Xingjie Ren

Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9

Significance Using the recently introduced Cas9/sgRNA technique, we have developed a method for specifically targeting Drosophila germ-line cells to generate heritable mutant alleles. We have established transgenic lines that stably express Cas9 in the germ line and compared different promoters and scaffolds of sgRNA in terms of their efficiency of mutagenesis. An overall mutagenesis rate of 74.2% was achieved with this optimized system, as determined by the number of mutant progeny out of all progeny screened. We also evaluated the off-targets associated with the method and established a Web-based resource, as well as a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Our results demonstrate that this optimized Cas9/sgRNA system in Drosophila is efficient, specific, and cost-effective and can be readily applied in a semi-high-throughput manner. The ability to engineer genomes in a specific, systematic, and cost-effective 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.

A Toolkit of CRISPR-Based Genome Editing Systems in Drosophila

The last couple of years have witnessed an explosion in development of CRISPR-based genome editing technologies in cell lines as well as in model organisms. In this review, we focus on the applications of this popular system in Drosophila. We discuss the effectiveness of the CRISPR/Cas9 systems in terms of delivery, mutagenesis detection, parameters affecting efficiency, and off-target issues, with an emphasis on how to apply this powerful tool to characterize gene functions.

Performance of the Cas9 Nickase System in Drosophila melanogaster

Recent studies of the Cas9/sgRNA system in Drosophila melanogaster genome editing have opened new opportunities to generate site-specific mutant collections in a high-throughput manner. However, off-target effects of the system are still a major concern when analyzing mutant phenotypes. Mutations converting Cas9 to a DNA nickase have great potential for reducing off-target effects in vitro. Here, we demonstrated that injection of two plasmids encoding neighboring offset sgRNAs into transgenic Cas9D10A nickase flies efficiently produces heritable indel mutants. We then determined the effective distance between the two sgRNA targets and their orientations that affected the ability of the sgRNA pairs to generate mutations when expressed in the transgenic nickase flies. Interestingly, Cas9 nickase greatly reduces the ability to generate mutants with one sgRNA, suggesting that the application of Cas9 nickase and sgRNA pairs can almost avoid off-target effects when generating indel mutants. Finally, a defined piwi mutant allele is generated with this system through homology-directed repair. However, Cas9D10A is not as effective as Cas9 in replacing the entire coding sequence of piwi with two sgRNAs.

Histone H1-mediated epigenetic regulation controls germline stem cell self-renewal by modulating H4K16 acetylation

Epigenetics plays critical roles in controlling stem cell self-renewal and differentiation. Histone H1 is one of the most critical chromatin regulators, but its role in adult stem cell regulation remains unclear. Here we report that H1 is intrinsically required in the regulation of germline stem cells (GSCs) in the Drosophila ovary. The loss of H1 from GSCs causes their premature differentiation through activation of the key GSC differentiation factor bam. Interestingly, the acetylated H4 lysine 16 (H4K16ac) is selectively augmented in the H1-depleted GSCs. Furthermore, overexpression of mof reduces H1 association on chromatin. In contrast, the knocking down of mof significantly rescues the GSC loss phenotype. Taken together, these results suggest that H1 functions intrinsically to promote GSC self-renewal by antagonizing MOF function. Since H1 and H4K16 acetylation are highly conserved from fly to human, the findings from this study might be applicable to stem cells in other systems.

Optimized strategy for in vivo Cas9-activation in Drosophila

Significance Recently, a number of approaches have been developed to repurpose the CRISPR/Cas9 system as a sequence-specific transcriptional activator for gain-of-function experiments (CRISPR activators, or “CRISPRa”). While multiple CRISPRa strategies have been characterized in cell culture, little is known about their performance in vivo. We present an optimized strategy for generating a large-scale CRISPRa resource in Drosophila and show that this system has a high success rate and generates easily recognizable phenotypes in vivo. We describe a growing collection of transgenic fly lines to facilitate large-scale in vivo CRISPRa experiments. While several large-scale resources are available for in vivo loss-of-function studies in Drosophila, an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.

Heterochromatin remodeling by CDK12 contributes to learning in Drosophila

Significance Eukaryotic genomes are compacted into chromosomes, in which heterochromatin is generally considered to be distinct from euchromatin in chromosomal packaging levels and locations. In Drosophila, heterochromatin is mainly found in pericentric and telomeric regions. In this study, we show that heterochromatin landscapes that interspersed in euchromatic arms are counteracted by CDK12, a major RNA polymerase II C-terminal domain kinase. After the depletion of CDK12, heterochromatin enrichment can be observed on euchromatic arms, especially on the X chromosome, which leads to transcriptional attenuation in targeted genes and defects in neuronal functions. Our findings provide insights into the regulation of heterochromatin domain in the natural chromosomal context and suggest a chromatin regulatory role of CDK12 in neuronal functions. Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. Here, we report that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors.

Next-generation CRISPR/Cas9 transcriptional activation in Drosophila using flySAM

Significance We present flySAM, a potent system for Cas9-based transcriptional activation (CRISPRa) in Drosophila. flySAM greatly improves on existing in vivo CRISPRa techniques in terms of potency, scalability, and ease of use, and provides a simple and general method for conducting overexpression experiments and screens. flySAM will now serve as the basis for our growing collection of publicly available CRISPRa transgenic fly lines. CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in Drosophila melanogaster. 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.

Genome editing in Drosophila melanogaster : from basic genome engineering to the multipurpose CRISPR-Cas9 system

Nowadays, genome editing tools are indispensable for studying gene function in order to increase our knowledge of biochemical processes and disease mechanisms. The extensive availability of mutagenesis and transgenesis tools make Drosophila melanogaster an excellent model organism for geneticists. Early mutagenesis tools relied on chemical or physical methods, ethyl methane sulfonate (EMS) and X-rays respectively, to randomly alter DNA at a nucleotide or chromosomal level. Since the discovery of transposable elements and the availability of the complete fly genome, specific genome editing tools, such as P-elements, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have undergone rapid development. Currently, one of the leading and most effective contemporary tools is the CRISPR-cas9 system made popular because of its low cost, effectiveness, specificity and simplicity of use. This review briefly addresses the most commonly used mutagenesis and transgenesis tools in Drosophila, followed by an in-depth review of the multipurpose CRISPR-Cas9 system and its current applications.

The Mediator CDK8-Cyclin C complex modulates vein patterning in Drosophila by stimulating Mad-dependent transcription

Dysregulations of CDK8 and its regulatory partner CycC, two subunits of the conserved Mediator complex, have been linked to diverse human diseases such as cancer, thus it is essential to understand the regulatory network mobilizing the CDK8-CycC complex in both normal development and tumorigenesis. To identify upstream regulators or downstream effectors of CDK8, we performed a dominant modifier genetic screen in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in wing imaginal discs. We identified 26 genomic loci whose haploinsufficiency can modify these CDK8-specific phenotypes. Further analysis of two deficiency lines and mutant alleles led us to identify interactions between CDK8-CycC and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFβ) signaling pathway. We observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFβ signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the Mediator complex have revealed six additional Mediator subunits that are required for Mad-dependent transcription in the wing discs, including Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, CDK9 and Yorkie also positively regulate Mad-dependent gene expression in vivo. These results suggest that the Mediator complex may coordinate with other transcription cofactors in regulating Mad-dependent transcription during the wing vein patterning in Drosophila. Significance CDK8 is a conserved subunit of the transcription cofactor Mediator complex that bridges transcription factors with RNA Polymerase II in eukaryotes. Here we explore the role of CDK8 in Drosophila by performing a dominant modifier genetic screen based on vein patterning defects caused by alteration of CDK8-specific activities. We show that components of the Dpp/TGFβ signaling pathway genetically interact with CDK8; CDK8 positively regulates gene expression activated by Mad, the key transcription factor downstream of Dpp/TGFβ signaling, by directly interacting with the linker region of Mad protein. Given the fundamental roles of Dpp/TGFβ signaling in regulating development and its misregulation in various diseases, understanding how Mad/Smad interacts the Mediator complex may have broad implications in understanding and treating these diseases.

The Lysine Demethylase dKDM2 Is Non-essential for Viability, but Regulates Circadian Rhythms in Drosophila

Post-translational modification of histones, such as histone methylation controlled by specific methyltransferases and demethylases, play critical roles in modulating chromatin dynamics and transcription in eukaryotes. Misregulation of histone methylation can lead to aberrant gene expression, thereby contributing to abnormal development and diseases such as cancer. As such, the mammalian lysine-specific demethylase 2 (KDM2) homologs, KDM2A and KDM2B, are either oncogenic or tumor suppressive depending on specific pathological contexts. However, the role of KDM2 proteins during development remains poorly understood. Unlike vertebrates, Drosophila has only one KDM2 homolog (dKDM2), but its functions in vivo remain elusive due to the complexities of the existing mutant alleles. To address this problem, we have generated two dKdm2 null alleles using the CRISPR/Cas9 technique. These dKdm2 homozygous mutants are fully viable and fertile, with no developmental defects observed under laboratory conditions. However, the dKdm2 null mutant adults display defects in circadian rhythms. Most of the dKdm2 mutants become arrhythmic under constant darkness, while the circadian period of the rhythmic mutant flies is approximately 1 h shorter than the control. Interestingly, lengthened circadian periods are observed when dKDM2 is overexpressed in circadian pacemaker neurons. Taken together, these results demonstrate that dKdm2 is not essential for viability; instead, dKDM2 protein plays important roles in regulating circadian rhythms in Drosophila. Further analyses of the molecular mechanisms of dKDM2 and its orthologs in vertebrates regarding the regulation of circadian rhythms will advance our understanding of the epigenetic regulations of circadian clocks.