Gene Tagging Research Articles

Identification of prognostic immune subtypes of lung squamous cell carcinoma by unsupervised consistent clustering.

We performed UCC on the expression data of lung squamous cell carcinoma tumor samples to identify the classification of lung squamous cell carcinoma (LUSC) tumor samples, and calculated the levels of different classified immune cells by single-sample gene enrichment analysis (ssGSEA) to obtain a set of immune-related subtype gene tags, which can be used for subtype classification of lung squamous cell carcinoma. TCGA-LUSC and GSE30219 data of lung squamous cell carcinoma were obtained from TCGA and GEO databases. Prognostic-associated subtypes were identified by unsupervised consensus clustering (UCC). Using ssGSEA analysis to calculate the level of immune cells of different subtypes, obtain the connection between subtypes and immunity, identify the gene signatures recognized by subtypes, and verify this group of gene signatures through GSE30219. We effectively identified 2 subtypes that were significantly associated with prognostic survival by UCC, and calculated according to ssGSEA, the 2 subtypes were significantly different at the level of immune cells, followed by introducing a This weighted thinking computes a set of gene signatures that are significantly associated with subtype 1. During validation, this set of gene signatures could efficiently and robustly identify distinct prognostic immune subtypes, demonstrated the validity of this set of gene signatures, as well as 2 subtypes of lung squamous cell carcinoma. We used lung squamous cell carcinoma data from public databases and identified 2 prognostic immunosubtypes of lung squamous cell carcinoma and a set of gene tags that can be used to classify immune subtypes of lung squamous cell carcinoma, which may provide effective evidence for accurate clinical treatment of lung squamous cell carcinoma.

Peroxisomal NAD(H) Homeostasis in the Yeast Debaryomyces hansenii Depends on Two Redox Shuttles and the NAD+ Carrier, Pmp47.

Debaryomyces hansenii is considered an unconventional yeast with a strong biotechnological potential, which can produce and store high amounts of lipids. However, relatively little is known about its lipid metabolism, and genetic tools for this yeast have been limited. The aim of this study was to explore the fatty acid β-oxidation pathway in D. hansenii. To this end, we employed recently developed methods to generate multiple gene deletions and tag open reading frames with GFP in their chromosomal context in this yeast. We found that, similar as in other yeasts, the β-oxidation of fatty acids in D. hansenii was restricted to peroxisomes. We report a series of experiments in D. hansenii and the well-studied yeast Saccharomyces cerevisiae that show that the homeostasis of NAD+ in D. hansenii peroxisomes is dependent upon the peroxisomal membrane protein Pmp47 and two peroxisomal dehydrogenases, Mdh3 and Gpd1, which both export reducing equivalents produced during β-oxidation to the cytosol. Pmp47 is the first identified NAD+ carrier in yeast peroxisomes.

Evaluating Atlantic bluefin tuna harvest strategies that use conventional genetic tagging data

An individual tagging model was implemented within the spatial, seasonal, multi-stock, multi-fleet operating models of the peer-reviewed Management Strategy Evaluation (MSE) framework for Atlantic bluefin tuna to evaluate the benefits of a harvest strategy that utilizes conventional gene tagging. A multi-year Brownie estimator was developed to test the accuracy and precision of exploitation rate estimates arising from gene tagging programs with various scenarios for spatial release distribution, release numbers and fishery exploitation rates. Harvest strategies that used the Brownie estimator were tested to evaluate yield and resource conservation performance relative to idealized management using perfect information. For the eastern stock, releasing 1,000 fish throughout the Atlantic and genotyping 27% of all landed fish at an estimated cost of US$2M was sufficient to obtain estimates of exploitation rate with a coefficient of variation of 20%. For the western stock, the same precision in exploitation rate estimates required the release of 1,300 fish and genotyping rate of 35% at an estimated cost of US$2.5M. Harvest strategies using the gene tagging data provided expected yield and resource conservation performance that was not substantially lower than a harvest strategy assuming using perfect information regarding vulnerable biomass. Reducing the number of releases most strongly affected the worst-case ‘lower-tail’ outcomes for West area yield and eastern stock biomass. Conventional gene tagging harvest strategies offer a promising basis for calculating management advice for Atlantic bluefin tuna that may be cheaper, simpler, and more robust than the current conventional stock assessment paradigm.

RNA Interference-Mediated Knockdown of Tryptophan 2,3-Dioxygenase and Kynurenine-3-Monooxygenase in Monochamus Alternatus: Implications for Insect Control

Monochamus alternatus Hope (Coleoptera: Cerambycidae), an invasive beetle that has caused billions of dollars in economic losses, is a serious pest of Pinus massoniana in many Asian countries. Clarifying the eye pigment gene and its knockdown phenotype of M. alternatus can provide functional gene identification and a marker for screening of gene editing, as well as help develop new control ideas. In this study, we first screened the transcriptome and found one homologous gene of tryptophan 2,3-dioxygenase (TDO) and one of kynurenine-3-monooxygenase (KMO). By measuring the expression levels of TDO and KMO in different developmental periods, it was indicated that TDO and KMO were expressed in various stages of M. alternatus. The gene expression of MaKMO was higher than MaTDO, showing high expression after pupation and decreasing at the beginning of eclosion. MaTDO and MaKMO were knocked down using RNA interference technology in different periods of expression, and the temporal expression changes were obtained using RT-qPCR technology. The results showed that the expressions of MaTDO and MaKMO were significantly inhibited by the injection of dsRNA; the expressions of MaTDO at 48 h, 96 h and after pupation were 21.9%, 32.3% and 59.2%, respectively, meanwhile, those of KMO were 23.4%, 25.0% and 69.7%, respectively. There was a significant change in eye color, and the beetles were able to pupate normally without their activity being affected. Therefore, both MaTDO and MaKMO can be used as tag genes for M. alternatus. A dominant marker system based on eye color can be developed for the genetic manipulation and control of M. alternatus.

SsDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid RPE1 and HCT116 cells

BackgroundRecent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells.ResultsIn this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration.ConclusionsThese results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells.

Efficient protein tagging and cis-regulatory element engineering via precise and directional oligonucleotide-based targeted insertion in plants.

Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient non-homologous end-joining-mediated targeted insertion method was recently developed by combining CRISPR/SpCas9 gene editing with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet this approach often leads to imprecise insertions with no control over the insertion direction. Here, we compared the influence of chemical protection of dsODNs on efficiency of targeted insertion. We observed that CRISPR/SpCas9 frequently induced staggered cleavages with 1-nucleotide 5´overhangs; we also evaluated the effect of donor end structures on the direction and precision of targeted insertions. We demonstrate that chemically protected dsODNs with 1-nucleotide 5´ overhangs significantly improved the precision and direction control of target insertions in all tested CRISPR targeted sites. We applied this method to endogenous gene tagging in green foxtail (Setaria viridis), and engineering of cis-regulatory elements for disease resistance in rice (Oryza sativa). We directionally inserted two distinct transcription activator-like effector binding elements into the promoter region of a recessive rice bacterial blight resistance gene with up to 24.4% efficiency. The resulting rice lines harboring heritable insertions exhibited strong resistance to infection by the pathogen Xanthomonas oryzae pv. oryzae in an inducible and strain-specific manner.

Abstract 6508: Eclipse, an automated CRISPR platform for the large-scale generation of cell models

Abstract Major advances in gene editing using CRISPR have improved accessibility for the creation of disease relevant in vitro models, allowing researchers to readily interrogate oncogenic driver mutations and elucidate novel targets to overcome challenges within cancer research such as drug resistance. Despite this, the rapid generation of disease relevant cell lines at the scale needed for successful and efficient therapeutic development remains challenging in part due to the sheer number of potential oncogenic driver mutations and cancer targets along with using traditional manual approaches. Specific challenges include: the efficient generation of gene knockouts and corresponding loss of protein; efficient knock-in of gene tags and single nucleotide variants; and controlling the zygosity of these genetic outcomes. To address these limitations, we describe the use of our automated, high throughput CRISPR editing platform, ECLIPSE, for the rapid generation of cell models such as immortalized cells and induced pluripotent stem cells. We leveraged our chemically modified synthetic sgRNAs, optimized nucleofection methods, and automated pipelines for the generation of CRISPR-edited cells at various scales to address the needs of researchers. This scale ranges from individual edited cell clones for drug target validation to the generation of hundreds of knockout cell pools in a ready-to-assay arrayed format for gene target identification in drug screening. The utilization of automated systems such as the ECLIPSE platform are critical catalysts for the rapid generation of engineered cell lines at relevant scales to address the challenges of cancer research. Citation Format: Peter Deng, Matthew Rowe, Anna King, Dana Sailor, Antinea Chair, Montse Morell, Andreia G. Sommer, Kevin Holden. Eclipse, an automated CRISPR platform for the large-scale generation of cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6508.

Metagenomic analyses of a microbial assemblage in a subglacial lake beneath the Vatnajökull ice cap, Iceland.

Skaftárkatlar are two subglacial lakes located beneath the Vatnajökull ice cap in Iceland associated with geothermal and volcanic activity. Previous studies of these lakes with ribosomal gene (16S rDNA) tag sequencing revealed a limited diversity of bacteria adapted to cold, dark, and nutrient-poor waters. In this study, we present analyses of metagenomes from the lake which give new insights into its microbial ecology. Analyses of the 16S rDNA genes in the metagenomes confirmed the existence of a low-diversity core microbial assemblage in the lake and insights into the potential metabolisms of the dominant members. Seven taxonomic genera, Sulfuricurvum, Sulfurospirillum, Acetobacterium, Pelobacter/Geobacter, Saccharibacteria, Caldisericum, and an unclassified member of Prolixibacteraceae, comprised more than 98% of the rDNA reads in the library. Functional characterisation of the lake metagenomes revealed complete metabolic pathways for sulphur cycling, nitrogen metabolism, carbon fixation via the reverse Krebs cycle, and acetogenesis. These results show that chemolithoautotrophy constitutes the main metabolism in this subglacial ecosystem. This assemblage and its metabolisms are not reflected in enrichment cultures, demonstrating the importance of in situ investigations of this environment.

Microsatellite Genome-Wide Database Development for the Commercial Blackhead Seabream (Acanthopagrus schlegelii)

Simple sequence repeats (SSRs), the markers with the highest polymorphism and co-dominance degrees, offer a crucial genetic research resource. Limited SSR markers in blackhead seabream have been reported. The availability of the blackhead seabream genome assembly provided the opportunity to carry out genome-wide identification for all microsatellite markers, and bioinformatic analyses open the way for developing a microsatellite genome-wide database in blackhead seabream. In this study, a total of 412,381 SSRs were identified in the 688.08 Mb genome by Krait software. Whole-genome sequences (10×) of 42 samples were aligned against the reference genome and genotyped using the HipSTR tools by comparing and counting repeat number variation across the SSR loci. A total of 156,086 SSRs with a 2-4 bp repeat were genotyped by HipSTR tools, which accounted for 55.78% of the 2-4 bp SSRs in the reference genome. High accuracy of genotyping was observed by comparing HipSTR tools and PCR amplification. A set of 109,131 loci with a number of alleles ≥ 3 and with a number of genotyped individuals ≥ 6 were reserved to constitute the polymorphic SSR database. Fifty-one polymorphic SSR loci were identified through PCR amplification. This strategy to develop polymorphic SSR markers not only obtained a large set of polymorphic SSRs but also eliminated the need for laborious experimental screening. SSR markers developed in this study may facilitate blackhead seabream research, which lays a certain foundation for further gene tagging and genetic linkage analysis, such as marker-assisted selection, genetic mapping, as well as comparative genomic analysis.

Illuminating dynamic structural states in hiPSC through endogenous gene tagging.

The Allen Institute for Cell Science aims to understand the principles by which human induced pluripotent stem cells (hiPSCs) establish and maintain robust dynamic localization of cellular structures, and how they transition between states during differentiation and disease. As a first step [JG1], we have created a collection of endogenously tagged hiPSC lines to illuminate cell organization. The tagged proteins include notable membrane-bound and membrane-less cellular organelles, signaling complexes, phase transition markers, transcription factors, and structural markers specific to differentiated cells. To date, the Allen Cell Collection consists of 54 single- or dual-edited lines that have undergone extensive quality control testing to ensure genomic, cell biological, and stem cell integrity. Here, we highlight our gene-editing and quality control workflow for mono- and biallelic editing of expressed or silent genes that are expressed specifically during differentiation and function as reporters of cellular state. Furthermore, we underscore our efforts to increase efficiency of the current workflow by utilizing Adeno-Associated Virus (AAV) to deliver donor DNAs and multiplexing transfection strategies for gene tagging at multiple loci. Our most recently released lines are CTCF-mEGFP and PCNA-mEGFP, for dynamic visualization of chromatin domains and replication foci, G3BP1-mEGFP to label stress granules, mono- and bi-allelic mEGFP-E-cadherin to visualize adherens junctions and DCP1a-mEGFP for P-bodies. We now have gene [RG2] [JG3]-edited cell populations for N- and VE-cadherin ready for single cell sorting. Our overarching goal is to make these tools openly available to the cell biology community to help accelerate biomedical research. In addition to our cell lines, the donor plasmids, segmented 3D images of about 200,000 cells from our lines, image analysis, visualization tools, integrated cell models and biological findings are all openly available to the research community (www.allencell.org).

Identification of therapeutic targets for osteosarcoma by integrating single-cell RNA sequencing and network pharmacology.

Background: Osteosarcoma (OS) is a common primary tumor with extensive heterogeneity. In this study, we used single-cell RNA sequencing (scRNA-seq) and network pharmacology to analyze effective targets for Osteosarcoma treatment. Methods: The cell heterogeneity of the Osteosarcoma single-cell dataset GSE162454 was analyzed using the Seurat package. The bulk-RNA transcriptome dataset GSE36001 was downloaded and analyzed using the CIBERSORT algorithm. The key targets for OS therapy were determined using Pearson's correlation analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed on key targets. The DeepDR algorithm was used to predict potential drugs for Osteosarcoma treatment. Molecular docking analysis was performed to verify the binding abilities of the predicted drugs and key targets. qRT-PCR assay was used to detect the expression of key targets in osteoblasts and OS cells. Results: A total of 21 cell clusters were obtained based on the GSE162454 dataset, which were labeled as eight cell types by marker gene tagging. Four cell types (B cells, cancer-associated fibroblasts (CAFs), endothelial cells, and plasmocytes) were identified in Osteosarcoma and normal tissues, based on differences in cell abundance. In total, 17 key targets were identified by Pearson's correlation analysis. GO and KEGG analysis showed that these 17 genes were associated with immune regulation pathways. Molecular docking analysis showed that RUNX2, OMD, and CD4 all bound well to vincristine, dexamethasone, and vinblastine. The expression of CD4, OMD, and JUN was decreased in Osteosarcoma cells compared with osteoblasts, whereas RUNX2 and COL9A3 expression was increased. Conclusion: We identified five key targets (CD4, RUNX2, OMD, COL9A3, and JUN) that are associated with Osteosarcoma progression. Vincristine, dexamethasone, and vinblastine may form a promising drug-target pair with RUNX2, OMD, and CD4 for Osteosarcoma treatment.

Genome Editing Mediated by Primordial Germ Cell in Chicken.

Genome editing technology has facilitated the studies on exploring specific gene functions in diverse living organisms. The technology has also contributed to creating high-value livestock in industry fields in terms of enhancing productivity or acquiring disease resistance. Particularly, applying genome editing technologies in avian species has been emphasized in both academic and industrial fields due to their unique developmental patterns as well as application possibilities. To accomplish genome editing in avian species, gene integration into chicken primordial germ cell (PGC) genome using a virus or transposition systems has been widely used, and recently developed programmable genome editing technologies including clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas9) systems enable to edit the genetic information precisely for maximizing the application potentials of avian species. In these regards, this chapter will cover the methods for producing genome-edited chickens, particularly by CRISPR/Cas9 technologies allowing targeted gene insertion, gene knockout, and gene tagging.

A marker-free genome editing method in S. pombe using the delitto perfetto approach.

The fission yeast, like budding yeast, offer an easy manipulation of their genome, despite their distinct biology. Most tools available in budding yeast are also available in fission yeast in versions taking into account the features of each organism. The delitto perfetto is a powerful approach, initially developed in S. cerevisiae , for in vivo site-directed mutagenesis. Here, we present an adaptation of the approach to S. pombe manipulation and demonstrate its applicability for a rapid, marker-free and efficient in vivo site-directed mutagenesis and N-terminal tagging of nonessential genes in fission yeast.

Immunohistochemical Analysis of Nuclear Lamina Structures in the Drosophila Ovary Using CRISPR-Tagged Genes.

The Drosophila ovary represents an outstanding model for investigating tissue homeostasis. Females continuously produce oocytes throughout their lifetime. However, as females age, fecundity declines, in part, due to changes in ovarian niche function and germline stem cell (GSC) homeostasis. Understanding the dynamics of GSC maintenance will provide needed insights into how coordinated tissue homeostasis is lost during aging. Critical regulators of GSC maintenance are proteins that reside in the nuclear lamina (NL), including the NL proteins emerin and Barrier-to-Autointegration Factor (BAF). Continued investigation of how emerin, BAF, and other NL proteins contribute to GSC function depends upon the availability of antibodies for NL proteins, a limiting resource. In this chapter, we discuss strategies for using clustered regularly interspaced short palindromic repeats (CRISPR) genomic editing to produce endogenously tagged NL genes to circumvent this obstacle, using the generation of the gfp-baf allele as an example. We describe strategies for validation of tagged alleles. Finally, we outline methods for immunohistochemical analysis of resulting tagged-NL proteins.

Molecular and Physiological Variability in Bread Wheat and Its Wild Relative (Aegilops tauschii Coss.) Species under Water-Deficit Stress Conditions.

Aegilops and Triticum spp. are two ideal gene pools for the breeding purposes of wheat. In this study, a set of Iranian accessions of Aegilops tauschii Coss. and Triticum aestivum L. species were evaluated in terms of some physiological and biochemical features under control and water-deficit stress conditions. Moreover, several simple sequence repeat (SSR) markers were employed to identify marker loci associated with the measured traits. The results indicated that water-deficit stress significantly affected all measured traits and the highest reductions due to water-deficit were recorded for shoot fresh and dry biomasses (SFB and SDB), stomatal conductance (Gs), leaf relative water content (RWC), and chlorophyll b content (Chl b). In molecular analysis, 25 SSR markers generated 50 fragments, out of which 49 fragments (98%) were polymorphic. Furthermore, the genetic variation observed within species is more than between species. The results of cluster and Bayesian model analysis classified all evaluated accessions into three main clusters. Under control and water-deficit stress conditions, 28 and 27 significant marker-trait associations (MTAs) were identified, respectively. Furthermore, 10 MTAs showed sufficiently stable expression across both growth conditions. Of these, the markers Xgwm-111, Xgwm-44, Xgwm-455, Xgwm-272, and Xgwm-292 were associated with multiple traits. Hence, these markers could serve as useful molecular tools for population characterization, gene tagging, and other molecular breeding studies.

STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity

Transcription is a dynamic process. To detect the dynamic relationship among protein clusters of RNA polymerase II and coactivators, gene loci, and transcriptional activity, we insert an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site. By optimizing the individual elements, we develop the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNA polymerase II and BRD4 are observed proximal to the transcription start site of Nanog when the gene is transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 tend to be located near the transcription start site, even without transcription activity. Thus, the STREAMING-tag system reveals the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding transcriptional regulation in living cells.

SCON—a Short Conditional intrON for conditional knockout with one-step zygote injection

The generation of conditional alleles using CRISPR technology is still challenging. Here, we introduce a Short Conditional intrON (SCON, 189 bp) that enables the rapid generation of conditional alleles via one-step zygote injection. In this study, a total of 13 SCON mouse lines were successfully generated by 2 different laboratories. SCON has conditional intronic functions in various vertebrate species, and its target insertion is as simple as CRISPR/Cas9-mediated gene tagging.

NanoTag Nanobody Tools for Drosophila In Vitro and In Vivo Studies.

Nanobodies have emerged as powerful protein-binding tools to uncover protein functions. Using functionalized protein binders, proteins of interest can be visualized, degraded, delocalized, or post-translationally modified in vivo. We recently reported the use of two short peptide tags, 10-aa 127D01 and 14-aa VHH05, and their corresponding nanobodies, Nb127D01 and NbVHH05, for both in vitro and in vivo studies in Drosophila. Here, we provide detailed protocols for nanobody production and for visualization of proteins of interest in either fixed or live samples. In addition, we include protocols for endogenous protein tagging using CRISPR-mediated genome engineering. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Nanobody production in S2 cells Basic Protocol 2: Nanobody expression and purification in bacterial cells Basic Protocol 3: Immunostaining with nanobodies Basic Protocol 4: Immunoblotting with nanobodies Basic Protocol 5: Immunoprecipitation with nanobodies prepared from S2 cells Basic Protocol 6: Immunoprecipitation with nanobodies prepared from bacteria Basic Protocol 7: NbVHH05 and Nb127D01 used as chromobodies Basic Protocol 8: NanoTag trap as a method to alter protein localization Support Protocol: CRISPR-mediated tagging of endogenous genes with NanoTags.

Highly Efficient One-Step Tagging of Endogenous Genes in Primary Cells Using CRISPR-Cas Ribonucleoproteins.

Genome editing tools have simplified the generation of knock-in gene fusions, which are widely used to study proteins in their natural context. However, strategies for tagging endogenous genes in primary cells are few and inefficient. In this study, we developed a one-step endogenous gene-tagging strategy by co-delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoprotein complexes and chemically modified donor DNA into cells. Upon CRISPR-Cas9 blunt-end double-strand breaks, highly efficient site-specific insertion of genetic materials (3 × FLAG or eGFP) was achieved in both cell lines and primary cells. We further optimized the gene-tagging efficiency and precision by using CRISPR-Cas12a, which produces a staggered cut with a 5' overhang and thus enables precise ligation of DNA donors with a complementary 3' overhang. With high efficiency and flexibility, this platform would be extremely useful for multiplex endogenous genes tagging and further exploration of protein functions in various cell types.

Fluorescent Tagging of Endogenous Beta-Globin Gene in S-HUDEP2 Cell Model for Live Cell Monitoring of Beta-Globin Expression and Regulation

Introduction: The translation of β-globin (HBB) gene is crucial for adult hemoglobin (HbA) formation, and naturally occurring mutations in HBB cause disorders of erythropoiesis. Therefore, understanding the mechanisms governing HBB regulation and expression has been an active area of research. We aim to develop a cell model to monitor HBB expression in live cells as a tool to gain better understanding of HBB expression, regulation and for the discovery of novel therapeutic targets. Methods: Sickle Human Umbilical Cord Derived Erythroid Progenitor cells (S-HUDEP2) are immortalized CD34+ hematopoietic stem and progenitor cells with homozygous sickle mutation that exclusively produce sickle hemoglobin upon erythroid differentiation. To generate the cell model in S-HUDEP2, we delivered HiFi SpCas9/gRNA targeting the C-terminus of HBB together with a double-stranded DNA donor template encoding self-cleaving peptide, eGFP, and β-globin polyA signal flanking homology arms for simultaneous expression of β-globin and eGFP under the HBB promoter. We created this clonal cell model and confirmed biallelic precise in-frame targeted integration by PCR, Sanger sequencing and digital droplet PCR copy number assay to ensure simultaneous monitoring of both HBB alleles. Results: To validate this model, we delivered HiFi SpCas9/R-66S sgRNA as ribonucleoprotein (RNP) targeting HBB currently in pre-clinical studies for sickle cell anemia (SCA). With RNP delivery, CRISPR/Cas9 double-strand breaks (DSBs) will lead to insertions and deletions (indels); we hypothesized that varying indel frequencies and patterns will result in change in GFP expression. After RNP delivery, we conducted 7 days of in vitro cellular expansion to allow for completion of editing and stabilization of GFP signal followed by 5 days of in vitro cellular differentiation. We identified 3 distinct populations based on GFP expression: GFP-, GFPlow, and GFPhigh. These cells were bulk sorted, genomic DNA was extracted and we analyzed indel patterns for each population through targeted amplicon Next Generation Sequencing (NGS). Each population had a unique indel pattern: confirming that GFP expression is based on genotype. In addition, an increase in GFP mean fluorescent intensity (MFI) was seen after in vitro differentiation with preservation of these distinct groups; indicative of expected increase in HBB transcription. Based on these observations, through use of ddPCR copy number assay, we determined that the creation of CRISPR/Cas9 mediated large deletion (>200bp) is the underlying mechanism leading to GFP loss seen in GFP- andGFPlow populations. Nonsense mutations occurring early in exon 1 of HBB resulting in premature translation-termination codons are resistant to nonsense mediated mRNA decay (NMD), a well-known cellular surveillance mechanism. We hypothesized that RNP induced small frameshift indels in exon 1 allow for NMD escape thus preserving HBB translation and GFP expression while large deletions do not, leading to GFP loss. To further validate this model and define its potential for broad applicability, we confirmed its ability to undergo normal differentiation based on erythroid specific cell markers when compared to untagged S-HUDEP2. We also confirmed our model's capacity to form Hb tetramer with α-chains using Hb native PAGE which allows for identification of intact Hb tetramers based on reference migration. Conclusion: We have successfully established and validated a clonal cell model with GFP tagged β-globin, which can be used to monitor HBB expression in live cells via GFP expression, eliminating the need for cellular fixation and HbA antibody staining. Our model may provide a useful tool for a broad range of applications, including the examination of cellular mechanisms underlying HBB expression and the screening of potential therapeutic targets for hemoglobinopathies and other disorders of erythropoiesis.