, we review the progress in the design of sgRNA from the original dual RNA guide for S. pyogenes and Staphylococcus aureus Cas9 (SpCas9 and SaCas9). New assays for genome-wide identification of off-targets have provided important insights into the issue of cleavage specificity in vivo. At the same time, the on-target activity of thousands of guides has been determined. These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences. It appears that for most basic research applications, cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions. Moreover, recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing. Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA, several variants of SpCas9 have recently been engineered, either with novel protospacer adjacent motifs (PAMs) or with drastically reduced off-targets. Novel Cas9 and Cas9-like proteins called Cpf1 have also been characterized from other bacteria and will benefit from the insights obtained from SpCas9. Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage.

Copy number variations (CNVs) of the human 16p11.2 locus are associated with several developmental/neurocognitive syndromes. Particularly, deletion and duplication of this genetic interval are found in patients with autism spectrum disorders, intellectual disability and other psychiatric traits. The high gene density associated with the region and the strong phenotypic variability of incomplete penetrance, make the study of the 16p11.2 syndromes extremely complex. To systematically study the effect of 16p11.2 CNVs and identify candidate genes and molecular mechanisms involved in the pathophysiology, mouse models were generated previously and showed learning and memory, and to some extent social deficits. To go further in understanding the social deficits caused by 16p11.2 syndromes, we engineered deletion and duplication of the homologous region to the human 16p11.2 genetic interval in two rat outbred strains, Sprague Dawley (SD) and Long Evans (LE). The 16p11.2 rat models displayed convergent defects in social behavior and in the novel object test in male carriers from both genetic backgrounds. Interestingly major pathways affecting MAPK1 and CUL3 were found altered in the rat 16p11.2 models with additional changes in males compared to females. Altogether, the consequences of the 16p11.2 genetic region dosage on social behavior are now found in three different species: humans, mice and rats. In addition, the rat models pointed to sexual dimorphism with lower severity of phenotypes in rat females compared to male mutants. This phenomenon is also observed in humans. We are convinced that the two rat models will be key to further investigating social behavior and understanding the brain mechanisms and specific brain regions that are key to controlling social behavior. [ABSTRACT FROM AUTHOR]

Bertrand, Anne, Valerie Ngo-Muller, Daniele Hentzen, Jean-Paul Concordet, Dominique Daegelen, and David Tuil. Muscle electrotransfer as a tool for studying muscle fiber-specific and nerve-dependent activity of promoters. Am J Physiol Cell Physiol 285: C1071-C1081, 2003. First published July 2, 2003; 10.1152/ajpcell.00104.2003.--Muscle electrotransfer has recently become a promising tool for efficient delivery of plasmids and transgene expression in skeletal muscle. This technology has been mainly applied to use of muscle as a bioreactor for production of therapeutic proteins. However, it remains to be determined whether muscle electrotransfer may also be accurately used as an alternative tool to transgenesis for studying aspects of muscle-specific gene control that must be explored in fully mature muscle fibers in vivo, such as fiber specificity and nerve dependence. It was also not known to what extent the initial electrical stimulations alter muscle physiology and gene expression. Therefore, optimized conditions of skeletal muscle electroporation were first tested for their effects on muscles of transgenic mice harboring a pM310-CAT transgene in which the CAT reporter gene was under control of the fast IIB fiber-specific and nerve-dependent aldolase A pM promoter. Surprisingly, electrostimulation led to a drastic but transient shutdown of pM310-CAT transgene expression concomitant with very transient activation of MyoD and, mostly, with activation of myogenin, suggesting profound alterations in transcriptional status of the electroporated muscle. Return to a normal transcriptional state was observed 7-10 days after electroporation. Therefore, we investigated whether a reporter construct placed under control of pM could exhibit fiber-specific expression 10 days after electrotransfer in either fast tibialis anterior or slow soleus muscle. We show that not only fiber specificity, but also nerve dependence, of a pM-driven construct can be reproduced. However, after electrotransfer, pM displayed a less tight control than previously observed for the same promoter when integrated in a chromatin context. fast or slow fibers; aldolase A; MyoD; myogenin