1. Genetics and Genomics

Closely related type II-C Cas9 orthologs recognize diverse PAMs

  1. Jingjing Wei
  2. Linghui Hou
  3. Jingtong Liu
  4. Ziwen Wang
  5. Siqi Gao
  6. Tao Qi
  7. Song Gao
  8. Shuna Sun  Is a corresponding author
  9. Yongming Wang  Is a corresponding author
  1. Fudan University, China
  2. Sun Yat-sen University Cancer Center, China
  3. Children's Hospital of Fudan University, China
https://doi.org/10.7554/eLife.77825
Share this article
Cite this article
  1. Jingjing Wei
  2. Linghui Hou
  3. Jingtong Liu
  4. Ziwen Wang
  5. Siqi Gao
  6. Tao Qi
  7. Song Gao
  8. Shuna Sun
  9. Yongming Wang
(2022)
Closely related type II-C Cas9 orthologs recognize diverse PAMs
eLife 11:e77825.
https://doi.org/10.7554/eLife.77825
  2. Download BibTeX
  3. Download .RIS

Abstract

The RNA-guided CRISPR/Cas9 system is a powerful tool for genome editing, but its targeting scope is limited by the protospacer-adjacent motif (PAM). To expand the target scope, it is crucial to develop a CRISPR toolbox capable of recognizing multiple PAMs. Here, using a GFP-activation assay, we tested the activities of 29 type II-C orthologs closely related to Nme1Cas9, 25 of which are active in human cells. These orthologs recognize diverse PAMs with variable length and nucleotide preference, including purine-rich, pyrimidine-rich, and mixed purine and pyrimidine PAMs. We characterized in depth the activity and specificity of Nsp2Cas9. We also generated a chimeric Cas9 nuclease that recognizes a simple N4C PAM, representing the most relaxed PAM preference for compact Cas9s to date. These Cas9 nucleases significantly enhance our ability to perform allele-specific genome editing.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file.

Article and author information

Author details

  1. Jingjing Wei

    State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.

  2. Linghui Hou

    State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.

  3. Jingtong Liu

    State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.

  4. Ziwen Wang

    State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, China
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1678-7624

  5. Siqi Gao

    State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.

  6. Tao Qi

    State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China
    Competing interests
    The authors declare that no competing interests exist.

  7. Song Gao

    State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, China
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7427-6681

  8. Shuna Sun

    National Children's Medical Center, Children's Hospital of Fudan University, Shanghai, China
    For correspondence
    sun_shuna@fudan.edu.cn
    Competing interests
    The authors declare that no competing interests exist.

  9. Yongming Wang

    State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China
    For correspondence
    ymw@fudan.edu.cn
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8269-5296

Funding

National Key Research and Development Program of China (2021YFA0910602,2021YFC2701103)

  • Yongming Wang

National Natural Science Foundation of China (82070258,81870199)

  • Yongming Wang

Open Research Fund of State Key Laboratory of Genetic Engineering, Fudan University (No. SKLGE-2104)

  • Yongming Wang

Science and Technology ReSearch Program of Shanghai (19DZ2282100)

  • Yongming Wang

Natural Science Fund of Shanghai Science and Technology Commission (19ZR1406300)

  • Yongming Wang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2022, Wei 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

  • 1,937
    views
  • 467
    downloads
  • 24
    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)

  1. Jingjing Wei
  2. Linghui Hou
  3. Jingtong Liu
  4. Ziwen Wang
  5. Siqi Gao
  6. Tao Qi
  7. Song Gao
  8. Shuna Sun
  9. Yongming Wang
(2022)
Closely related type II-C Cas9 orthologs recognize diverse PAMs
eLife 11:e77825.
https://doi.org/10.7554/eLife.77825

Share this article

https://doi.org/10.7554/eLife.77825

Categories and tags

Further reading

    1. Genetics and Genomics
    2. Microbiology and Infectious Disease

    Control of pili synthesis and putrescine homeostasis in Escherichia coli

    Iti Mehta, Jacob B Hogins ... Larry Reitzer
    Research Article

    Polyamines are biologically ubiquitous cations that bind to nucleic acids, ribosomes, and phospholipids and, thereby, modulate numerous processes, including surface motility in Escherichia coli. We characterized the metabolic pathways that contribute to polyamine-dependent control of surface motility in the commonly used strain W3110 and the transcriptome of a mutant lacking a putrescine synthetic pathway that was required for surface motility. Genetic analysis showed that surface motility required type 1 pili, the simultaneous presence of two independent putrescine anabolic pathways, and modulation by putrescine transport and catabolism. An immunological assay for FimA—the major pili subunit, reverse transcription quantitative PCR of fimA, and transmission electron microscopy confirmed that pili synthesis required putrescine. Comparative RNAseq analysis of a wild type and ΔspeB mutant which exhibits impaired pili synthesis showed that the latter had fewer transcripts for pili structural genes and for fimB which codes for the phase variation recombinase that orients the fim operon promoter in the ON phase, although loss of speB did not affect the promoter orientation. Results from the RNAseq analysis also suggested (a) changes in transcripts for several transcription factor genes that affect fim operon expression, (b) compensatory mechanisms for low putrescine which implies a putrescine homeostatic network, and (c) decreased transcripts of genes for oxidative energy metabolism and iron transport which a previous genetic analysis suggests may be sufficient to account for the pili defect in putrescine synthesis mutants. We conclude that pili synthesis requires putrescine and putrescine concentration is controlled by a complex homeostatic network that includes the genes of oxidative energy metabolism.

    1. Genetics and Genomics

    UBR-1 deficiency leads to ivermectin resistance in Caenorhabditis elegans

    Yi Li, Long Gong ... Shangbang Gao
    Research Article

    Resistance to anthelmintics, particularly the macrocyclic lactone ivermectin (IVM), presents a substantial global challenge for parasite control. We found that the functional loss of an evolutionarily conserved E3 ubiquitin ligase, UBR-1, leads to IVM resistance in Caenorhabditis elegans. Multiple IVM-inhibiting activities, including viability, body size, pharyngeal pumping, and locomotion, were significantly ameliorated in various ubr-1 mutants. Interestingly, exogenous application of glutamate induces IVM resistance in wild-type animals. The sensitivity of all IVM-affected phenotypes of ubr-1 is restored by eliminating proteins associated with glutamate metabolism or signaling: GOT-1, a transaminase that converts aspartate to glutamate, and EAT-4, a vesicular glutamate transporter. We demonstrated that IVM-targeted GluCls (glutamate-gated chloride channels) are downregulated and that the IVM-mediated inhibition of serotonin-activated pharynx Ca2+ activity is diminished in ubr-1. Additionally, enhancing glutamate uptake in ubr-1 mutants through ceftriaxone completely restored their IVM sensitivity. Therefore, UBR-1 deficiency-mediated aberrant glutamate signaling leads to ivermectin resistance in C. elegans.

    1. Genetics and Genomics

    Dimeric R25CPTH(1–34) activates the parathyroid hormone-1 receptor in vitro and stimulates bone formation in osteoporotic female mice

    Minsoo Noh, Xiangguo Che ... Sihoon Lee
    Research Article

    Osteoporosis, characterized by reduced bone density and strength, increases fracture risk, pain, and limits mobility. Established therapies of parathyroid hormone (PTH) analogs effectively promote bone formation and reduce fractures in severe osteoporosis, but their use is limited by potential adverse effects. In the pursuit of safer osteoporosis treatments, we investigated R25CPTH, a PTH variant wherein the native arginine at position 25 is substituted by cysteine. These studies were prompted by our finding of high bone mineral density in a hypoparathyroidism patient with the R25C homozygous mutation, and we explored its effects on PTH type-1 receptor (PTH1R) signaling in cells and bone metabolism in mice. Our findings indicate that R25CPTH(1–84) forms dimers both intracellularly and extracellularly, and the synthetic dimeric peptide, R25CPTH(1–34), exhibits altered activity in PTH1R-mediated cyclic AMP (cAMP) response. Upon a single injection in mice, dimeric R25CPTH(1–34) induced acute calcemic and phosphaturic responses comparable to PTH(1–34). Furthermore, repeated daily injections increased calvarial bone thickness in intact mice and improved trabecular and cortical bone parameters in ovariectomized (OVX) mice, akin to PTH(1–34). The overall results reveal a capacity of a dimeric PTH peptide ligand to activate the PTH1R in vitro and in vivo as PTH, suggesting a potential path of therapeutic PTH analog development.