Gene-edited Cell Lines Research Articles

The insufficiency of ATG4A in macroautophagy

During autophagy, LC3 and GABARAP proteins become covalently attached to phosphatidylethanolamine on the growing autophagosome. This attachment is also reversible. Deconjugation (or delipidation) involves the proteolytic cleavage of an isopeptide bond between LC3 or GABARAP and the phosphatidylethanolamine headgroup. This cleavage is carried about by the ATG4 family of proteases (ATG4A, B, C, and D). Many studies have established that ATG4B is the most active of these proteases and is sufficient for autophagy progression in simple cells. Here we examined the second most active protease, ATG4A, to map out key regulatory motifs on the protein and to establish its activity in cells. We utilized fully in vitro reconstitution systems in which we controlled the attachment of LC3/GABARAP members and discovered a role for a C-terminal LC3-interacting region on ATG4A in regulating its access to LC3/GABARAP. We then used a gene-edited cell line in which all four ATG4 proteases have been knocked out to establish that ATG4A is insufficient to support autophagy and is unable to support GABARAP proteins removal from the membrane. As a result, GABARAP proteins accumulate on membranes other than mature autophagosomes. These results suggest that to support efficient production and consumption of autophagosomes, additional factors are essential including possibly ATG4B itself or one of its proteolytic products in the LC3 family.

Loss of 5'-Methylthioadenosine Phosphorylase (MTAP) is Frequent in High-Grade Gliomas; Nevertheless, it is Not Associated with Higher Tumor Aggressiveness.

The 5’-methylthioadenosine phosphorylase (MTAP) gene is located in the chromosomal region 9p21. MTAP deletion is a frequent event in a wide variety of human cancers; however, its biological role in tumorigenesis remains unclear. The purpose of this study was to characterize the MTAP expression profile in a series of gliomas and to associate it with patients’ clinicopathological features. Moreover, we sought to evaluate, through glioma gene-edited cell lines, the biological impact of MTAP in gliomas. MTAP expression was evaluated in 507 glioma patients by immunohistochemistry (IHC), and the expression levels were associated with patients’ clinicopathological features. Furthermore, an in silico study was undertaken using genomic databases totalizing 350 samples. In glioma cell lines, MTAP was edited, and following MTAP overexpression and knockout (KO), a transcriptome analysis was performed by NanoString Pan-Cancer Pathways panel. Moreover, MTAP’s role in glioma cell proliferation, migration, and invasion was evaluated. Homozygous deletion of 9p21 locus was associated with a reduction of MTAP mRNA expression in the TCGA (The Cancer Genome Atlas) - glioblastoma dataset (p < 0.01). In addition, the loss of MTAP expression was markedly high in high-grade gliomas (46.6% of cases) determined by IHC and Western blotting (40% of evaluated cell lines). Reduced MTAP expression was associated with a better prognostic in the adult glioblastoma dataset (p < 0.001). Nine genes associated with five pathways were differentially expressed in MTAP-knockout (KO) cells, with six upregulated and three downregulated in MTAP. Analysis of cell proliferation, migration, and invasion did not show any significant differences between MTAP gene-edited and control cells. Our results integrating data from patients as well as in silico and in vitro models provide evidence towards the lack of strong biological importance of MTAP in gliomas. Despite the frequent loss of MTAP, it seems not to have a clinical impact in survival and does not act as a canonic tumor suppressor gene in gliomas.

79 CRISPR gene editing in bovine zygotes — mutation confirmation by integration of protein expression and DNA sequencing analyses

Confirmation of gene editing in livestock following CRISPR/Cas9 zygotic microinjection is often limited to either protein expression or gene sequencing analyses of unfixed embryos, but not both, due to the challenges of extracting DNA from fixed samples suitable for genotyping. Bovine embryos have been derived from gene-edited cell lines to screen for mutations followed by nuclear transfer, but these techniques limit embryo production efficiency, include technical challenges, and alter physiological relevance. Herein we report an integrative approach to evaluate both protein expression and the genotype of individual, fixed bovine embryos subjected to CRISPR/Cas9 microinjection. Bovine zygotes were derived from IVF and parthenogenetic activation of in vitro-matured oocytes followed by intracytoplasmic injection of CRISPR/Cas9 targeting embryonic POU5F1. Embryos were cultured for 7 days and fixed in 4% paraformaldehyde and stored at 5°C for up to 4 weeks. Fixed embryos were first subjected to immunohistochemistry to determine POU5F1 mutation success by protein expression. Binder reinforcement labels were affixed to glass slides as a substitute for adhesive spacers. Single embryos were added to 9µL of PBS with a coverslip for imaging. Individual embryos were then recovered and placed in 10µL of QuickExtract (Lucigen, Middleton, WI, USA) for DNA extraction by heating samples to 65°C for 6min and 95°C for 2min. Two rounds of PCR were applied to templates in 20-µL reactions consisting of 10µL of GoTaq Hot Start Green Master Mix (2×; Promega, Madison, WI, USA) with the addition of 0.4µL (10µM) of forward and reverse primer each and 9.2µL of template. The second PCR reaction contained 5µL of PCR product from the first reaction, nested primers, and 4.2µL of H2O. The PCR conditions were |95°C, 3 min| and 35 cycles of |95°C, 30s |56°C, 30 s|72°C, 30 s|72°C, 7 min|. The PCR product was run on an agarose gel to confirm DNA amplification of a single band, and the remainder was purified (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany) and submitted (100ng) for Sanger sequencing. The CRISPR-injected embryos were aligned to wild-type embryo sequences using SnapGene (http://www.snapgene.com/) and TIDE software (https://tide.nki.nl/). Both DNA extraction (n=18/19) and Sanger sequencing (n=19/23) proved highly efficient and repeatable (83 and 95%, respectively). These methods provide an efficient, cost effective, and reproducible approach for confirmation of CRISPR gene editing in individual and fixed bovine embryos by both protein expression and gene sequencing. In addition, these techniques complement the production of gene editing in livestock after zygotic CRISPR microinjection by conclusively determining mutation efficiency through both proteomic and genomic analyses. This research was supported by a USDA NIFA AFRI ELI Postdoctoral Fellowship 2016-67012-25254 to BWD and NIFA multistate research project W3171 to PJR. BD is currently supported by a grant from the Eunice Kennedy Shiver National Institute of Child Health and Human Development of the National Institutes of Health under the award numbers T32HD087166.

CRISPR editing validation, immunostaining and DNA sequencing of individual fixed bovine embryos.

CRISPR technologies used for mammalian embryology have wide implications from basic research to applications in agriculture and biomedicine. Confirmation of successful gene editing following CRISPR/Cas9 delivery is often limited to either protein expression or sequencing analyses of embryos but not both, due to technical challenges. Herein we report an integrative approach for evaluating both protein expression and genotype of single embryos from fixed bovine embryos previously subjected to CRISPR/Cas9 microinjection. The techniques described facilitate investigation of functional genomics in bovine embryos compatible with gene editing in livestock after zygotic CRISPR microinjection. These methods avoid traditional avenues that necessitate the use of gene-edited cell lines followed by nuclear transfer that hinder efficiency, limit physiological relevance and contribute to technical challenges.

PO-170 Dissecting the role of IFITM1 in response to anticancer treatment

IntroductionConstitutive expression of interferons (IFNs) and their downstream signalling pathways play a critical role in host responses to cell transformation in the tumour microenvironment. Induction of IFNs initiates the transcription of a variety of genes, so called IFN- stimulated genes (ISGs). Although expression of ISGs is classically associated with tumour suppression, a subset defined as the IRDS (Interferon - Related DNA- Damage Resistance Signature), is elevated in response to endogenous IFN, self-DNA and RNA exposure in the tumour microenvironment acquiring radiation and chemotherapy resistance. INF induced transmembrane receptor (IFITM1) is thought to participate in proliferating signalling and oncogenesis. Three out of five IFITM genes (IFITM1,2,3) share high amino-acid homology, however, IFITM1 has a unique C-terminal domain, a 13 amino-acid extension, and a shorter N-terminal amino-acid sequence.Material and methodsTo analyse the impact of IFITM1 loss, or mutation, on signalling and phenotypic events such as growth, viability and drug resistance, knock – out, knock – in and domain mutants were generated using Crispr/Cas9 mediated gene – editing. To identify the effects of radiation in the growth of wild type and mutant IFITM1 cells, proliferation assays were conducted while immunoassays were used to check for activation of the IRDS pathway. The gene edited cell lines were used in Mass Spectrometry proteomic approaches to discover IFITM1 interacting proteins under normal growth conditions as well as in INF treated and/or radiated cells.Results and discussionsIrradiation of glioma stem cells, results in a substantial increase in the expression of IFITM1 and an activation of the IRDS pathway which suggests a potential mechanism by which cancer stem cells escape chemotherapy. Deletion of IFITM1 in SiHa cells results in sensitivity to chemo- and radiation therapy, while loss of both IFITM1 and IFITM3 function generates chemoresistant cancer cells suggesting a potential interaction between IFITM1 and IFITM3.Structure-function analysis has shown that the C-terminal regulatory domain of IFITM1 is required for its ability to promote cell growth and to localise to the membrane.ConclusionWe have identified IFITM1 as an upstream regulator of the IRDS which promotes cancer cell survival and mediates chemoresistance. The C-terminal domain of IFITM1 is important for its proliferative activity in cancer and lay the foundation for future research aiming to determine IFITM1’s potential as a therapeutic target in cancer.

Correction of PTEN mutations in glioblastoma cell lines via AAV-mediated gene editing.

PTEN is among the most commonly mutated tumor suppressor genes in human cancer. However, studying the role of PTEN in the pathogenesis of cancer has been limited, in part, by the paucity of human cell-based isogenic systems that faithfully model PTEN loss. In an effort to remedy this problem, gene editing was used to correct an endogenous mutant allele of PTEN in two human glioblastoma multiforme (GBM) cell lines– 42MGBA and T98G. PTEN correction resulted in reduced cellular proliferation that was Akt-dependent in 42MGBA cells and Akt-independent in T98G cells. This is the first report of human cancer cell lines in which mutant PTEN has been corrected by gene editing. The isogenic sets of gene edited cell lines reported here will likely prove useful for further study of PTEN mutations in the pathogenesis of cancer, and for the discovery and validation of novel therapeutics targeting the PTEN pathway.

A Cas9 Variant for Efficient Generation of Indel-Free Knockin or Gene-Corrected Human Pluripotent Stem Cells.

SummaryWhile Cas9 nucleases permit rapid and efficient generation of gene-edited cell lines, the CRISPR-Cas9 system can introduce undesirable “on-target” mutations within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address this, we fused the Streptococcus pyogenes Cas9 (SpCas9) nuclease to a peptide derived from the human Geminin protein (SpCas9-Gem) to facilitate its degradation during the G1 phase of the cell cycle, when DNA repair by NHEJ predominates. We also use mRNA transfection to facilitate low and transient expression of modified and unmodified versions of Cas9. Although the frequency of homologous recombination was similar for SpCas9-Gem and SpCas9, we observed a marked reduction in the capacity for SpCas9-Gem to induce NHEJ-mediated indels at the target locus. Moreover, in contrast to native SpCas9, we demonstrate that transient SpCas9-Gem expression enables reliable generation of both knockin reporter cell lines and genetically repaired patient-specific induced pluripotent stem cell lines free of unwanted mutations at the targeted locus.

575. High Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells

Gene-edited human cells are important resources for drug target identification, regulatory science, regenerative medicine and basic biology. Recently, use of the CRISPR-Cas9 system drastically cut the time required to produce gene-edited cell lines down to a few months. Targeted gene disruption in a population of human cells followed by selection and next-generation sequencing can identify drug targets, however many of these methods destroy all mutant clones, so a subsequent gene-editing experiment is required to obtain living mutant cells for downstream analysis. Overall, there is a need to increase the speed, multiplexing and precision in generating CRISPR-Cas9 mutants. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs (see FigureFigure), separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells from all three major germ layers. This allows for the real-time observation of mutations causing phenotypic differences that can be measured in in vitro tissues and organoids during culture as opposed to defined end points. The live cell and tissue assays on ArrayEdit are compatible with standard screening platforms and a variety of human cell types, thus permitting robust and sensitive detection of differences in proliferation, differentiation and other downstream effects of gene editing. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.FigureArrayEdit provides a new window into the process of gene editing human cells. Overview of ArrayEdit assembly and key components. Top: Schematic of one-pot PCR and T7 transcription. Bottom: Surface modification to the bottom of multiwell plates generates cell-adhesive µFeatures on a glass bottom. Each µFeature can be tracked over time via high-content imaging and stitched together to form a time-lapse visualization of edited cell phenotypes.View Large Image | Download PowerPoint Slide