Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split–Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split–Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.

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The mechanism of action responsible for the motor depressant effects of cannabinoids, which operate through centrally expressed cannabinoid CB 1 receptors, is still a matter of debate. In the present study, we report that CB 1 and adenosine A 2A receptors form heteromeric complexes in co-transfected HEK-293T cells and rat striatum, where they colocalize in fibrilar structures. In a human neuroblastoma cell line, CB 1 receptor signaling was found to be completely dependent on A 2A receptor activation. Accordingly, blockade of A 2A receptors counteracted the motor depressant effects produced by the intrastriatal administration of a cannabinoid CB 1 receptor agonist. These biochemical and behavioral findings demonstrate that the profound motor effects of cannabinoids depend on physical and functional interactions between striatal A 2A and CB 1 receptors.

Recent evidence suggests that glutamatergic and dopaminergic afferents must be activated to induce persistent long-term potentiation (LTP) in the hippocampus. Whereas extensive evidence supports the role of glutamate receptors in long-lasting synaptic plasticity and spatial learning and memory, there is less evidence regarding the role of dopamine receptors in these processes. Here, we used dopamine D(1) receptor knockout (D(1)R(-/-)) mice to explore the role of D(1)R in hippocampal LTP and its associated gene expression. We show that the magnitude of early and late phases of LTP (E-LTP and L-LTP) was markedly reduced in hippocampal slices from D(1)R(-/-) mice compared with wild-type mice. SCH23390, a D(1)/D(5)R antagonist, did not further reduce L-LTP in D(1)R(-/-) mice, suggesting that D(5)Rs are not involved. D(1)R(-/-) mice also showed a significant reduction of D(1)R-induced potentiation of N-Methyl-D-aspartic acid-mediated currents, via protein kinase activated by cyclic adenosine 3',5'-monophosphate activation. Finally, LTP-induced expression of the immediate early genes zif268 and arc in the hippocampal CA1 area was abolished in D(1)R(-/-) mice, and these mice showed impaired learning. These results indicate that D(1)R but not D(5)R are critical for hippocampal LTP and for the induction of Zif268 and Arc, proteins required for the transition from E-LTP to L-LTP and for memory consolidation in mammals.

The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by highthroughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of HermanskyPudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions.gene targeting | mouse genetics | knockout | knockin G ene targeting in embryonic stem (ES) cells is routinely applied to modify the mouse genome and establish the mouse as the most commonly used genetic disease model (1). Nevertheless, the production of targeted mutants is a laborious and time-consuming task that requires the construction of targeting vectors with selection markers, the isolation of mutant ES cells, and the generation of germ-line chimaeras. Moreover, advanced allele design strategies, like the creation of missense mutations, require an additional working step to eliminate selection marker genes from targeted loci (2). Therefore, the establishment of such mutants is presently a low-throughput and long-term procedure, restricting rapid advancements in disease modeling. In contrast, a burst of nucleotide replacements, small deletions and insertions in coding regions that underlie Mendelian or complex human diseases are discovered by high-throughput genomic analysis such as whole-exome sequencing (3-5). The faithful reproduction of such mutations in mouse models will be instrumental in understanding disease mechanisms and to develop therapeutic interventions. This demand is neither covered by available mutagenesis technologies nor by large-scale mouse mutagenesis programs that aim for complete gene inactivation in classical or conditional knockout mice (6). To cope with this challenge, we aimed to create mutations within the shortest time directly in the genome of one-cell mouse embryos, avoiding the time-consuming handling of ES cells, gene-targeting vectors, selection markers, and chimaeras. Our approach is based on transcription activator-like (TAL) effector nucleases (TALENs) to create targeted double-strand breaks that are either sealed by homologous recombination (HR) with mutant, synthetic oligodeoxynucleotides (ODNs) or closed by nonhomologous endj...

Associative learning depends on multiple cortical and subcortical structures, including striatum, hippocampus, and amygdala. Both glutamatergic and dopaminergic neurotransmitter systems have been implicated in learning and memory consolidation. While the role of glutamate is well established, the role of dopamine and its receptors in these processes is less clear. In this study, we used two models of dopamine D 1 receptor (D 1 R, Drd1a) loss, D 1 R knock-out mice (Drd1a

Gene targeting by zinc-finger nucleases in one-cell embryos provides an expedite mutagenesis approach in mice, rats, and rabbits. This technology has been recently used to create knockout and knockin mutants through the deletion or insertion of nucleotides. Here we apply zinc-finger nucleases in one-cell mouse embryos to generate disease-related mutants harboring single nucleotide or codon replacements. Using a gene-targeting vector or a synthetic oligodesoxynucleotide as template for homologous recombination, we introduced missense and silent mutations into the Rab38 gene, encoding a small GTPase that regulates intracellular vesicle trafficking. These results demonstrate the feasibility of seamless gene editing in one-cell embryos to create genetic disease models and establish synthetic oligodesoxynucleotides as a simplified mutagenesis tool.

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