Genome-Scale CRISPR Screens Identify Human Pluripotency-Specific Genes

Human pluripotent stem cells (hPSCs) generate a variety of disease-relevant cells that can be used to improve the translation of preclinical research. Despite the potential of hPSCs, their use for genetic screening has been limited by technical challenges. We developed a scalable and renewable Cas9 and sgRNA-hPSC library in which loss-of-function mutations can be induced at will. Our inducible mutant hPSC library can be used for multiple genome-wide CRISPR screens in a variety of hPSC-induced cell types. As proof of concept, we performed three screens for regulators of properties fundamental to hPSCs: their ability to self-renew and/or survive (fitness), their inability to survive as single-cell clones, and their capacity to differentiate. We identified the majority of known genes and pathways involved in these processes, as well as a plethora of genes with unidentified roles. This resource will increase the understanding of human development and genetics. This approach will be a powerful tool to identify disease-modifying genes and pathways

Editing the genome of chicken primordial germ cells to introduce alleles and study gene function

With continuing advances in genome sequencing technology, the chicken genome assembly is now better annotated with improved accuracy to the level of single nucleotide polymorphisms. Additionally, the genomes of other birds such as the duck, turkey and zebra finch have now been sequenced. A great opportunity exists in avian biology to use genome editing technology to introduce small and defined sequence changes to create specific haplotypes in chicken to investigate gene regulatory function, and also perform rapid and seamless transfer of specific alleles between chicken breeds. The methods for performing such precise genome editing are well established for mammalian species but are not readily applicable in birds due to evolutionary differences in reproductive biology. A significant leap forward to address this challenge in avian biology was the development of long-term culture methods for chicken primordial germ cells (PGCs). PGCs present a cell line in which to perform targeted genetic manipulations that will be heritable. Chicken PGCs have been successfully targeted to generate genetically modified chickens. However, genome editing to introduce small and defined sequence changes has not been demonstrated in any avian species. To address this deficit, the application of CRISPR/Cas9 and short oligonucleotide donors in chicken PGCs for performing small and defined sequence changes was investigated in this thesis. Specifically, homology-directed DNA repair (HDR) using oligonucleotide donors along with wild-type CRISPR/Cas9 (SpCas9-WT) or high fidelity CRISPR/Cas9 (SpCas9-HF1) was investigated in cultured chicken PGCs. The results obtained showed that small sequences changes ranging from a single to a few nucleotides could be precisely edited in many loci in chicken PGCs. In comparison to SpCas9-WT, SpCas9-HF1 increased the frequency of biallelic and single allele editing to generate specific homozygous and heterozygous genotypes. This finding demonstrates the utility of high fidelity CRISPR/Cas9 variants for performing sequence editing with high efficiency in PGCs. Since PGCs can be converted into pluripotent stem cells that can potentially differentiate into many cell types from the three germ layers, genome editing of PGCs can, therefore, be used to generate PGC-derived avian cell types with defined genetic alterations to investigate the host-pathogen interactions of infectious avian diseases. To investigate this possibility, the chicken ANP32A gene was investigated as a target for genetic resistance to avian influenza virus in PGC-derived chicken cell lines. Targeted modification of ANP32A was performed to generate clonal lines of genome-edited PGCs. Avian influenza minigenome replication assays were subsequently performed in the ANP32A-mutant PGC-derived cell lines. The results verified that ANP32A function is crucial for the function of both avian virus polymerase and human-adapted virus polymerase in chicken cells. Importantly, an asparagine to isoleucine mutation at position 129 (N129I) in chicken ANP32A failed to support avian influenza polymerase function. This genetic change can be introduced into chickens and validated in virological studies. Importantly, the results of my investigation demonstrate the potential to use genome editing of PGCs as an approach to generate many types of unique cell models for the study of avian biology. Genome editing of PGCs may also be applied to unravel the genes that control the development of the avian germ cell lineage. In the mouse, gene targeting has been extensively applied to generate loss-of-function mouse models to use the reverse genetics approach to identify key genes that regulate the migration of specified PGCs to the genital ridges. Avian PGCs express similar cytokine receptors as their mammalian counterparts. However, the factors guiding the migration of avian PGCs are largely unknown. To address this, CRISPR/Cas9 was used in this thesis to generate clonal lines of chicken PGCs with loss-of-function deletions in the CXCR4 and c-Kit genes which have been implicated in controlling mouse PGC migration. The results showed that CXCR4-deficient PGCs are absent from the gonads whereas c-Kit-deficient PGCs colonise the developing gonads in reduced numbers and are significantly reduced or absent from older stages. This finding shows a conserved role for CXCR4 and c-Kit signalling in chicken PGC development. Importantly, other genes suspected to be involved in controlling the development of avian germ cells can be investigated using this approach to increase our understanding of avian reproductive biology. Finally, the methods developed in this thesis for editing of the chicken genome may be applied in other avian species once culture methods for the PGCs from these species are develope

Translational Control by the DEAD Box RNA Helicase <em>belle</em> Regulates Ecdysone-Triggered Transcriptional Cascades

<div><p>Steroid hormones act, through their respective nuclear receptors, to regulate target gene expression. Despite their critical role in development, physiology, and disease, however, it is still unclear how these systemic cues are refined into tissue-specific responses. We identified a mutation in the evolutionarily conserved DEAD box RNA helicase <em>belle/DDX3</em> that disrupts a subset of responses to the steroid hormone ecdysone during <em>Drosophila melanogaster</em> metamorphosis. We demonstrate that <em>belle</em> directly regulates translation of <em>E74A</em>, an ets transcription factor and critical component of the ecdysone-induced transcriptional cascade. Although <em>E74A</em> mRNA accumulates to abnormally high levels in <em>belle</em> mutant tissues, no E74A protein is detectable, resulting in misregulation of E74A-dependent ecdysone response genes. The accumulation of <em>E74A</em> mRNA in <em>belle</em> mutant salivary glands is a result of auto-regulation, fulfilling a prediction made by Ashburner nearly 40 years ago. In this model, Ashburner postulates that, in addition to regulating secondary response genes, protein products of primary response genes like <em>E74A</em> also inhibit their own ecdysone-induced transcription. Moreover, although ecdysone-triggered transcription of <em>E74A</em> appears to be ubiquitous during metamorphosis, <em>belle</em>-dependent translation of <em>E74A</em> mRNA is spatially restricted. These results demonstrate that translational control plays a critical, and previously unknown, role in refining transcriptional responses to the steroid hormone ecdysone.</p> </div

Transcription of <i>E74A</i> is not properly repressed in <i>belle</i> salivary glands.

<p>Fluorescent <i>in situ</i> hybridizations for <i>E74A</i> mRNA in control (A–C) and <i>bel^psg9</i> mutant (D–F) salivary glands shown in red with DAPI costained nuclei in blue. Two hours before head eversion (−2 AHE), <i>E74A</i> mRNA is detected as strong nuclear bands indicative of active transcription at ecdysone-induced chromosomal puffs in both control (A) and mutant (D) glands. In control glands at head eversion (HE) (B), <i>E74A</i> mRNA is primarily cytoplasmic and nuclear puff staining is no longer detectable. In contrast, <i>bel^psg9</i> mutant glands at HE (E) show continued nuclear puff staining (arrowheads) and increased cytoplasmic staining. At +1.5 AHE, <i>E74A</i> mRNA is barely detectable in control glands (C). Similarly staged mutant glands (F) show continued accumulation of cytoplasmic <i>E74A</i> RNA. HE: head eversion.</p

Translational control determines spatial distribution of E74A protein.

<p>Tissues at the onset of metamorphosis were dissected and imaged to detect <i>E74A</i> mRNA (in red, first column), E74A protein (in red, second through fifth columns) or Belle protein (in green, fifth column). Although ecdysone-induced transcription of <i>E74A</i> mRNA appears to be ubiquitously expressed (first column), E74A protein is not (second column). This spatial distribution of E74A protein is disrupted in <i>bel^psg9</i> mutant animals (third column) and becomes ubiquitous after expression of E74A protein from the <i>hs-E74A</i> transgene (fourth column). E74A protein is detected in the large cells of the proventriculus (G) and <i>repo</i>-positive glial cells in the ventral nerve cord (L and data not shown), but absent in the entire imaginal wing discs (B), the imaginal ring of the proventriculus (G) and neurons in the ventral nerve cord (L). Endogenous Belle protein, visualized with the <i>bel^GFP-ZCL1911</i> exon trap, is expressed in all cells including those that do not express E74A protein (fifth column). Staining with antibodies directed to Belle protein show identical expression patterns (data not shown). For <i>in situ</i> hybridizations, tissues were dissected from animals a few hours before puparium formation (clear gut stage) when <i>E74A</i> mRNA levels are their highest. For E74A protein and BEL-GFP imaging, tissues were dissected at puparium formation when E74A protein levels are at their highest. DAPI costained nuclei in blue. WD: imaginal wing discs, PV: proventriculus, VNC: ventral nerve cords.</p

Mutation in <i>belle</i> disrupts a subset of ecdysone-triggered responses during metamorphosis.

<p>(A) Schematic of conserved motifs in the DEAD-box RNA helicase (adapted from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003085#pgen.1003085-Linder1" target="_blank">[8]</a>) and sequence alignment of the DEAD-box containing motif II and the <i>belle/DDX3</i> family-specific post II region. <i>bel^psg9</i> has a missense mutation that changes a phenylalanine to a serine at residue 469 within the post II region. <i>bel⁶</i> has a nonsense mutation at residue 288 introducing an early stop codon. (B–C) Live pupae at 12 AHE with salivary gland specific expression of GFP. Salivary gland GFP (<i>fkh-GAL4</i>, <i>UAS-GFP</i>) is no longer present in control animals at 12 AHE (B) but persists in similarly staged <i>bel^psg9</i> mutant glands (C), indicating a block in the ecdysone-triggered destruction of this tissue. (D–E) Control (D) and <i>bel^psg9</i> mutant (E) pupae dissected out of their pupal cases 12 hours after head eversion (AHE). Both control and mutant pupae have everted heads and fully extended legs and wings, suggesting that the global ecdysone-induced genetic hierarchy is intact. (F) Lethal phase analysis of <i>bel^psg9</i> hemizygous animals. Most (70%) <i>bel^psg9</i> mutant animals arrest as newly formed pupae (n = 215). PP: prepupae, P: pupae, PA: pharate adults, A: adults eclosed.</p

<i>belle</i> selectively disrupts regulation of the ecdysone early response gene <i>E74A</i>.

<p>qPCR analysis of ecdysone hierarchy genes in control (solid lines) and <i>bel^psg9</i> mutant (dashed lines) larval salivary glands. Expression profiles of the ecdysone hierarchy genes <i>E75A</i>, <i>BR-C</i>, <i>E93</i>, <i>FTZ-F1 and E74B</i> are relatively normal in <i>bel^psg9</i> mutant salivary glands. In contrast, the early response gene <i>E74A</i> is not properly repressed. y-axis plots relative expression, normalized to <i>rp49</i>; x-axis plots developmental stage relative to head eversion. Each time point represents three independently isolated salivary gland samples. AHE: after head eversion.</p

E74A protein is sufficient to inhibit its own ecdysone-induced transcription.

<p>(A) Experimental paradigm for expressing E74A protein prior to the prepupal pulse of ecdysone. Expression profile of endogenous <i>E74A</i> mRNA (in gray) and E74A protein (in blue) in salivary glands shown relative to hours after head eversion (AHE). Arrows mark timing of the 30 minute heat-shock treatment and the subsequent dissection of larval salivary glands. (B) Precocious expression of E74A protein (gray bars) is sufficient to repress induction of endogenous <i>E74A</i> mRNA in control and <i>bel^psg9</i> mutant salivary glands. Control salivary glands show over 100-fold induction of <i>E74A</i> in response to the prepupal pulse of ecdysone (first bar from left). In contrast, ectopic expression of E74A protein (second bar), but not E75A protein (third bar), inhibits endogenous <i>E74A</i> transcription. Similarly, ectopic E74A protein inhibits the ecdysone-induced expression of <i>E74A</i> in <i>bel^psg9</i> mutant salivary glands (fourth and fifth bars). (C) Although precocious expression of E74A protein is sufficient to repress the ecdysone-induced transcription of <i>E74A</i>, it has a minor effect on the expression of ecdysone early response genes <i>E75A</i> and <i>BR-C Z1</i>. Salivary glands dissected from heat-shock-treated control (white bars) and <i>hs-E74A</i> carrying (gray bars) animals. (D) <i>E74A</i> is not necessary for the regression of other ecdysone early response genes. Salivary glands dissected from control (white bars) and <i>E74A^neo24/Df</i> mutant (gray bars) animals at +1.5 AHE, when endogenous expression of early response genes <i>E75A</i> and <i>BR-C</i> have regressed to levels prior to the ecdysone pulse. <i>E74A^neo24</i> allele is an RNA null, hence the barely detectable levels of endogenous <i>E74A</i> mRNA. y-axis plots relative expression, normalized to <i>rp49</i>. Expression ratios in 5B calculated relative to −4 AHE control samples to show induction in response to the prepupal pulse of ecdysone. All samples are in triplicate; asterisks indicate p-values <0.05. <i>bel/Df</i>: <i>bel^psg9/Df</i>. AHE: after head eversion.</p

Belle directly regulates <i>E74A</i> mRNA translation.

<p>(A–I) Salivary glands dissected from animals staged relative to head eversion and stained with antibodies directed to E74A protein shown in red (A–C and E–H) or to BROAD Z1 protein in green (D, I) with DAPI costained nuclei in blue. (A–C) In control salivary glands, E74A protein is detected at head eversion (HE)(B), but not 2 hours before HE (A) or 1.5 hours AHE (C) (N/A: control glands are too fragile to dissect after +1.5 AHE). (E–H) In <i>bel^psg9</i> mutant salivary glands, E74A protein is barely detectable at stages before, during or after head eversion. (D–I) In contrast, both control and <i>bel^psg9</i> mutant salivary glands express BROAD Z1 protein at head eversion. (J) Ribonucleoprotein (RNP) immunoprecipitation (RIP) experiments with BEL-GFP fusion protein, followed by qPCR analysis for target mRNAs. <i>E74A</i> and <i>BR-C Z1</i> transcripts showed significant (37-fold and 18-fold, respectively) enrichment in BEL-GFP containing RNP complexes, while <i>E75A</i> and <i>UbcD6</i> transcripts did not show significant enrichment. Data represent average qPCR results from three independent RIP experiments; asterisks indicate p-values <0.05. HE: head eversion, <i>Z1</i>: <i>BR-C Z1</i>.</p

Translational control by Belle regulates E74A-dependent ecdysone-triggered responses.

<p>A model of how Belle protein regulates a subset of ecdysone-triggered transcriptional responses. E74A protein has two functions: to induce expression of E74A-dependent target genes and to inhibit its own ecdysone-induced transcription. Given that Belle is required for translation of <i>E74A</i> mRNA, Belle activity effectively regulates E74A-dependent targets within the ecdysone-triggered transcriptional cascade. Moreover, Belle-dependent translational control of a ubiquitous transcriptional target of ecdysone like <i>E74A</i> occurs in a tissue-specific manner, adding a novel regulatory layer to steroid hormone signaling. Thus, Belle-dependent translational control helps refine global ecdysone signals into distinct tissue-specific transcriptional responses.</p