Gene knock-outs in human CD34+ hematopoietic stem and progenitor cells and in the human immune system of mice

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Abstract

Human CD34 ⁺ hematopoietic stem and progenitor cells (HSPCs) are a standard source of cells for clinical HSC transplantations as well as experimental xenotransplantation to generate “humanized mice”. To further extend the range of applications of these humanized mice, we developed a protocol to efficiently edit the genomes of human CD34 ⁺ HSPCs before transplantation. In the past, manipulating HSPCs has been complicated by the fact that they are inherently difficult to transduce with lentivectors, and rapidly lose their stemness and engraftment potential during in vitro culture. However, with optimized nucleofection of sgRNA:Cas9 ribonucleoprotein complexes, we are now able to edit a candidate gene in CD34 ⁺ HSPCs with almost 100% efficiency, and transplant these modified cells in immunodeficient mice with high engraftment levels and multilineage hematopoietic differentiation. The result is a humanized mouse from which we knocked out a gene of interest from their human immune system.

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m6A-dependent regulation of messenger RNA stability

Xiao Wang, Zhike Lu, Adrián Gómez-Brandón … (2013)

N6 -methyladenosine (m6A) is the most prevalent internal (non-cap) modification present in the messenger RNA (mRNA) of all higher eukaryotes 1,2 . Although essential to cell viability and development 3–5 , the exact role of m6A modification remains to be determined. The recent discovery of two m6A demethylases in mammalian cells highlighted the importance of m6A in basic biological functions and disease 6–8 . Here we show that m6A is selectively recognized by the human YTH domain family 2 (YTHDF2) protein to regulate mRNA degradation. We identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but which also include non-coding RNAs, with a conserved core motif of G(m6A)C. We further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies 9 . The C-terminal domain of YTHDF2 selectively binds to m6A-containing mRNA whereas the N-terminal domain is responsible for the localization of the YTHDF2-mRNA complex to cellular RNA decay sites. Our results indicate that the dynamic m6A modification is recognized by selective-binding proteins to affect the translation status and lifetime of mRNA.

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Computational correction of copy-number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells

Robin Meyers, Jordan G Bryan, James McFarland … (2017)

The CRISPR-Cas9 system has revolutionized gene editing both on single genes and in multiplexed loss-of-function screens, enabling precise genome-scale identification of genes essential to proliferation and survival of cancer cells 1,2 . However, previous studies reported that a gene-independent anti-proliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, leading to false positive results in copy number amplified regions 3,4 . We developed CERES, a computational method to estimate gene dependency levels from CRISPR-Cas9 essentiality screens while accounting for the copy-number-specific effect. As part of our efforts to define a cancer dependency map, we performed genome-scale CRISPR-Cas9 essentiality screens across 342 cancer cell lines and applied CERES to this dataset. We found that CERES reduced false positive results and estimated sgRNA activity for both this dataset and previously published screens performed with different sgRNA libraries. Here, we demonstrate the utility of this collection of screens, upon CERES correction, in revealing cancer-type-specific vulnerabilities.

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N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions

Nian Liu, Chao-Qing Dai, Guanqun Zheng … (2015)

RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBM) 1-3 . However, RBMs can be buried within their local RNA structures 4-7 , thus inhibiting RNA-protein interactions. N 6-methyladenosine (m6A), the most abundant and dynamic internal modification in eukaryotic messenger RNA 8-19 , can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs 15 , but how m6A achieves wide-ranging physiological significance needs further exploration. Here we show that m6A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism “m6A-switch”. We found that m6A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (hnRNP C), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing 20-24 . Combining PAR-CLIP and m6A/MeRIP approaches enabled us to identify 39,060 m6A-switches among hnRNP C binding sites; and global m6A reduction decreased hnRNP C binding at 2,798 high confidence m6A-switches. We determined that these m6A-switch-regulated hnRNP C binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m6A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m6A-dependent RNA structural remodeling, and provide a new direction for investigating RNA-modification-coded cellular biology.

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Author and article information

Contributors

Daniel A. Kuppers: Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: Writing – original draftRole: Writing – review & editing

Jonathan Linton: Role: InvestigationRole: MethodologyRole: Validation

Sergio Ortiz Espinosa:

ORCID: https://orcid.org/0000-0001-9192-2956

Role: InvestigationRole: MethodologyRole: Validation

Kelly M. McKenna: Role: InvestigationRole: MethodologyRole: Validation

Anthony Rongvaux: Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing

Patrick J. Paddison:

ORCID: https://orcid.org/0000-0002-7144-0228

Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: MethodologyRole: ResourcesRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing

Chen Ling: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS One

Journal ID (publisher-id): plos

Title: PLOS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 28 June 2023

Publication date Collection: 2023

Volume: 18

Issue: 6

Electronic Location Identifier: e0287052

Affiliations

[1 ] Human Biology Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America

[2 ] Translational Science and Therapeutics Division, Program in Immunology, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America

[3 ] Department of Immunology, University of Washington, Seattle, Washington, United States of America

Fudan University, CHINA

Author notes

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: rongvaux@ 123456fredhutch.org (AR); paddison@ 123456fredhutch.org (PJP)

Author information

Sergio Ortiz Espinosa https://orcid.org/0000-0001-9192-2956

Patrick J. Paddison https://orcid.org/0000-0002-7144-0228

Article

Publisher ID: PONE-D-22-34997

DOI: 10.1371/journal.pone.0287052

PMC ID: 10306193

PubMed ID: 37379309

SO-VID: a069c7a7-b93a-48c4-bcb8-b953dba39071

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 12 January 2023

Date accepted : 26 May 2023