The 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. We 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. To 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.

https://hal.archives-ouvertes.fr/hal-01346049/document

Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.

Major advances in genome editing have recently been made possible with the development of the TALEN and CRISPR/Cas9 methods. The speed and ease of implementing these technologies has led to an explosion of mutant and transgenic organisms. A rate-limiting step in efficiently applying TALEN and CRISPR/Cas9 methods is the selection and design of targeting constructs. We have developed an online tool, CHOPCHOP (https://chopchop.rc.fas.harvard.edu), to expedite the design process. CHOPCHOP accepts a wide range of inputs (gene identifiers, genomic regions or pasted sequences) and provides an array of advanced options for target selection. It uses efficient sequence alignment algorithms to minimize search times, and rigorously predicts off-target binding of single-guide RNAs (sgRNAs) and TALENs. Each query produces an interactive visualization of the gene with candidate target sites displayed at their genomic positions and color-coded according to quality scores. In addition, for each possible target site, restriction sites and primer candidates are visualized, facilitating a streamlined pipeline of mutant generation and validation. The ease-of-use and speed of CHOPCHOP make it a valuable tool for genome engineering.

/pdf/chopchop-a-crispr-cas9-and-talen-web-tool-for-genome-editing-56ly6s8s4f.pdf

CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing

The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line–restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25–100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.

/pdf/optimized-crispr-cas-tools-for-efficient-germline-and-3r631oh62l.pdf

Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.

Seminal fluid proteins (SFPs) produced in reproductive tract tissues of male insects and transferred to females during mating induce numerous physiological and behavioral postmating changes in females. These changes include decreasing receptivity to remating; affecting sperm storage parameters; increasing egg production; and modulating sperm competition, feeding behaviors, and mating plug formation. In addition, SFPs also have antimicrobial functions and induce expression of antimicrobial peptides in at least some insects. Here, we review recent identification of insect SFPs and discuss the multiple roles these proteins play in the postmating processes of female insects.

/pdf/insect-seminal-fluid-proteins-identification-and-function-8a29hdq0i9.pdf

Insect seminal fluid proteins: identification and function.

The core mission of National Institute of Neurological Disorders and Stroke (NINDS) is twofold. First, NINDS seeks fundamental knowledge about the brain and nervous system. Second, NINDS aims to use that knowledge to reduce the burden of neurological diseases. In support of its mission, NINDS performs and funds basic, translational, and clinical neuroscience research on more than 600 neurological diseases, including genetic diseases (e.g. Huntington’s disease; muscular dystrophy), developmental disorders (e.g. cerebral palsy), neurodegenerative diseases (e.g. Parkinson’s disease; Alzheimer’s disease; multiple sclerosis), metabolic diseases (e.g. Gaucher’s disease), cerebrovascular diseases (e.g. stroke; vascular dementia), trauma (e.g. spinal cord and head injury), convulsive disorders (e.g. epilepsy), infectious diseases (e.g. AIDS dementia) and brain tumors. Common marmosets (Callithrix jacchus) offer unique, powerful advantages to both components of the NINDS mission. In support of the first component, marmosets are particularly well suited for neuroanatomical and functional brain studies, as their brains retain the typical anatomical and functional organization of the primate brain. A major advantage is that the marmoset is a lissencephalic primate, which greatly facilitates the mapping of functional brain areas by neuroimaging techniques, such as fMRI and optical imaging, as well as by electrophysiology, with high spatial resolution. In support of the second component, marmosets are excellent models of neurological disorders. Unlike rodents, marmosets are outbred and every individual is genetically different. Further, the marmoset brain has a gray-to-white matter ratio comparable to humans, which strongly facilitates modeling diseases such as multiple sclerosis and small vessel disease. The species also exhibits the breadth of cognitive sophistication that distinguishes primates from other taxonomic groups. Finally, geneedited marmosets can be generated with an intergeneration time and establishment of transgenic lines 2-3 times faster than other primate species, which makes marmosets be the ideal primate species for the development of genetically engineered lines. For all of the above reasons, marmosets are poised to be a central player to advance the core mission of the NINDS.

National Institute of Neurological Disorders and Stroke

We and others recently demonstrated that the readily programmable CRISPR/Cas9 system can be used to edit the Drosophila genome. However, most applications to date have relied on aberrant DNA repair to stochastically generate frameshifting indels and adoption has been limited by a lack of tools for efficient identification of targeted events. Here we report optimized tools and techniques for expanded application of the CRISPR/Cas9 system in Drosophila through homology-directed repair (HDR) with double-stranded DNA (dsDNA) donor templates that facilitate complex genome engineering through the precise incorporation of large DNA sequences, including screenable markers. Using these donors, we demonstrate the replacement of a gene with exogenous sequences and the generation of a conditional allele. To optimize efficiency and specificity, we generated transgenic flies that express Cas9 in the germline and directly compared HDR and off-target cleavage rates of different approaches for delivering CRISPR components. We also investigated HDR efficiency in a mutant background previously demonstrated to bias DNA repair toward HDR. Finally, we developed a web-based tool that identifies CRISPR target sites and evaluates their potential for off-target cleavage using empirically rooted rules. Overall, we have found that injection of a dsDNA donor and guide RNA-encoding plasmids into vasa-Cas9 flies yields the highest efficiency HDR and that target sites can be selected to avoid off-target mutations. Efficient and specific CRISPR/Cas9-mediated HDR opens the door to a broad array of complex genome modifications and greatly expands the utility of CRISPR technology for Drosophila research.

https://www.genetics.org/content/genetics/196/4/961.full.pdf

Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila

The CRISPR-Cas9 system has transformed genome engineering of model organisms from possible to practical. CRISPR-Cas9 can be readily programmed to generate sequence-specific double-strand breaks that disrupt targeted loci when repaired by error-prone non-homologous end joining (NHEJ) or to catalyze precise genome modification through homology-directed repair (HDR). Here we describe a streamlined approach for rapid and highly efficient engineering of the Drosophila genome via CRISPR-Cas9-mediated HDR. In this approach, transgenic flies expressing Cas9 are injected with plasmids to express guide RNAs (gRNAs) and positively marked donor templates. We detail target-site selection; gRNA plasmid generation; donor template design and construction; and the generation, identification, and molecular confirmation of engineered lines. We also present alternative approaches and highlight key considerations for experimental design. The approach outlined here can be used to rapidly and reliably generate a variety of engineered modifications, including genomic deletions and replacements, precise sequence edits, and incorporation of protein tags.

/pdf/crispr-cas9-genome-editing-in-drosophila-rgfmgzsy42.pdf

CRISPR-Cas9 Genome Editing in Drosophila

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.

https://www.pnas.org/content/pnas/110/43/17420.full.pdf

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Studies of social behavior generally focus on interactions between two or more individual animals. However, these interactions are not simply between whole animals, but also occur between molecules that were produced by the interacting individuals. Such "molecular social interactions" can both influence and be influenced by the organismal-level social interactions. We illustrate this by reviewing the roles played by seminal fluid proteins (Sfps) in molecular social interactions between males and females of the fruit fly Drosophila melanogaster. Sfps, which are produced by males and transferred to females during mating, are involved in inherently social interactions with female-derived molecules, and they influence social interactions between males and females and between a female's past and potential future mates. Here, we explore four examples of molecular social interactions involving D. melanogaster Sfps: processes that influence mating, sperm storage, ovulation, and ejaculate transfer. We consider the molecular and organismal players involved in each interaction and the consequences of their interplay for the reproductive success of both sexes. We conclude with a discussion of the ways in which Sfps can both shape and be shaped by (in an evolutionary sense) the molecular social interactions in which they are involved.

/pdf/molecular-social-interactions-drosophila-melanogaster-46q26l435s.pdf

C. Dustin Rubinstein

Papers

Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila

Insect seminal fluid proteins: identification and function.

CRISPR-Cas9 Genome Editing in Drosophila

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Molecular Social Interactions: Drosophila melanogaster Seminal Fluid Proteins as a Case Study