The ability to engineer genomes in a specific, systematic, and costeffective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.nanos-Cas9 | HRMA M uch of our knowledge of the mechanisms underlying biological processes relies on genetic approaches, whereby gene activity is perturbed and the phenotypic consequences of perturbation are analyzed in detail. In recent years, several major advances have been made in the design of methods for specifically and efficiently perturbing genomes. Arguably, the most exciting advances rely on the ability to induce double-strand breaks (DSBs) by targeting a nuclease to a specific genomic sequence. Repair of DSBs by the error-prone nonhomologous endjoining (NHEJ) mechanism allows for the recovery of small deletions; moreover, repair of DSBs by homologous recombination (HR) in the presence of a donor template opens the door to a wide range of specifically engineered changes at the targeted site (1).Two nuclease-based systems, the zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) systems, work effectively in a number of organisms (2-7). But because these approaches require the production of a construct encoding a unique DNA-binding protein fused to the nuclease domain, they can be both cumbersome and costly. In contrast, the recent approach based on the bacterial CRISPR-associated single-guide RNA (Cas9/sgRNA) system does not require production of specific fusion proteins for each targeted sequence (8-10).Cas9 was first identified in type II Streptococcus pyogenes as an RNA-guided defense system against invading viruses and plasmids (11-13). This adaptive immune-like system contains three components: CRISPR RNA (crRNA), trans-activating CRISPR RNA (tracrRNA), and Cas9. The tracrRNA triggers Cas9 nuclease activity and the crRNA guides Cas9 to cleave the specific foreign dsDNA sequence via base-pairing between the crRNA and the target DNA. Importantly, a single-guide RNA (sgRNA, also known as chiRNA), comprising the minimal crRNA and tracrRNA, can function similarly to the crRNA and tracrRNA, thereby providing a simplified method for genome editing (8)(9)(10)(14)(15)(16)(17)(18)(19)(20).Given the great promise of the Cas9/sgRNA method for genome engineering, we set out to test the sys...

Summary The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, sgRNA parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for the first time a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair, and achieved an overall mutagenesis rate significantly higher than previously reported. Our work presents the most comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.

Mutations in gene regulatory elements have been associated with a wide range of complex neuropsychiatric disorders. However, due to their cell-type specificity and difficulties in characterizing their regulatory targets, the ability to identify causal genetic variants has remained limited. To address these constraints, we perform integrative analysis of chromatin interactions using promoter capture Hi-C (pcHi-C), open chromatin regions using ATAC-seq, and transcriptomes using RNA-seq in four functionally distinct neural cell types: iPSC-induced excitatory neurons and lower motor neurons, iPSC-derived hippocampal dentate gyrus (DG)-like neurons, and primary astrocytes. We identify hundreds of thousands of long-range cis interactions between promoters and distal promoter-interacting regions (PIRs), enabling us to link regulatory elements to their target genes and reveal putative processes that are dysregulated in disease. Finally, we validate several PIRs using CRISPR techniques in human excitatory neurons, demonstrating that CDK5RAP3 , STRAP , and DRD2 are transcriptionally regulated by physically linked enhancers.

CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE.

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