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Using CRISPR base editing to functionalize the human spliceosome

Applicant Beusch Irene
Number 191929
Funding scheme Early Postdoc.Mobility
Research institution Madhani Lab Department of Biochemistry and Biophysics University of California San Francisco
Institution of higher education Institution abroad - IACH
Main discipline Molecular Biology
Start/End 01.01.2020 - 30.06.2021
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All Disciplines (2)

Discipline
Molecular Biology
Genetics

Keywords (4)

spliceosome; CRISPR; base editing; splicing mechanism

Lay Summary (German)

Lead
Das Spliceosom katalysiert einen wichtigen Schritt der Verarbeitung der Ribonukleinsäure in höheren Eukaryoten. Wie seine Substrate ausgewählt werden, und auf welche Weise die Genauigkeit des Prozesses gewährleistet wird, ist für das menschliche Spliceosom noch nicht erschlossen. Dieses Projekt soll sich dieser Fragenstellung widmen.
Lay summary

Inhalt und Ziele des Forschungsprojekts

Das menschliche Genom kodiert ungefähr 20000 Gene. Im Durchschnitt werden diese Gene durch acht Introne geteilt und ein einzelnes Gen kann bis zu dreihundert Introne enthalten. Paradoxerweise können sich Intronsignale für das Splicen stark unterscheiden und trotzdem wird das Splicen sehr präzise durchgeführt. Das Splicen wird durch das Spliceosom katalysiert. Der katalytische Kern des Spliceosoms wird durch RNA gebildet, während Proteine wichtige strukturelle Funktionen übernehmen und der Spezifität beitragen.

Ich werde Mechanismen dieser Spezifität des Spliceosoms entschlüsseln, indem ich programmierte Mutagenese auf die Proteine des Spliceosoms anwenden werde. Dazu verwende ich CRISPR Technologie gekoppelt mit Reportern, die die Genauigkeit des Splicings wiedergeben. Dies erlaubt mir ein genaues kartieren funktioneller Komponente des Spliceosoms.

 

Wissenschaftlicher und gesellschaftlicher Kontext des Forschungsprojekts

Viele, vor allem vererbbare Krankheiten, haben ihren Ursprung in einem Defekt im Splicing des betroffenen Genes. Obwohl einige dieser Krankheiten therapierbar sind, bleiben aber viele andere nicht behandelbar. Ein grundlegendes Verständnis dieses Prozesses bleibt daher unabdingbar. Mein Ansatz, das Spliceosom unvoreingenommen und gesamtheitlich zu untersuchen, kann daher dazu beitragen diesen Prozess besser zu verstehen und die Basis zur Erforschung weiterer Behandlungsansätze liefern.

 

Direct link to Lay Summary Last update: 13.01.2020

Responsible applicant and co-applicants

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
RNA 2021 Talk given at a conference Base editing the human spliceosome in haploid cells reveals a role for SUGP1 in drug resistance 25.05.2021 online, Singapore Beusch Irene;
SpliceCon 2021 Talk given at a conference Base editing the human spliceosome in haploid cells reveals a role for SUGP1 in drug resistance 22.04.2021 online, United States of America Beusch Irene;


Associated projects

Number Title Start Funding scheme
203008 Using CRISPR base editing to functionalise the human spliceosome 01.07.2021 Postdoc.Mobility

Abstract

Rationale: The human genome encodes approximately 20,000 protein-coding genes. On average, genes are split by eight introns, and a single gene can harbor three hundred introns. Almost all genes encode multiple mRNA isoforms mediated by alternative splicing, and many isoforms are tissue-specific (Wang et al. 2008, Tapial et al. 2017). Paradoxically, intron signals for splicing are highly degenerate, yet splicing must be accomplished with precision. Splicing is catalyzed by the spliceosome, a dynamic ribonucleoprotein (RNP). Spliceosomes assemble de novo onto each intron substrate in a highly orchestrated assembly process that requires at least 8 ATPases (Jurica & Moore 2003, Will & Lührmann 2011, Papasaikas & Valcárcel 2016). Some 90 proteins in the yeast S. cerevisiae and at least 150 in human cells compose the spliceosome. The catalytic core of the spliceosome is formed by RNA, while proteins are instrumental in its assembly, remodeling and in the positioning of active site RNAs (Galej et al. 2014, Scheres & Nagai 2017). Studies in yeast have shown that ATP hydrolysis rates act as a molecular timer for proofreading mechanisms enabled by discard pathways that compete with substrate progression (Burke et al. 2018). However, mutant splicing substrates that are rejected by yeast spliceosomes often correspond to allowed natural substrates in human cells as the latter often display poor matches to consensus signals. Thus, despite the availability of high-resolution cryo-EM structures, how human spliceosomes accept or reject substrates is unknown. Addressing these key questions has been hampered by a lack of tools to interrogate individual residues of essential cellular machines in human cells.Objective: I propose to decipher mechanisms of spliceosomal fidelity by combining programmed mutagenesis of the entire spliceosome using CRISPR base editing technology coupled with high-throughput splicing measurements.Aim 1: Computational design and generation of sgRNA libraries for a range of available CRISPR base editors. The current state of the art corresponds to nickase Cas9 (nCas9) fusions to a number of cytosine and adenine deaminases with additional fusion partners to enhance editing (Rees & Liu 2018). I will optimize, test and evaluate a range of promising base editors for use in our system of choice. Concomitantly, I will design high-fidelity guides that target all available spliceosomal residues.Aim 2: Testing the sgRNA libraries and base editing in human cells using a U5 snRNP sublibrary. Each base editor will be introduced into the HAP1 haploid human immortalized cell line and then a sgRNA sublibrary targeting the U5 snRNP will be transduced. I will assess our experimental approach by targeted quantitative high-depth sequencing of the U5 snRNP associated protein genes’ coding exons using exome capture and sequencing technology. Next, libraries containing guides for all targetable spliceosomal residues will be generated.Aim 3: Interrogating the human spliceosome’s fidelity by targeted mutagenesis. I will generate a panel of haploid cell lines with both the desired base editor(s) and two-color reporters of splicing efficiency in which one reporter is driven by an optimal consensus intron while the second is driven by nonoptimal variant. These will be mutagenized with sgRNA libraries and flow cytometry will be used to sort cells based on changes in splicing efficiency over time. Sequencing of the guides and spliceosome component exomes of the sorted cells will reveal mutable spliceosomal residues that determine acceptance versus rejection of each substrate, while also revealing residues essential for viability and growth.Significance: Understanding how human spliceosomes select the correct substrates is critical for understanding human disease. 9% of known inherited human disease is caused by mutations in splicing signals which often trigger aberrant splicing (Abramowicz & Gos 2018). Inherited spliceosome mutations account for a substantial fraction of the incurable progressive blindness disease retinitis pigmentosa. Driver spliceosome mutations occur in 10% of all tumors with extraordinarily high frequencies in myelodysplasia, chronic myelomonocytic leukemia and uveal melanoma. Mechanistic understanding of the spliceosome is critical for devising therapies for these largely incurable diseases (Lee et al. 2018).
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