RESUMEN
Biopharmaceuticals like therapeutic monoclonal antibodies (mAbs) and other derived proteins are popular for treating various diseases. Transient gene expression (TGE) is typically used as a fast yet efficient method to generate moderate amounts of material. It has been used to support early stage research and discovery processes. Introduction of a robust high yielding and predictive TGE platform in Chinese hamster ovary (CHO) is crucial. It maintains the consistency in cell lines and processes throughout the early drug discovery and downstream manufacturing processes. This helps researchers to identify the issues at an early stage for timely resolution. In this study, we have demonstrated a simple high-titer platform for TGE in CHO based on a dilution process of seeding cells. We achieved titers ranging from 0.8 to 1.9 g/L for eight model mAbs at three scales (1, 30, 100 mL) in 10 days using our new platform. The ability to seed by dilution significantly streamlined the process and dramatically enhanced platform throughput. We observed a modest reduction in titer ranging from 11% to 28% when cells were seeded using dilution compared to when cells were seeded using medium exchange. Further studies revealed that carry over of spent medium into transfection negatively affected the DNA uptake and transcription processes, while the translation and secretion was minimally impacted. In summary, our transient CHO platform using cells prepared by dilution at high densities can achieve high titers of up to 1.9 g/L, which can be further improved by targeting the bottlenecks of transfection and transcription.
Asunto(s)
Anticuerpos Monoclonales , Cricetulus , Células CHO , Animales , Anticuerpos Monoclonales/química , Cricetinae , Recuento de Células , Técnicas de Cultivo de Célula/métodos , Medios de Cultivo/químicaRESUMEN
Approximately 3% of the human genome is composed of short tandem repeat (STR) DNA sequence known as microsatellites, which can be found in both coding and non-coding regions. When associated with genic regions, expansion of microsatellite repeats beyond a critical threshold causes dozens of neurological repeat expansion disorders. To better understand the molecular pathology of repeat expansion disorders, precise cloning of microsatellite repeat sequence and expansion size is highly valuable. Unfortunately, cloning repeat expansions is often challenging and presents a significant bottleneck to practical investigation. Here, we describe a clear method for seamless and systematic cloning of practically any microsatellite repeat expansion. We use cloning and expansion of GGGGCC repeats, which are the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as an example. We employ a recursive directional ligation (RDL) technique to build multiple GGGGCC repeat-containing vectors. We describe methods to validate repeat expansion cloning, including diagnostic restriction digestion, PCR across the repeat, and next-generation long-read MinION nanopore sequencing. Validated cloning of microsatellite repeats beyond the critical expansion threshold can facilitate step-by-step characterization of disease mechanisms at the cellular and molecular level.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Clonación Molecular/métodos , Expansión de las Repeticiones de ADN , Demencia Frontotemporal/genética , Repeticiones de Microsatélite , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Secuencia de Bases , Proteína C9orf72/metabolismo , Enzimas de Restricción del ADN/química , Escherichia coli/genética , Escherichia coli/metabolismo , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Genoma Humano , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
DNA repeat expansion sequences cause a myriad of neurological diseases when they expand beyond a critical threshold. Previous electrochemical approaches focused on the detection of trinucleotide repeats (CAG, CGG, and GAA) and relied on labeling of the probe and/or target strands or enzyme-linked assays. However, detection of expanded GC-rich sequences is challenging because they are prone to forming secondary structures such as cruciforms and quadruplexes. Here, we present label-free detection of hexanucleotide GGGGCC repeat sequences, which cause the leading genetic form of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The approach relies on capturing targets by surface-bound oligonucleotide probes with a different number of complementary repeats, which proportionately translates the length of the target strands into charge transfer resistance (RCT) signal measured by electrochemical impedance spectroscopy. The probe carrying three tandem repeats transduces the number of repeats into RCT with a 3× higher calibration sensitivity and detection limit. Chronocoulometric measurements show a decrease in surface density with increasing repeat length, which is opposite of the impedance trend. This implies that the length of the target itself can contribute to amplification of the impedance signal independent of the surface density. Moreover, the probe can distinguish between a control and patient sequences while remaining insensitive to non-specific Huntington's disease (CAG) repeats in the presence of a complementary target. This label-free strategy might be applied to detect the length of other neurodegenerative repeat sequences using short probes with a few complementary repeats. Graphical abstract Short oligomeric probes with multiple complementary repeats detect long neurodegenerative targets with high sensitivity and transduce into higher impedance signal.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Expansión de las Repeticiones de ADN , Sondas de Oligonucleótidos/genética , Secuencia de Bases , Técnicas Biosensibles/métodos , Espectroscopía Dieléctrica/métodos , Humanos , Enfermedad de Huntington/genética , ARN/genéticaRESUMEN
Clustered regularly interspaced short palindromic repeat (CRISPR) RNAs and their associated effector (Cas) enzymes are being developed into promising therapeutics to treat disease. However, CRISPR-Cas enzymes might produce unwanted gene editing or dangerous side effects. Drug-like molecules that can inactivate CRISPR-Cas enzymes could help facilitate safer therapeutic development. Based on the requirement of guide RNA and target DNA interaction by Cas enzymes, we rationally designed small nucleic acid-based inhibitors (SNuBs) of Streptococcus pyogenes (Sp) Cas9. Inhibitors were initially designed as 2'-O-methyl-modified oligonucleotides that bound the CRISPR RNA guide sequence (anti-guide) or repeat sequence (anti-tracr), or DNA oligonucleotides that bound the protospacer adjacent motif (PAM)-interaction domain (anti-PAM) of SpCas9. Coupling anti-PAM and anti-tracr modules together was synergistic and resulted in high binding affinity and efficient inhibition of Cas9 DNA cleavage activity. Incorporating 2'F-RNA and locked nucleic acid nucleotides into the anti-tracr module resulted in greater inhibition as well as dose-dependent suppression of gene editing in human cells. CRISPR SNuBs provide a platform for rational design of CRISPR-Cas enzyme inhibitors that should translate to other CRISPR effector enzymes and enable better control over CRISPR-based applications.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Proteínas de Unión al ADN/genética , Edición Génica , Proteína 9 Asociada a CRISPR/antagonistas & inhibidores , Proteína 9 Asociada a CRISPR/farmacología , Sistemas CRISPR-Cas/efectos de los fármacos , ADN/efectos de los fármacos , ADN/genética , Proteínas de Unión al ADN/efectos de los fármacos , Humanos , Motivos de Nucleótidos/efectos de los fármacos , Motivos de Nucleótidos/genética , Oligonucleótidos/genética , Oligonucleótidos/farmacología , ARN Guía de Kinetoplastida/efectos adversos , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/farmacología , Streptococcus pyogenes/enzimología , Secuencias Repetidas en Tándem/efectos de los fármacos , Secuencias Repetidas en Tándem/genéticaRESUMEN
CRISPR (clustered regularly interspaced short palindromic repeat) endonucleases are at the forefront of biotechnology, synthetic biology and gene editing. Methods for controlling enzyme properties promise to improve existing applications and enable new technologies. CRISPR enzymes rely on RNA cofactors to guide catalysis. Therefore, chemical modification of the guide RNA can be used to characterize structure-activity relationships within CRISPR ribonucleoprotein (RNP) enzymes and identify compatible chemistries for controlling activity. Here, we introduce chemical modifications to the sugar-phosphate backbone of Streptococcus pyogenes Cas9 CRISPR RNA (crRNA) to probe chemical and structural requirements. Ribose sugars that promoted or accommodated A-form helical architecture in and around the crRNA 'seed' region were tolerated best. A wider range of modifications were acceptable outside of the seed, especially D-2'-deoxyribose, and we exploited this property to facilitate exploration of greater chemical diversity within the seed. 2'-fluoro was the most compatible modification whereas bulkier O-methyl sugar modifications were less tolerated. Activity trends could be rationalized for selected crRNAs using RNP stability and DNA target binding experiments. Cas9 activity in vitro tolerated most chemical modifications at predicted 2'-hydroxyl contact positions, whereas editing activity in cells was much less tolerant. The biochemical principles of chemical modification identified here will guide CRISPR-Cas9 engineering and enable new or improved applications.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , ARN Bacteriano/química , División del ADN , ADN de Forma A/química , ARN Bacteriano/metabolismo , Ribonucleoproteínas/metabolismo , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/genética , Relación Estructura-ActividadRESUMEN
DNA substitutions in RNA can probe the importance of A-form structure, 2'-hydroxyl contacts, and conformational constraints within RNA-guided enzymes. Using this approach, we found that Cas9 biochemical activity tolerated significant substitution with DNA nucleotides in the clustered regularly interspaced short palindromic repeat RNA (crRNA). Only minimal RNA content was needed in or near the seed region. Simultaneous substitution at all positions with predicted crRNA-Cas9 2'-hydroxyl contacts had no effect on enzyme activity. The trans-activating crRNA (tracrRNA) also tolerated >50% substitution with DNA. DNA substitutions in the tracrRNA-pairing region of crRNA consistently enhanced cleavage activity while maintaining or improving target specificity. Together, results point to a prominent role for guide:target A-form-like helical structure and a possible regulatory role for the crRNA-tracrRNA pairing motif. A model chimeric crRNA with high activity did not significantly alter RNP assembly or target binding but did reduce Cas9 ribonucleoprotein stability, suggesting effects through conformation or dynamics. Cas9 directed by chimeric RNA-DNA guides may represent a cost-effective synthetic or molecular biology tool for robust and specific DNA cleavage.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Endonucleasas/química , Endonucleasas/genética , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/genética , División del ADN , Endonucleasas/metabolismo , ARN Bacteriano/química , ARN Guía de KinetoplastidaRESUMEN
Microsatellites, or simple tandem repeat sequences, occur naturally in the human genome and have important roles in genome evolution and function. However, the expansion of microsatellites is associated with over two dozen neurological diseases. A common denominator among the majority of these disorders is the expression of expanded tandem repeat-containing RNA, referred to as xtrRNA in this review, which can mediate molecular disease pathology in multiple ways. This review focuses on the potential impact that simple tandem repeat expansions can have on the biology and metabolism of RNA that contain them and underscores important gaps in understanding. Merging the molecular biology of repeat expansion disorders with the current understanding of RNA biology, including splicing, transcription, transport, turnover and translation, will help clarify mechanisms of disease and improve therapeutic development.
Asunto(s)
Expansión de las Repeticiones de ADN/fisiología , Repeticiones de Microsatélite , ARN/metabolismo , Animales , Humanos , Empalme del ARN , Transcripción GenéticaRESUMEN
The spinach family of RNA aptamers are RNA mimics of green fluorescent protein (GFP) that have previously been designed to address the challenges of imaging RNA inside living cells. However, relatively low levels of free intracellular magnesium limited the practical use of these aptamers. Recent cell-based selections identified the broccoli RNA aptamer, which requires less magnesium for fluorescence, but the basis for magnesium preference remained unclear. Here, we find that the broccoli RNA structure is very similar to that of baby spinach, a truncated version of the spinach aptamer. Differences in stability and metal ion preferences between these two aptamers, and among broccoli mutants, are primarily associated with the sequence and structure of predicted quadruplex-flanking stem structures. Mutation of purine-purine pairs in broccoli at the terminal stem-quadruplex transition caused reversion of broccoli to a higher magnesium dependence. Unique duplex-to-quadruplex transitions in GFP-mimic RNAs likely explain their sensitivity to magnesium for stability and fluorescence. Thus, optimizations designed to improve aptamers should take into consideration the role of metal ions in stabilizing the transitions and interactions between independently folding RNA structural motifs.