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

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.

Reviewing Editor

  1. Jeremy J Day, University of Alabama at Birmingham, United States

Publication history

  1. Received: February 11, 2022
  2. Preprint posted: February 21, 2022 (view preprint)
  3. Accepted: August 11, 2022
  4. Accepted Manuscript published: August 12, 2022 (version 1)
  5. Version of Record published: August 31, 2022 (version 2)

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

  • 605
    Page views
  • 319
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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

Further reading

    1. Evolutionary Biology
    2. Genetics and Genomics
    Yi Feng, Rafik Neme ... Laura F Landweber
    Research Article

    Ciliates are microbial eukaryotes that undergo extensive programmed genome rearrangement, a natural genome editing process that converts long germline chromosomes into smaller gene-rich somatic chromosomes. Three well-studied ciliates include Oxytricha trifallax, Tetrahymena thermophila and Paramecium tetraurelia, but only the Oxytricha lineage has a massively scrambled genome, whose assembly during development requires hundreds of thousands of precise programmed DNA joining events, representing the most complex genome dynamics of any known organism. Here we study the emergence of such complex genomes by examining the origin and evolution of discontinuous and scrambled genes in the Oxytricha lineage. This study compares six genomes from three species, the germline and somatic genomes for Euplotes woodruffi, Tetmemena sp., and the model ciliate Oxytricha trifallax. To complement existing data, we sequenced, assembled and annotated the germline and somatic genomes of Euplotes woodruffi, which provides an outgroup, and the germline genome of Tetmemena sp.. We find that the germline genome of Tetmemena is as massively scrambled and interrupted as Oxytricha's : 13.6% of its gene loci require programmed translocations and/or inversions, with some genes requiring hundreds of precise gene editing events during development. This study revealed that the earlier-diverged spirotrich, E. woodruffi, also has a scrambled genome, but only roughly half as many loci (7.3%) are scrambled. Furthermore, its scrambled genes are less complex, together supporting the position of Euplotes as a possible evolutionary intermediate in this lineage, in the process of accumulating complex evolutionary genome rearrangements, all of which require extensive repair to assemble functional coding regions. Comparative analysis also reveals that scrambled loci are often associated with local duplications, supporting a gradual model for the origin of complex, scrambled genomes via many small events of DNA duplication and decay.

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics
    Joseph V Geisberg, Zarmik Moqtaderi ... Kevin Struhl
    Research Advance

    Alternative polyadenylation yields many mRNA isoforms whose 3' termini occur disproportionately in clusters within 3' UTRs. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. Within yeast isoform clusters, these shifts occur steadily from one isoform to the next across nucleotide distances. In contrast, the shift between clusters from the last isoform of one cluster to the first isoform of the next - is much less pronounced, even over large distances. GC content in a region 13-30 nt downstream from isoform clusters correlates with their sensitivity to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at the nucleotide level within clusters, but not between them. Pol II occupancy increases just downstream of the most speed-sensitive poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that 1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, 2) poly(A) site clusters are linked to the local elongation rate and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, 3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and 4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.