Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification, and Desirable "clean" Mutant Selection in Clostridium beijerinckii as an Example

Yi Wang, Zhong Tian Zhang, Seung Oh Seo, Patrick Lynn, Ting Lu, Yong-Su Jin, Hans-Peter M Blaschek

Research output: Contribution to journalArticle

Abstract

CRISPR-Cas9 has been demonstrated as a transformative genome engineering tool for many eukaryotic organisms; however, its utilization in bacteria remains limited and ineffective. Here we explored Streptococcus pyogenes CRISPR-Cas9 for genome editing in Clostridium beijerinckii (industrially significant but notorious for being difficult to metabolically engineer) as a representative attempt to explore CRISPR-Cas9 for genome editing in microorganisms that previously lacked sufficient genetic tools. By combining inducible expression of Cas9 and plasmid-borne editing templates, we successfully achieved gene deletion and integration with high efficiency in single steps. We further achieved single nucleotide modification by applying innovative two-step approaches, which do not rely on availability of Protospacer Adjacent Motif sequences. Severe vector integration events were observed during the genome engineering process, which is likely difficult to avoid but has never been reported by other researchers for the bacterial genome engineering based on homologous recombination with plasmid-borne editing templates. We then further successfully employed CRISPR-Cas9 as an efficient tool for selecting desirable "clean" mutants in this study. The approaches we developed are broadly applicable and will open the way for precise genome editing in diverse microorganisms.

Original languageEnglish (US)
Pages (from-to)721-732
Number of pages12
JournalACS synthetic biology
Volume5
Issue number7
DOIs
StatePublished - Jul 15 2016

Fingerprint

Clostridium beijerinckii
Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Genomes
Clostridium
Nucleotides
Genes
Plasmids
Genome
Streptococcus pyogenes
Homologous Recombination
Gene Deletion
Microorganisms
Research Personnel
Bacteria
Gene Editing
Availability
Engineers

Keywords

  • CRISPR-Cas9
  • genome engineering
  • homologous recombination
  • single nucleotide modification (SNM)
  • synthetic biology
  • vector integration event (VIE)

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

Cite this

@article{2fcfc74d7dc1465d9d5f3c48d6e1f519,
title = "Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification, and Desirable {"}clean{"} Mutant Selection in Clostridium beijerinckii as an Example",
abstract = "CRISPR-Cas9 has been demonstrated as a transformative genome engineering tool for many eukaryotic organisms; however, its utilization in bacteria remains limited and ineffective. Here we explored Streptococcus pyogenes CRISPR-Cas9 for genome editing in Clostridium beijerinckii (industrially significant but notorious for being difficult to metabolically engineer) as a representative attempt to explore CRISPR-Cas9 for genome editing in microorganisms that previously lacked sufficient genetic tools. By combining inducible expression of Cas9 and plasmid-borne editing templates, we successfully achieved gene deletion and integration with high efficiency in single steps. We further achieved single nucleotide modification by applying innovative two-step approaches, which do not rely on availability of Protospacer Adjacent Motif sequences. Severe vector integration events were observed during the genome engineering process, which is likely difficult to avoid but has never been reported by other researchers for the bacterial genome engineering based on homologous recombination with plasmid-borne editing templates. We then further successfully employed CRISPR-Cas9 as an efficient tool for selecting desirable {"}clean{"} mutants in this study. The approaches we developed are broadly applicable and will open the way for precise genome editing in diverse microorganisms.",
keywords = "CRISPR-Cas9, genome engineering, homologous recombination, single nucleotide modification (SNM), synthetic biology, vector integration event (VIE)",
author = "Yi Wang and Zhang, {Zhong Tian} and Seo, {Seung Oh} and Patrick Lynn and Ting Lu and Yong-Su Jin and Blaschek, {Hans-Peter M}",
year = "2016",
month = "7",
day = "15",
doi = "10.1021/acssynbio.6b00060",
language = "English (US)",
volume = "5",
pages = "721--732",
journal = "ACS Synthetic Biology",
issn = "2161-5063",
publisher = "American Chemical Society",
number = "7",

}

TY - JOUR

T1 - Bacterial Genome Editing with CRISPR-Cas9

T2 - Deletion, Integration, Single Nucleotide Modification, and Desirable "clean" Mutant Selection in Clostridium beijerinckii as an Example

AU - Wang, Yi

AU - Zhang, Zhong Tian

AU - Seo, Seung Oh

AU - Lynn, Patrick

AU - Lu, Ting

AU - Jin, Yong-Su

AU - Blaschek, Hans-Peter M

PY - 2016/7/15

Y1 - 2016/7/15

N2 - CRISPR-Cas9 has been demonstrated as a transformative genome engineering tool for many eukaryotic organisms; however, its utilization in bacteria remains limited and ineffective. Here we explored Streptococcus pyogenes CRISPR-Cas9 for genome editing in Clostridium beijerinckii (industrially significant but notorious for being difficult to metabolically engineer) as a representative attempt to explore CRISPR-Cas9 for genome editing in microorganisms that previously lacked sufficient genetic tools. By combining inducible expression of Cas9 and plasmid-borne editing templates, we successfully achieved gene deletion and integration with high efficiency in single steps. We further achieved single nucleotide modification by applying innovative two-step approaches, which do not rely on availability of Protospacer Adjacent Motif sequences. Severe vector integration events were observed during the genome engineering process, which is likely difficult to avoid but has never been reported by other researchers for the bacterial genome engineering based on homologous recombination with plasmid-borne editing templates. We then further successfully employed CRISPR-Cas9 as an efficient tool for selecting desirable "clean" mutants in this study. The approaches we developed are broadly applicable and will open the way for precise genome editing in diverse microorganisms.

AB - CRISPR-Cas9 has been demonstrated as a transformative genome engineering tool for many eukaryotic organisms; however, its utilization in bacteria remains limited and ineffective. Here we explored Streptococcus pyogenes CRISPR-Cas9 for genome editing in Clostridium beijerinckii (industrially significant but notorious for being difficult to metabolically engineer) as a representative attempt to explore CRISPR-Cas9 for genome editing in microorganisms that previously lacked sufficient genetic tools. By combining inducible expression of Cas9 and plasmid-borne editing templates, we successfully achieved gene deletion and integration with high efficiency in single steps. We further achieved single nucleotide modification by applying innovative two-step approaches, which do not rely on availability of Protospacer Adjacent Motif sequences. Severe vector integration events were observed during the genome engineering process, which is likely difficult to avoid but has never been reported by other researchers for the bacterial genome engineering based on homologous recombination with plasmid-borne editing templates. We then further successfully employed CRISPR-Cas9 as an efficient tool for selecting desirable "clean" mutants in this study. The approaches we developed are broadly applicable and will open the way for precise genome editing in diverse microorganisms.

KW - CRISPR-Cas9

KW - genome engineering

KW - homologous recombination

KW - single nucleotide modification (SNM)

KW - synthetic biology

KW - vector integration event (VIE)

UR - http://www.scopus.com/inward/record.url?scp=84978699037&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84978699037&partnerID=8YFLogxK

U2 - 10.1021/acssynbio.6b00060

DO - 10.1021/acssynbio.6b00060

M3 - Article

C2 - 27115041

AN - SCOPUS:84978699037

VL - 5

SP - 721

EP - 732

JO - ACS Synthetic Biology

JF - ACS Synthetic Biology

SN - 2161-5063

IS - 7

ER -