A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene
- Mayo Clinic, United States
- University of Wisconsin-Madison, United States
- University of California, Davis, United States
- University of California, Irvine, United States
- University of Maryland, Baltimore, United States
- The University of Texas Health Science Center at Houston, United States
- Qingdao University, China
- Shanghai Jiao Tong University, China
Abstract
Previously we showed the generation of a protein trap library made with the gene-break transposon (GBT) in zebrafish (Danio rerio) that could be used to facilitate novel functional genome annotation towards understanding molecular underpinnings of human diseases (Ichino et al, 2020). Here, we report a significant application of this library for discovering essential genes for heart rhythm disorders such as sick sinus syndrome (SSS). SSS is a group of heart rhythm disorders caused by malfunction of the sinus node, the heart's primary pacemaker. Partially owing to its aging-associated phenotypic manifestation and low expressivity, molecular mechanisms of SSS remain difficult to decipher. From 609 GBT lines screened, we generated a collection of 35 zebrafish insertional cardiac (ZIC) mutants in which each mutant traps a gene with cardiac expression. We further employed electrocardiographic measurements to screen these 35 ZIC lines and identified three GBT mutants with SSS-like phenotypes. More detailed functional studies on one of the arrhythmogenic mutants, GBT411, in both zebrafish and mouse models unveiled Dnajb6 as a novel SSS causative gene with a unique expression pattern within the subpopulation of sinus node pacemaker cardiomyocytes that partially overlaps with the expression of hyperpolarization activated cyclic nucleotide gated channel 4 (Hcn4), supporting heterogeneity of the cardiac pacemaker cells.
Data availability
All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figure 4 and Figure 7.
Article and author information
Author details
Funding
Mayo Foundation for Medical Education and Research
- Xiaolei Xu
Wisconsin Partnership Program 4140
- Alexey V Glukhov
American Heart Association (17POST33370089)
- Di Lang
American Heart Association (846898)
- Di Lang
National Institute of Health (R00HL138160)
- Stefano Morotti
National Institute of Health (1OT2OD026580-01)
- Eleonora Grandi
Science and Technology Innovation Action Plan of Shanghai (201409005600)
- Yigang Li
National Institute of Health (GM063904)
- Stephen C Ekker
American Heart Association (16SDG29120011)
- Alexey V Glukhov
National Institute of Health (R00HL138160)
- Stefano Morotti
National Institute of Health (R01HL131517,P01HL141084)
- Eleonora Grandi
American Heart Association (15SDG24910015)
- Eleonora Grandi
American Heart Association (20POST35120462)
- Haibo Ni
National Institute of Health (NIH R01HL141214)
- Alexey V Glukhov
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication No. 85-23, revised 1996). All animal procedures and protocols used in these studies (for zebrafish, #: A00005409-20; for mouse, #: A00003511-20 and M005490-R02) have been approved by the Mayo Clinic Institutional Animal Care and Use Committee (Permit number: D16-00187) and by the Animal Care and Use Committee of University of Wisconsin-Madison (Permit number: D16-00239).
Copyright
© 2022, Ding et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,067
- views
-
- 199
- downloads
-
- 9
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
A phenotype-based forward genetic screen identifies Dnajb6 as a sick sinus syndrome gene
eLife 11:e77327.
https://doi.org/10.7554/eLife.77327
Further reading
-
- Chromosomes and Gene Expression
- Genetics and Genomics
A new method for mapping torsion provides insights into the ways that the genome responds to the torsion generated by RNA polymerase II.
-
- Genetics and Genomics
- Microbiology and Infectious Disease
The ability to analyze the function of all genes in a genome is highly desirable, yet challenging in Leishmania due to a repetitive genome, limited DNA repair mechanisms, and lack of RNA interference in most species. While our introduction of a cytosine base editor (CBE) demonstrated potential to overcome these limitations (Engstler and Beneke, 2023), challenges remained, including low transfection efficiency, variable editing rates across species, parasite growth effects, and competition between deleterious and non-deleterious mutations. Here, we present an optimized approach addressing these issues. We identified a T7 RNAP promoter variant ensuring high editing rates across Leishmania species without compromising growth. A revised CBE single-guide RNAs (sgRNAs) scoring system was developed to prioritize STOP codon generation. Additionally, a triple-expression construct was created for stable integration of CBE sgRNA expression cassettes into a Leishmania safe harbor locus using AsCas12a ultra-mediated DNA double-strand breaks, increasing transfection efficiency by ~400-fold to 1 transfectant per 70 transfected cells. Using this improved system for a small-scale proof-of-principle pooled screen, we successfully confirmed the essential and fitness-associated functions of CK1.2, CRK2, CRK3, AUK1/AIRK, TOR1, IFT88, IFT139, IFT140, and RAB5A in Leishmania mexicana, demonstrating a significant improvement over our previous method. Lastly, we show the utility of co-expressing AsCas12a ultra, T7 RNAP, and CBE for hybrid CRISPR gene replacement and base editing within the same cell line. Overall, these improvements will broaden the range of possible gene editing applications in Leishmania species and will enable a variety of loss-of-function screens in the near future.
