CRISPR/Cas12a toolbox for genome editing in Methanosarcina acetivorans

Ping Zhu, Tejas Somvanshi, Jichen Bao*, Silvan Scheller*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

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Methanogenic archaea play an important role in the global carbon cycle and may serve as host organisms for the biotechnological production of fuels and chemicals from CO2 and other one-carbon substrates. Methanosarcina acetivorans is extensively studied as a model methanogen due to its large genome, versatile substrate range, and available genetic tools. Genome editing in M. acetivorans via CRISPR/Cas9 has also been demonstrated. Here, we describe a user-friendly CRISPR/Cas12a toolbox that recognizes T-rich (5′-TTTV) PAM sequences. The toolbox can manage deletions of 3,500 bp (i.e., knocking out the entire frhADGB operon) and heterologous gene insertions with positive rates of over 80%. Cas12a-mediated multiplex genome editing was used to edit two separate sites on the chromosome in one round of editing. Double deletions of 100 bp were achieved, with 8/8 of transformants being edited correctly. Simultaneous deletion of 100 bp at one site and replacement of 100 bp with the 2,400 bp uidA expression cassette at a separate site yielded 5/6 correctly edited transformants. Our CRISPR/Cas12a toolbox enables reliable genome editing, and it can be used in parallel with the previously reported Cas9-based system for the genetic engineering of the Methanosarcina species.

Original languageEnglish
Article number1235616
Number of pages9
JournalFrontiers in Microbiology
Publication statusPublished - 12 Dec 2023
MoE publication typeA1 Journal article-refereed


  • CRISPR/Cas12a
  • genome editing
  • methanogens
  • Methanosarcina acetivorans
  • synthetic biology


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