The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5′ adenine were improved by rescuing 5′ end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.

In just 3 years CRISPR genome editing has transformed biology, and its popularity and potency continue to grow. New CRISPR effectors and rules for locating optimum targets continue to be reported, highlighting the need for computational CRISPR targeting tools to compile these rules and facilitate target selection and design. CHOPCHOP is one of the most widely used web tools for CRISPR- and TALEN-based genome editing. Its overarching principle is to provide an intuitive and powerful tool that can serve both novice and experienced users. In this major update we introduce tools for the next generation of CRISPR advances, including Cpf1 and Cas9 nickases. We support a number of new features that improve the targeting power, usability and efficiency of CHOPCHOP. To increase targeting range and specificity we provide support for custom length sgRNAs, and we evaluate the sequence composition of the whole sgRNA and its surrounding region using models compiled from multiple large-scale studies. These and other new features, coupled with an updated interface for increased usability and support for a continually growing list of organisms, maintain CHOPCHOP as one of the leading tools for CRISPR genome editing. CHOPCHOP v2 can be found at http://chopchop.cbu.uib.no

The Rosetta software suite for macromolecular modeling, docking, and design is widely used in pharmaceutical, industrial, academic, non-profit, and government laboratories. Despite its broad modeling capabilities, Rosetta remains consistently among leading software suites when compared to other methods created for highly specialized protein modeling and design tasks. Developed for over two decades by a global community of over 60 laboratories, Rosetta has undergone multiple refactorings, and now comprises over three million lines of code. Here we discuss methods developed in the last five years in Rosetta, involving the latest protocols for structure prediction; protein-protein and protein-small molecule docking; protein structure and interface design; loop modeling; the incorporation of various types of experimental data; modeling of peptides, antibodies and proteins in the immune system, nucleic acids, non-standard chemistries, carbohydrates, and membrane proteins. We briefly discuss improvements to the energy function, user interfaces, and usability of the software. Rosetta is available at www.rosettacommons.org.

Genetic methods of manipulating or eradicating disease vector populations have long been discussed as an attractive alternative to existing control measures because of their potential advantages in terms of effectiveness and species specificity1–3. The development of genetically engineered malaria-resistant mosquitoes has shown, as a proof-of-principle, the possibility of targeting the mosquito’s ability to serve as a disease vector4–7. The translation of these achievements into control measures requires an effective technology to spread a genetic modification from laboratory mosquitoes to field populations8. We have previously suggested that homing endonuclease genes (HEGs), a class of simple selfish genetic elements, could be exploited for this purpose9. Here we demonstrate that a synthetic genetic element, consisting of mosquito regulatory regions10 and the homing endonuclease gene I-SceI11–13, can substantially increase its transmission to the progeny in transgenic mosquitoes of the human malaria vector Anopheles gambiae. We show that the I-SceI element is able to rapidly invade receptive mosquito cage populations, validating mathematical models for the transmission dynamics of HEGs. Molecular analyses confirm that expression of I-SceI in the male germline induces high rates of site-specific chromosomal cleavage and gene conversion, which results in the gain of the I-SceI gene, and underlies the observed genetic drive. These findings demonstrate a new mechanism by which genetic control measures can be implemented. Our results also show in principle how sequence-specific genetic drive elements like HEGs could be used to take the step from the genetic engineering of individuals to the genetic engineering of populations.

Copy-number variants of chromosome 16 region 16p11.2 are linked to neuropsychiatric disorders1–6 and are among the most prevalent in autism spectrum disorders1,2,7. Of many 16p11.2 genes, Kctd13 has been implicated as a major driver of neurodevelopmental phenotypes8,9. The function of KCTD13 in the mammalian brain, however, remains unknown. Here we delete the Kctd13 gene in mice and demonstrate reduced synaptic transmission. Reduced synaptic transmission correlates with increased levels of Ras homolog gene family, member A (RhoA), a KCTD13/CUL3 ubiquitin ligase substrate, and is reversed by RhoA inhibition, suggesting increased RhoA as an important mechanism. In contrast to a previous knockdown study8, deletion of Kctd13 or kctd13 does not increase brain size or neurogenesis in mice or zebrafish, respectively. These findings implicate Kctd13 in the regulation of neuronal function relevant to neuropsychiatric disorders and clarify the role of Kctd13 in neurogenesis and brain size. Our data also reveal a potential role for RhoA as a therapeutic target in disorders associated with KCTD13 deletion.

Graphical AbstractHighlights d 132 zebrafish mutants for genes located in schizophreniaassociated genomic regions d Phenotypes for many understudied genes with previously unknown functions d Phenotype atlas for abnormal behavior and brain activity d More than 30 genes prioritized for future study

The CRISPR/Cas system uses guide RNAs (gRNAs) to direct sequence-specific DNA cleavage. Not every gRNA elicits cleavage and the mechanisms that govern gRNA activity have not been resolved. Low activity could result from either failure to form a functional Cas9–gRNA complex or inability to recognize targets in vivo. Here we show that both phenomena influence Cas9 activity by comparing mutagenesis rates in zebrafish embryos with in vitro cleavage assays. In vivo, our results suggest that genomic factors such as CTCF inhibit mutagenesis. Comparing near-identical gRNA sequences with different in vitro activities reveals that internal gRNA interactions reduce cleavage. Even though gRNAs containing these structures do not yield cleavage-competent complexes, they can compete with active gRNAs for binding to Cas9. These results reveal that both genomic context and internal gRNA interactions can interfere with Cas9-mediated cleavage and illuminate previously uncharacterized features of Cas9–gRNA complex formation.

Enzymes utilize substrate binding energy both to promote ground state association and to selectively lower the energy of the reaction transition state.i The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in the center of a 20 base-pair DNA target site, with the N-terminal domain of the enzyme making extensive binding interactions with the left (−) side of the target site and the similarly structured C-terminal domain interacting with the right (+) side.ii Despite the approximate two-fold symmetry of the enzyme-DNA complex, we find that there is almost complete segregation of interactions responsible for substrate binding to the (−) side of the interface and interactions responsible for transition state stabilization to the (+) side. While single base-pair substitutions throughout the entire DNA target site reduce catalytic efficiency, mutations in the (−) DNA half-site almost exclusively increase KD and KM*, and those in the (+) half-site primarily decrease kcat*. The reduction of activity produced by mutations on the (−) side, but not mutations on the (+) side, can be suppressed by tethering the substrate to the endonuclease displayed on the surface of yeast. This dramatic asymmetry in the utilization of enzyme-substrate binding energy for catalysis has direct relevance to the redesign of endonucleases to cleave genomic target sites for gene therapy and other applications. Computationally redesigned enzymes that achieve new specificities on the (−) side do so by modulating KM*, while redesigns with altered specificities on the (+) side modulate kcat*. Our results illustrate how classical enzymology and modern protein design can each inform the other.