tgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9.

CRISPR-interference (CRISPRi), a highly effective method for silencing genes in mammalian cells, employs an enzymatically dead form of Cas9 (dCas9) complexed with one or more guide RNAs (gRNAs) with 20 nucleotides (nt) of complementarity to transcription initiation sites of target genes. Such gRNA/dCas9 complexes bind to DNA, impeding transcription of the targeted locus. Here, we present an alternative gene-suppression strategy using active Cas9 complexed with truncated gRNAs (tgRNAs). Cas9/tgRNA complexes bind to specific target sites without triggering DNA cleavage. When targeted near transcriptional start sites, these short 14-15 nts tgRNAs efficiently repress expression of several target genes throughout somatic tissues in Drosophila melanogaster without generating any detectable target site mutations. tgRNAs also can activate target gene expression when complexed with a Cas9-VPR fusion protein or modulate enhancer activity, and can be incorporated into a gene-drive, wherein a traditional gRNA sustains drive while a tgRNA inhibits target gene expression.

A comprehensive Drosophila resource to identify key functional interactions between SARS-CoV-2 factors and host proteins.

Development of effective therapies against SARS-CoV-2 infections relies on mechanistic knowledge of virus-host interface. Abundant physical interactions between viral and host proteins have been identified, but few have been functionally characterized. Harnessing the power of fly genetics, we develop a comprehensive Drosophila COVID-19 resource (DCR) consisting of publicly available strains for conditional tissue-specific expression of all SARS-CoV-2 encoded proteins, UAS-human cDNA transgenic lines encoding established host-viral interacting factors, and GAL4 insertion lines disrupting fly homologs of SARS-CoV-2 human interacting proteins. We demonstrate the utility of the DCR to functionally assess SARS-CoV-2 genes and candidate human binding partners. We show that NSP8 engages in strong genetic interactions with several human candidates, most prominently with the ATE1 arginyltransferase to induce actin arginylation and cytoskeletal disorganization, and that two ATE1 inhibitors can reverse NSP8 phenotypes. The DCR enables parallel global-scale functional analysis of SARS-CoV-2 components in a prime genetic model system.

Cas9/Nickase-induced allelic conversion by homologous chromosome-templated repair in Drosophila somatic cells.

Repair of double-strand breaks (DSBs) in somatic cells is primarily accomplished by error-prone nonhomologous end joining and less frequently by precise homology-directed repair preferentially using the sister chromatid as a template. Here, a Drosophila system performs efficient somatic repair of both DSBs and single-strand breaks (SSBs) using intact sequences from the homologous chromosome in a process we refer to as homologous chromosome-templated repair (HTR). Unexpectedly, HTR-mediated allelic conversion at the white locus was more efficient (40 to 65%) in response to SSBs induced by Cas9-derived nickases D10A or H840A than to DSBs induced by fully active Cas9 (20 to 30%). Repair phenotypes elicited by Nickase versus Cas9 differ in both developmental timing (late versus early stages, respectively) and the production of undesired mutagenic events (rare versus frequent). Nickase-mediated HTR represents an efficient and unanticipated mechanism for allelic correction, with far-reaching potential applications in the field of gene editing.

Reversing insecticide resistance with allelic-drive in Drosophila melanogaster.

A recurring target-site mutation identified in various pests and disease vectors alters the voltage gated sodium channel (vgsc) gene (often referred to as knockdown resistance or kdr) to confer resistance to commonly used insecticides, pyrethroids and DDT. The ubiquity of kdr mutations poses a major global threat to the continued use of insecticides as a means for vector control. In this study, we generate common kdr mutations in isogenic laboratory Drosophila strains using CRISPR/Cas9 editing. We identify differential sensitivities to permethrin and DDT versus deltamethrin among these mutants as well as contrasting physiological consequences of two different kdr mutations. Importantly, we apply a CRISPR-based allelic-drive to replace a resistant kdr mutation with a susceptible wild-type counterpart in population cages. This successful proof-of-principle opens-up numerous possibilities including targeted reversion of insecticide-resistant populations to a native susceptible state or replacement of malaria transmitting mosquitoes with those bearing naturally occurring parasite resistant alleles.

Dissecting the evolutionary role of the Hox gene proboscipedia in Drosophila mouthpart diversification by full locus replacement.

Hox genes determine positional codes along the head-to-tail axis. Here, we replaced the entire Drosophila melanogaster proboscipedia (pb) Hox locus, which controls the development of the proboscis and maxillary palps, with that from Drosophila mimica, a related species with highly modified mouthparts. The D. mimica replacement rescues most aspects of adult proboscis morphology; however, the shape and orientation of maxillary palps were modified, resembling D. mimica and closely related species. Expressing the D. mimica Pb protein in the D. melanogaster pattern fully rescued D. melanogaster morphology. However, the expression pattern directed by D. mimica pb cis-regulatory sequences differed from that of D. melanogaster pb in cells that produce altered maxillary structures, indicating that pb regulatory sequences can evolve in related species to alter morphology.

CopyCatchers are versatile active genetic elements that detect and quantify inter-homolog somatic gene conversion.

CRISPR-based active genetic elements, or gene-drives, copied via homology-directed repair (HDR) in the germline, are transmitted to progeny at super-Mendelian frequencies. Active genetic elements also can generate widespread somatic mutations, but the genetic basis for such phenotypes remains uncertain. It is generally assumed that such somatic mutations are generated by non-homologous end-joining (NHEJ), the predominant double stranded break repair pathway active in somatic cells. Here, we develop CopyCatcher systems in Drosophila to detect and quantify somatic gene conversion (SGC) events. CopyCatchers inserted into two independent genetic loci reveal unexpectedly high rates of SGC in the Drosophila eye and thoracic epidermis. Focused RNAi-based genetic screens identify several unanticipated loci altering SGC efficiency, one of which (c-MYC), when downregulated, promotes SGC mediated by both plasmid and homologous chromosome-templates in human HEK293T cells. Collectively, these studies suggest that CopyCatchers can serve as effective discovery platforms to inform potential gene therapy strategies.

Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives.

CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.

The Hox proteins Ubx and AbdA collaborate with the transcription pausing factor M1BP to regulate gene transcription.

In metazoans, the pausing of RNA polymerase II at the promoter (paused Pol II) has emerged as a widespread and conserved mechanism in the regulation of gene transcription. While critical in recruiting Pol II to the promoter, the role transcription factors play in transitioning paused Pol II into productive Pol II is, however, little known. By studying how Drosophila Hox transcription factors control transcription, we uncovered a molecular mechanism that increases productive transcription. We found that the Hox proteins AbdA and Ubx target gene promoters previously bound by the transcription pausing factor M1BP, containing paused Pol II and enriched with promoter-proximal Polycomb Group (PcG) proteins, yet lacking the classical H3K27me3 PcG signature. We found that AbdA binding to M1BP-regulated genes results in reduction in PcG binding, the release of paused Pol II, increases in promoter H3K4me3 histone marks and increased gene transcription. Linking transcription factors, PcG proteins and paused Pol II states, these data identify a two-step mechanism of Hox-driven transcription, with M1BP binding leading to Pol II recruitment followed by AbdA targeting, which results in a change in the chromatin landscape and enhanced transcription.

Role of Homothorax in region specific regulation of Deformed in embryonic neuroblasts.

The expression and regulation of Hox genes in developing central nervous system (CNS) lack important details like specific cell types where Hox genes are expressed and the transcriptional regulatory players involved in these cells. In this study we have investigated the expression and regulation of Drosophila Hox gene Deformed (Dfd) in specific cell types of embryonic CNS. Using Dfd neural autoregulatory enhancer we find that Dfd autoregulates itself in cells of mandibular neuromere. We have also investigated the role of a Hox cofactor Homothorax (Hth) for its role in regulating Dfd expression in CNS. We find that Hth exhibits a region specific role in controlling the expression of Dfd, but has no direct role in mandibular Dfd neural autoregulatory circuit. Our results also suggest that homeodomain of Hth is not required for regulating Dfd expression in embryonic CNS.