Highly reliable creation of floxed alleles by electroporating single-cell embryos.

BACKGROUND: Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking. RESULTS: Here, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specific and efficient second-round targeting ensures the generation of floxed F1 animals in roughly five months for a wide range of sequence lengths (448 bp to 160 kb reported here). CONCLUSIONS: Floxed alleles can be reliably obtained in a predictable timeline using the improved method of electroporation of two gRNA/Cas9 ribonucleoprotein particles (RNPs) and two ssODNs.

Lrp1 is a host entry factor for Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.

The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program to control neurite outgrowth.

The mitotic deacetylase complex (MiDAC) is a recently identified histone deacetylase (HDAC) complex. While other HDAC complexes have been implicated in neurogenesis, the physiological role of MiDAC remains unknown. Here, we show that MiDAC constitutes an important regulator of neural differentiation. We demonstrate that MiDAC functions as a modulator of a neurodevelopmental gene expression program and binds to important regulators of neurite outgrowth. MiDAC upregulates gene expression of pro-neural genes such as those encoding the secreted ligands SLIT3 and NETRIN1 (NTN1) by a mechanism suggestive of H4K20ac removal on promoters and enhancers. Conversely, MiDAC inhibits gene expression by reducing H3K27ac on promoter-proximal and -distal elements of negative regulators of neurogenesis. Furthermore, loss of MiDAC results in neurite outgrowth defects that can be rescued by supplementation with SLIT3 and/or NTN1. These findings indicate a crucial role for MiDAC in regulating the ligands of the SLIT3 and NTN1 signaling axes to ensure the proper integrity of neurite development.

A Survey of Validation Strategies for CRISPR-Cas9 Editing.

The T7 endonuclease 1 (T7E1) mismatch detection assay is a widely used method for evaluating the activity of site-specific nucleases, such as the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. To determine the accuracy and sensitivity of this assay, we compared the editing estimates derived by the T7E1 assay with that of targeted next-generation sequencing (NGS) in pools of edited mammalian cells. Here, we report that estimates of nuclease activity determined by T7E1 most often do not accurately reflect the activity observed in edited cells. Editing efficiencies of CRISPR-Cas9 complexes with similar activity by T7E1 can prove dramatically different by NGS. Additionally, we compared editing efficiencies predicted by the Tracking of Indels by Decomposition (TIDE) assay and the Indel Detection by Amplicon Analysis (IDAA) assay to that observed by targeted NGS for both cellular pools and single-cell derived clones. We show that targeted NGS, TIDE, and IDAA assays predict similar editing efficiencies for pools of cells but that TIDE and IDAA can miscall alleles in edited clones.

A distinct type of heterochromatin at the telomeric region of the Drosophila melanogaster Y chromosome.

Heterochromatin assembly and its associated phenotype, position effect variegation (PEV), provide an informative system to study chromatin structure and genome packaging. In the fruit fly Drosophila melanogaster, the Y chromosome is entirely heterochromatic in all cell types except the male germline; as such, Y chromosome dosage is a potent modifier of PEV. However, neither Y heterochromatin composition, nor its assembly, has been carefully studied. Here, we report the mapping and characterization of eight reporter lines that show male-specific PEV. In all eight cases, the reporter insertion sites lie in the telomeric transposon array (HeT-A and TART-B2 homologous repeats) of the Y chromosome short arm (Ys). Investigations of the impact on the PEV phenotype of mutations in known heterochromatin proteins (i.e., modifiers of PEV) show that this Ys telomeric region is a unique heterochromatin domain: it displays sensitivity to mutations in HP1a, EGG and SU(VAR)3-9, but no sensitivity to Su(z)2 mutations. It appears that the endo-siRNA pathway plays a major targeting role for this domain. Interestingly, an ectopic copy of 1360 is sufficient to induce a piRNA targeting mechanism to further enhance silencing of a reporter cytologically localized to the Ys telomere. These results demonstrate the diversity of heterochromatin domains, and the corresponding variation in potential targeting mechanisms.

Ectopic assembly of heterochromatin in Drosophila melanogaster triggered by transposable elements.

A persistent question in biology is how cis-acting sequence elements influence trans-acting factors and the local chromatin environment to modulate gene expression. We reported previously that the DNA transposon 1360 can enhance silencing of a reporter in a heterochromatic domain of Drosophila melanogaster. We have now generated a collection of variegating phiC31 landing-pad insertion lines containing 1360 and a heat-shock protein 70 (hsp70)-driven white reporter to explore the mechanism of 1360-sensitive silencing. Many 1360-sensitive sites were identified, some in apparently euchromatic domains, although all are close to heterochromatic masses. One such site (line 1198; insertion near the base of chromosome arm 2L) has been investigated in detail. ChIP analysis shows 1360-dependent Heterochromatin Protein 1a (HP1a) accumulation at this otherwise euchromatic site. The phiC31 landing pad system allows different 1360 constructs to be swapped with the full-length element at the same genomic site to identify the sequences that mediate 1360-sensitive silencing. Short deletions over sites with homology to PIWI-interacting RNAs (piRNAs) are sufficient to compromise 1360-sensitive silencing. Similar results were obtained on replacing 1360 with Invader4 (a retrotransposon), suggesting that this phenomenon likely applies to a broader set of transposable elements. Our results suggest a model in which piRNA sequence elements behave as cis-acting targets for heterochromatin assembly, likely in the early embryo, where piRNA pathway components are abundant, with the heterochromatic state subsequently propagated by chromatin modifiers present in somatic tissue.