Roles for CEP170 in cilia function and dynein-2 assembly

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ABSTRACT

Primary cilia are essential eukaryotic organelles required for signalling and secretion. Dynein-2 is a microtubule-motor protein complex and is required for ciliogenesis via its role in facilitating retrograde intraflagellar transport (IFT) from the cilia tip to the cell body. Dynein-2 must be assembled and loaded onto IFT trains for entry into cilia for this process to occur, but how dynein-2 is assembled and how it is recycled back into a cilium remain poorly understood. Here, we identify centrosomal protein of 170 kDa (CEP170) as a dynein-2-interacting protein in mammalian cells. We show that loss of CEP170 perturbs intraflagellar transport and hedgehog signalling, and alters the stability of dynein-2 holoenzyme complex. Together, our data indicate a role for CEP170 in supporting cilia function and dynein-2 assembly.

Abstract

Summary: Intraflagellar transport (IFT) is required for the function of primary cilia. Centrosomal protein 170 (CEP170) interacts with the IFT motor dynein-2, and loss of CEP170 causes defects in dynein-2 assembly and cilia function.

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Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.

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In just 3 years CRISPR genome editing has transformed biology, and its popularity and potency continue to grow. New CRISPR effectors and rules for locating optimum targets continue to be reported, highlighting the need for computational CRISPR targeting tools to compile these rules and facilitate target selection and design. CHOPCHOP is one of the most widely used web tools for CRISPR- and TALEN-based genome editing. Its overarching principle is to provide an intuitive and powerful tool that can serve both novice and experienced users. In this major update we introduce tools for the next generation of CRISPR advances, including Cpf1 and Cas9 nickases. We support a number of new features that improve the targeting power, usability and efficiency of CHOPCHOP. To increase targeting range and specificity we provide support for custom length sgRNAs, and we evaluate the sequence composition of the whole sgRNA and its surrounding region using models compiled from multiple large-scale studies. These and other new features, coupled with an updated interface for increased usability and support for a continually growing list of organisms, maintain CHOPCHOP as one of the leading tools for CRISPR genome editing. CHOPCHOP v2 can be found at http://chopchop.cbu.uib.no

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Author and article information

Contributors

David J. Stephens:

ORCID: http://orcid.org/0000-0001-5297-3240

Journal

Journal ID (nlm-ta): J Cell Sci

Journal ID (iso-abbrev): J Cell Sci

Journal ID (publisher-id): JCS

Title: Journal of Cell Science

Publisher: The Company of Biologists Ltd

ISSN (Print): 0021-9533

ISSN (Electronic): 1477-9137

Publication date Collection: 15 April 2024

Publication date (Electronic): 1 May 2024

Publication date PMC-release: 1 May 2024

Volume: 137

Issue: 8

Electronic Location Identifier: jcs261816

Affiliations

[ ¹ ]Cell Biology Laboratories, School of Biochemistry, Faculty of Life Sciences, University of Bristol , Bristol BS8 1TD, UK

[ ² ]Sir William Dunn School of Pathology, University of Oxford , Oxford OX1 3RE, UK

[ ³ ]Wolfson Bioimaging Facility, Faculty of Life Sciences, University Walk, University of Bristol , Bristol BS8 1TD, UK

[ ⁴ ]Proteomics Facility, Faculty of Life Sciences, University of Bristol , Bristol BS8 1TD, UK

Author notes

[*]

These authors contributed equally to this work

[‡ ]Author for correspondence ( david.stephens@ 123456bristol.ac.uk )

Handling Editor: Guangshuo Ou

Competing interests

The authors declare no competing or financial interests.

Author information

Johannes F. Weijman http://orcid.org/0000-0002-2082-5777

Laura Vuolo http://orcid.org/0000-0002-9801-9206

Caroline Shak http://orcid.org/0000-0002-2109-1058

Aakash G. Mukhopadhyay http://orcid.org/0000-0001-9397-9702

Lorna R. Hodgson http://orcid.org/0000-0001-6373-4405

Kate J. Heesom http://orcid.org/0000-0002-5418-5392

Anthony J. Roberts http://orcid.org/0000-0001-5277-6730

David J. Stephens http://orcid.org/0000-0001-5297-3240

Article

Publisher ID: JCS261816

DOI: 10.1242/jcs.261816

PMC ID: 11112123

PubMed ID: 38533689

SO-VID: ee9b9dd7-0bbb-4674-b509-a79b19bb84ac

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

History

Date received : 21 November 2023

Date accepted : 19 March 2024

Funding

Funded by: Biotechnology and Biological Sciences Research Council, http://dx.doi.org/10.13039/501100000268;

Award ID: BB/S005390/1

Funded by: Wellcome Trust, http://dx.doi.org/10.13039/100010269;

Award ID: 217186/Z/19/Z

Funded by: University of Bristol, http://dx.doi.org/10.13039/501100000883;