CRISPR/Cas12a toolbox for genome editing in Methanosarcina acetivorans

Ping Zhu; Tejas Somvanshi; Jichen Bao; Silvan Scheller

doi:10.3389/fmicb.2023.1235616

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 journal › Article › Scientific › peer-review

7 Citations (Scopus)

62 Downloads (Pure)

Abstract

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 language	English
Article number	1235616
Number of pages	9
Journal	Frontiers in Microbiology
Volume	14
DOIs	https://doi.org/10.3389/fmicb.2023.1235616
Publication status	Published - 12 Dec 2023
MoE publication type	A1 Journal article-refereed

Funding

This work was supported by Novo Nordisk Foundation (grant NNF19OC0054329 to SS; grant NNF20OC0065032 to JB) and the Academy of Finland (grant 326020 to SS). We thank the Novo Nordisk Foundation (grant NNF19OC0054329 to SS; grant NNF20OC0065032 to JB) and the Academy of Finland (grant 326020 to SS) for funding this research. We thank the Aalto Science Institute (AScI) project student Shreyash Borkar for helping with the plasmid transformation. We thank Prof. Michael Rother and Dr. Christian Schöne for testing the editing efficiency of our CRISPR/Cas12a system in their lab. We thank Dr. Ingemar von Ossowski for his helpful comments and suggestions.

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 7 Affordable and Clean Energy

Keywords

CRISPR/Cas12a
genome editing
methanogens
Methanosarcina acetivorans
synthetic biology

Access to Document

10.3389/fmicb.2023.1235616Licence: CC BY

CHEM_Zhu_et_al_CRISPRCas12a_toolbox_2023_Frontiers_in_MicrobiologyFinal published version, 1.38 MBLicence: CC BY

Cite this

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title = "CRISPR/Cas12a toolbox for genome editing in Methanosarcina acetivorans",

abstract = "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.",

keywords = "CRISPR/Cas12a, genome editing, methanogens, Methanosarcina acetivorans, synthetic biology",

author = "Ping Zhu and Tejas Somvanshi and Jichen Bao and Silvan Scheller",

note = "Funding Information: This work was supported by Novo Nordisk Foundation (grant NNF19OC0054329 to SS; grant NNF20OC0065032 to JB) and the Academy of Finland (grant 326020 to SS). Funding Information: We thank the Novo Nordisk Foundation (grant NNF19OC0054329 to SS; grant NNF20OC0065032 to JB) and the Academy of Finland (grant 326020 to SS) for funding this research. We thank the Aalto Science Institute (AScI) project student Shreyash Borkar for helping with the plasmid transformation. We thank Prof. Michael Rother and Dr. Christian Sch{\"o}ne for testing the editing efficiency of our CRISPR/Cas12a system in their lab. We thank Dr. Ingemar von Ossowski for his helpful comments and suggestions. Publisher Copyright: Copyright {\textcopyright} 2023 Zhu, Somvanshi, Bao and Scheller.",

year = "2023",

month = dec,

day = "12",

doi = "10.3389/fmicb.2023.1235616",

language = "English",

volume = "14",

journal = "Frontiers in Microbiology",

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AU - Zhu, Ping

AU - Somvanshi, Tejas

AU - Bao, Jichen

AU - Scheller, Silvan

N1 - Funding Information: This work was supported by Novo Nordisk Foundation (grant NNF19OC0054329 to SS; grant NNF20OC0065032 to JB) and the Academy of Finland (grant 326020 to SS). Funding Information: We thank the Novo Nordisk Foundation (grant NNF19OC0054329 to SS; grant NNF20OC0065032 to JB) and the Academy of Finland (grant 326020 to SS) for funding this research. We thank the Aalto Science Institute (AScI) project student Shreyash Borkar for helping with the plasmid transformation. We thank Prof. Michael Rother and Dr. Christian Schöne for testing the editing efficiency of our CRISPR/Cas12a system in their lab. We thank Dr. Ingemar von Ossowski for his helpful comments and suggestions. Publisher Copyright: Copyright © 2023 Zhu, Somvanshi, Bao and Scheller.

PY - 2023/12/12

Y1 - 2023/12/12

N2 - 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.

AB - 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.

KW - CRISPR/Cas12a

KW - genome editing

KW - methanogens

KW - Methanosarcina acetivorans

KW - synthetic biology

UR - https://www.scopus.com/pages/publications/85180725324

U2 - 10.3389/fmicb.2023.1235616

DO - 10.3389/fmicb.2023.1235616

M3 - Article

AN - SCOPUS:85180725324

SN - 1664-302X

VL - 14

JO - Frontiers in Microbiology

JF - Frontiers in Microbiology

M1 - 1235616

ER -

CRISPR/Cas12a toolbox for genome editing in Methanosarcina acetivorans

Abstract

Funding

UN SDGs

Keywords

Access to Document

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BioEle: Biochemical interconversion of alkanes to CO2 and electricity

Cite this

CRISPR/Cas12a toolbox for genome editing in Methanosarcina acetivorans

Abstract

Funding

UN SDGs

Keywords

Access to Document

Other files and links

Fingerprint

Projects

BioEle: Biochemical interconversion of alkanes to CO2 and electricity

Cite this