The Application of CRISPR Technology to High Content Screening in Primary Neurons

Axon growth is coordinated by multiple interacting proteins that remain incompletely characterized. High content screening (HCS), in which manipulation of candidate genes is combined with rapid image analysis of phenotypic effects, has emerged as a powerful technique to identify key regulators of axon outgrowth. Here we explore the utility of a genome editingapproach referred to as CRISPR (Clustered Regularly Interspersed Palindromic Repeats) for knockout screening in primary neurons. In the CRISPR approach a DNA-cleaving Cas enzyme is guided to genomic target sequences by user-created guide RNA (sgRNA), where it initiates a double-stranded break that ultimately results in frameshift mutation and loss of protein production. Using electroporation of plasmid DNA that co-expresses Cas9enzyme and sgRNA, we first verified the ability of CRISPR targeting to achieve protein-level knockdown in cultured postnatal cortical neurons. Targeted proteins included NeuN (RbFox3) and PTEN, a well-studied regulator of axon growth. Effective knockdown lagged at least four days behind transfection, but targeted proteins were eventually undetectable by immunohistochemistry in \u3e 80% of transfected cells. Consistent with this, anti-PTEN sgRNA produced no changes in neurite outgrowth when assessed three days post-transfection. When week-long cultures were replated, however, PTEN knockdown consistently increased neurite lengths. These CRISPR-mediated PTEN effects were achieved using multi-well transfection and automated phenotypic analysis, indicating the suitability of PTEN as a positive control for future CRISPR-based screening efforts. Combined, these data establish an example of CRISPR-mediated protein knockdown in primary cortical neurons and its compatibility with HCS workflows

Persistence of RNAi-Mediated Knockdown in Drosophila Complicates Mosaic Analysis Yet Enables Highly Sensitive Lineage Tracing.

RNA interference (RNAi) has emerged as a powerful way of reducing gene function in Drosophila melanogaster tissues. By expressing synthetic short hairpin RNAs (shRNAs) using the Gal4/UAS system, knockdown is efficiently achieved in specific tissues or in clones of marked cells. Here we show that knockdown by shRNAs is so potent and persistent that even transient exposure of cells to shRNAs can reduce gene function in their descendants. When using the FLP-out Gal4 method, in some instances we observed unmarked "shadow RNAi" clones adjacent to Gal4-expressing clones, which may have resulted from brief Gal4 expression following recombination but prior to cell division. Similarly, Gal4 driver lines with dynamic expression patterns can generate shadow RNAi cells after their activity has ceased in those cells. Importantly, these effects can lead to erroneous conclusions regarding the cell autonomy of knockdown phenotypes. We have investigated the basis of this phenomenon and suggested experimental designs for eliminating ambiguities in interpretation. We have also exploited the persistence of shRNA-mediated knockdown to design a sensitive lineage-tracing method, i-TRACE, which is capable of detecting even low levels of past reporter expression. Using i-TRACE, we demonstrate transient infidelities in the expression of some cell-identity markers near compartment boundaries in the wing imaginal disc

Design of RNAi reagents for invertebrate model organisms and human disease vectors

RNAi has become an important tool to silence gene expression in a variety of organisms, in particular when classical genetic methods are missing. However, application of this method in functional studies has raised new challenges in the design of RNAi reagents in order to minimize false positive and false negative results. Since the performance of reagents can be rarely validated on a genome-wide scale, improved computational methods are required that consider experimentally derived design parameters. Here, we describe computational methods for the design of RNAi reagents for invertebrate model organisms and human disease vectors, such as Anopheles. We describe procedures on how to design short and long double-stranded RNAs for single genes, and evaluate their predicted specificity and efficiency. Using a bioinformatics pipeline we also describe how to design a genome-wide RNAi library for Anopheles gambiae

Design of small molecule-responsive microRNAs based on structural requirements for Drosha processing

MicroRNAs (miRNAs) are prevalent regulatory RNAs that mediate gene silencing and play key roles in diverse cellular processes. While synthetic RNA-based regulatory systems that integrate regulatory and sensing functions have been demonstrated, the lack of detail on miRNA structure–function relationships has limited the development of integrated control systems based on miRNA silencing. Using an elucidated relationship between Drosha processing and the single-stranded nature of the miRNA basal segments, we developed a strategy for designing ligand-responsive miRNAs. We demonstrate that ligand binding to an aptamer integrated into the miRNA basal segments inhibits Drosha processing, resulting in titratable control over gene silencing. The generality of this control strategy was shown for three aptamer–small molecule ligand pairs. The platform can be extended to the design of synthetic miRNAs clusters, cis-acting miRNAs and self-targeting miRNAs that act both in cis and trans, enabling fine-tuning of the regulatory strength and dynamics. The ability of our ligand-responsive miRNA platform to respond to user-defined inputs, undergo regulatory performance tuning and display scalable combinatorial control schemes will help advance applications in biological research and applied medicine

Silencing disease genes in the laboratory and the clinic

Synthetic nucleic acids are commonly used laboratory tools for modulating gene expression and have the potential to be widely used in the clinic. Progress towards nucleic acid drugs, however, has been slow and many challenges remain to be overcome before their full impact on patient care can be understood. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are the two most widely used strategies for silencing gene expression. We first describe these two approaches and contrast their relative strengths and weaknesses for laboratory applications. We then review the choices faced during development of clinical candidates and the current state of clinical trials. Attitudes towards clinical development of nucleic acid silencing strategies have repeatedly swung from optimism to depression during the past 20 years. Our goal is to provide the information needed to design robust studies with oligonucleotides, making use of the strengths of each oligonucleotide technology

Expression of Multiple Artificial MicroRNAs from a Chicken miRNA126-Based Lentiviral Vector

Background: The use of RNAi in both basic and translational research often requires expression of multiple siRNAs from the same vector. Methods/Principal Findings: We have developed a novel chicken miR126-based artificial miRNA expression system that can express one, two or three miRNAs from a single cassette in a lentiviral vector. We show that each of the miRNAs expressed from the same lentiviral vector is capable of potent inhibition of reporter gene expression in transient transfection and stable integration assays in chicken fibroblast DF-1 cells. Transduction of Vero cells with lentivirus expressing two or three different anti-influenza miRNAs leads to inhibition of influenza virus production. In addition, the chicken miR126-based expression system effectively inhibits reporter gene expression in human, monkey, dog and mouse cells. These results demonstrate that the flanking regions of a single primary miRNA can support processing of three different stem-loops in a single vector. Conclusions/Significance: This novel design expands the means to express multiple miRNAs from the same vector for potent and effective silencing of target genes and influenza virus.National Institutes of Health (U.S.) (Grant R01AI056267)Cobb-Vantress, inc

Dynamic telomerase gene suppression via network effects of GSK3 inhibition

<b>Background</b>: Telomerase controls telomere homeostasis and cell immortality and is a promising anti-cancer target, but few small molecule telomerase inhibitors have been developed. Reactivated transcription of the catalytic subunit hTERT in cancer cells controls telomerase expression. Better understanding of upstream pathways is critical for effective anti-telomerase therapeutics and may reveal new targets to inhibit hTERT expression. <b>Methodology/Principal Findings</b>: In a focused promoter screen, several GSK3 inhibitors suppressed hTERT reporter activity. GSK3 inhibition using 6-bromoindirubin-3′-oxime suppressed hTERT expression, telomerase activity and telomere length in several cancer cell lines and growth and hTERT expression in ovarian cancer xenografts. Microarray analysis, network modelling and oligonucleotide binding assays suggested that multiple transcription factors were affected. Extensive remodelling involving Sp1, STAT3, c-Myc, NFκB, and p53 occurred at the endogenous hTERT promoter. RNAi screening of the hTERT promoter revealed multiple kinase genes which affect the hTERT promoter, potentially acting through these factors. Prolonged inhibitor treatments caused dynamic expression both of hTERT and of c-Jun, p53, STAT3, AR and c-Myc. <b>Conclusions/Significance</b>: Our results indicate that GSK3 activates hTERT expression in cancer cells and contributes to telomere length homeostasis. GSK3 inhibition is a clinical strategy for several chronic diseases. These results imply that it may also be useful in cancer therapy. However, the complex network effects we show here have implications for either setting

Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases.

We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or in vitro transcribed gRNA (gR.1). Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs

Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements.

Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements

Multiplex Gene Editing in Hepatocytes as a Therapeutic Approach for Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is a genetic disorder associated with elevated levels of low-density lipoprotein cholesterol (LDL-C) that increases the risk of atherosclerotic cardiovascular disease. FH affects 1 in 250 people.1 Most FH cases are caused by loss-of-function mutations in the either one or both copies of the LDLR gene, which encodes for the LDL-C receptor protein and is responsible for removing excess LDL from the circulating blood plasma.2 Current treatments for FH include medications that reduce LDL from the bloodstream. Angiopoietin-like 3 (ANGPTL3) is a lipoprotein lipase inhibitor. Studies show that loss-of-function mutations in ANGPTL3 are associated with reduced plasma LDL-C, triglyceride, and high-density lipoprotein levels. Scientists are currently investigating therapeutic drugs targeting the inhibition of ANGPTL3 for management of atherosclerosis. Evinacumab is an FDA approved monoclonal antibody against ANGPTL3 for the management of homozygous FH. It is administered every four weeks by IV infusion for 60 minutes.3 We propose an ex vivo gene therapy in hepatocytes that are isolated from a patient’s liver, followed by transplantation of gene edited cells to repopulate the liver with healthy hepatocytes. CRISPR-Cas can allow for a one-time curative treatment for FH by disrupting ANGPTL3 in hepatocytes. A challenge in ex vivo gene editing is selecting for edited cells when transplanted to the liver, since ANGPTL3-deficient hepatocytes will not have a selective advantage over native cells. Acetaminophen (APAP) selection is a novel method that can be used to provide this selective advantage for edited cells to repopulate the liver. Knockdown of ANGPTL3 along with NADPH-cytochrome p450 reductase (CYPOR), followed by transient APAP administration can be used to select for edited cells.4 Therefore, we propose to use Cas9 and Cas12a for multiplex gene knockdown in ANGPTL3 and CYPOR. The combination of Cas9 and Cas12a is beneficial because both endonucleases make different types of cuts in the DNA, which lowers the probability for unwanted translocation events in the genome and increases the safety of multiplex gene editing. The first aim of this study is to optimize the design of CRISPR-Cas12a gRNAs targeting Angptl3 in Hepa 1-6 cells and quantify editing efficiency. This study also evaluates the specificity of each guide design using Cas-OFFinder. The second aim is to optimize the multiplex delivery of CRISPR-Cas12a and Cas9 ribonucleoproteins (RNPs) to target Angptl3 and Cypor in Hepa 1-6 cells