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Genome Engineering by RNA-Guided Transposition for Anabaena sp. PCC 7120.

Sergio Arévalo1,2,3,4, Daniel Pérez Rico1, Dolores Abarca1

  • 1Biology Department, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

ACS Synthetic Biology
|March 6, 2024
PubMed
Summary

CRISPR-associated transposase (CAST) enables precise DNA insertion in filamentous cyanobacteria like Anabaena. This RNA-guided transposition method offers a new foundation for genome editing in these important microorganisms.

Keywords:
AnabaenaCRISPR-associated transposon (CAST)RNA-guided transpositionde novo genome assemblygenome engineeringminion sequencing

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genome Engineering

Background:

  • DNA integration in genome engineering is limited by cell division, hindering efficiency and accuracy.
  • RNA-guided transposition using CRISPR-associated transposase (CAST) has shown high effectiveness and specificity in E. coli.

Purpose of the Study:

  • To develop and test CAST for genome editing in filamentous cyanobacteria.
  • To establish Golden Gate vectors for CAST application in Anabaena sp. strain PCC 7120.

Main Methods:

  • Development of Golden Gate vectors for CAST and engineered transposons.
  • Transfer of plasmids into Anabaena via conjugation.
  • Testing of single-guide (sg) RNA and protospacer-associated motif (PAM) requirements.
  • Analysis of insertion sites and transposon cargo integrity.

Main Results:

  • CAST was confirmed effective in Anabaena sp. strain PCC 7120.
  • Successful transfer of large plasmids containing CAST and transposons via conjugation.
  • sgRNA encoding the leading strand and PAM sequence inclusion were effective.
  • Precise DNA insertion occurred 63 bases after the PAM in most cases.
  • CAST on a replicating plasmid exhibited toxicity, useful for plasmid curing.
  • Only the engineered transposon cargo was inserted, demonstrating a 'cut and paste' mechanism.
  • No mobilization of endogenous transposons was observed.

Conclusions:

  • This study establishes RNA-guided transposition with CAST as a viable genome editing tool in filamentous cyanobacteria.
  • The developed methods provide a foundational approach for precise genome engineering in Anabaena and other cyanobacteria.
  • The findings pave the way for advanced genetic manipulation of cyanobacteria in various environments.