Bacterial Genome Editing with CRISPR-Cas9: Taking Clostridium beijerinckii as an Example

2018 ◽  
pp. 297-325 ◽  
Author(s):  
Zhong-Tian Zhang ◽  
Pablo Jiménez-Bonilla ◽  
Seung-Oh Seo ◽  
Ting Lu ◽  
Yong-Su Jin ◽  
...  
Keyword(s):  
Genome Editing ◽  
Bacterial Genome ◽  
Clostridium Beijerinckii

scholarly journals Generalized bacterial genome editing using mobile group II introns and Cre‐ lox

2013 ◽  
Vol 9 (1) ◽  
pp. 685 ◽  
Author(s):  
Peter J Enyeart ◽  
Steven M Chirieleison ◽  
Mai N Dao ◽  
Jiri Perutka ◽  
Erik M Quandt ◽  
...  
Keyword(s):  
Genome Editing ◽  
Bacterial Genome ◽  
Group Ii Introns ◽  
Group Ii

scholarly journals Characterizing a thermostable Cas9 for bacterial genome editing and silencing

2017 ◽  
Author(s):  
Ioannis Mougiakos ◽  
Prarthana Mohanraju ◽  
Elleke F. Bosma ◽  
Valentijn Vrouwe ◽  
Max Finger Bou ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Elevated Temperatures ◽  
Bacterial Genome ◽  
Thermophilic Bacterium ◽  
Geobacillus Thermodenitrificans ◽  
Temperature Range ◽  
Fundamental Research

AbstractCRISPR-Cas9 based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here, we identify and characterize ThermoCas9: an RNA-guided DNA-endonuclease from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that ThermoCas9 is active in vitro between 20°C and 70°C, a temperature range much broader than that of the currently used Cas9 orthologues. Additionally, we demonstrate that ThermoCas9 activity at elevated temperatures is strongly associated with the structure of the employed sgRNA. Subsequently, we develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55°C in Bacillus smithii and for gene deletion at 37°C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish the first Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

scholarly journals Characterizing a thermostable Cas9 for bacterial genome editing and silencing

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Ioannis Mougiakos ◽  
Prarthana Mohanraju ◽  
Elleke F. Bosma ◽  
Valentijn Vrouwe ◽  
Max Finger Bou ◽  
...  
Keyword(s):  
Genome Editing ◽  
Bacterial Genome

scholarly journals A standardized workflow for surveying recombinases expands bacterial genome-editing capabilities

2017 ◽  
Vol 11 (1) ◽  
pp. 176-188 ◽  
Author(s):  
Deirdre E. Ricaurte ◽  
Esteban Martínez-García ◽  
Ákos Nyerges ◽  
Csaba Pál ◽  
Víctor de Lorenzo ◽  
...  
Keyword(s):  
Genome Editing ◽  
Bacterial Genome

Bacterial Genome Editing via a Designed Toxin–Antitoxin Cassette

2017 ◽  
Vol 7 (3) ◽  
pp. 822-831 ◽  
Author(s):  
Jie Wu ◽  
Aihua Deng ◽  
Qinyun Sun ◽  
Hua Bai ◽  
Zhaopeng Sun ◽  
...  
Keyword(s):  
Genome Editing ◽  
Bacterial Genome

scholarly journals Recent advances of Cas12a applications in bacteria

2021 ◽  
Author(s):  
Meliawati Meliawati ◽  
Christoph Schilling ◽  
Jochen Schmid
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Genome Engineering ◽  
Bacterial Genome ◽  
Adaptive Immune System ◽  
Eukaryotic Cell ◽  
Promising Alternative ◽  
Guide Rna ◽  
Recent Advances ◽  
Versatile Tool

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome engineering and related technologies have revolutionized biotechnology over the last decade by enhancing the efficiency of sophisticated biological systems. Cas12a (Cpf1) is an RNA-guided endonuclease associated to the CRISPR adaptive immune system found in many prokaryotes. Contrary to its more prominent counterpart Cas9, Cas12a recognizes A/T rich DNA sequences and is able to process its corresponding guide RNA directly, rendering it a versatile tool for multiplex genome editing efforts and other applications in biotechnology. While Cas12a has been extensively used in eukaryotic cell systems, microbial applications are still limited. In this review, we highlight the mechanistic and functional differences between Cas12a and Cas9 and focus on recent advances of applications using Cas12a in bacterial hosts. Furthermore, we discuss advantages as well as current challenges and give a future outlook for this promising alternative CRISPR-Cas system for bacterial genome editing and beyond. Key points • Cas12a is a powerful tool for genome engineering and transcriptional perturbation • Cas12a causes less toxic side effects in bacteria than Cas9 • Self-processing of crRNA arrays facilitates multiplexing approaches

Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability

2018 ◽  
pp. 27-37 ◽  
Author(s):  
Ida Lauritsen ◽  
Virginia Martínez ◽  
Carlotta Ronda ◽  
Alex Toftgaard Nielsen ◽  
Morten H. H. Nørholm
Keyword(s):  
Protein Stability ◽  
Genome Editing ◽  
Bacterial Genome

Harnessing CRISPR–Cas systems for bacterial genome editing

2015 ◽  
Vol 23 (4) ◽  
pp. 225-232 ◽  
Author(s):  
Kurt Selle ◽  
Rodolphe Barrangou
Keyword(s):  
Genome Editing ◽  
Bacterial Genome

scholarly journals Alteration of Salmonella enterica Virulence and Host Pathogenesis through Targeting sdiA by Using the CRISPR-Cas9 System

2021 ◽  
Vol 9 (12) ◽  
pp. 2564
Author(s):  
Momen Askoura ◽  
Ahmad J. Almalki ◽  
Amr S. Abu Lila ◽  
Khaled Almansour ◽  
Farhan Alshammari ◽  
...  
Keyword(s):  
Genome Editing ◽  
Salmonella Enterica ◽  
Bacterial Genome ◽  
Infection Model ◽  
Wild Type ◽  
Signal Molecule ◽  
Enteric Infections ◽  
The Impact

Salmonella enterica is a common cause of many enteric infections worldwide and is successfully engineered to deliver heterologous antigens to be used as vaccines. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA-guided Cas9 endonuclease is a promising genome editing tool. In the current study, a CRISPR-Cas9 system was used to target S.enterica sdiA that encodes signal molecule receptor SdiA and responds to the quorum sensing (QS) signaling compounds N-acylhomoserine lactones (AHLs). For this purpose, sdiA was targeted in both S.enterica wild type (WT) and the ΔssaV mutant strain, where SsaV has been reported to be an essential component of SPI2-T3SS. The impact of sdiA mutation on S. enterica virulence was evaluated at both early invasion and later intracellular replication in both the presence and absence of AHL. Additionally, the influence of sdiA mutation on the pathogenesis S. enterica WT and mutants was investigated in vivo, using mice infection model. Finally, the minimum inhibitory concentrations (MICs) of various antibiotics against S. enterica strains were determined. Present findings show that mutation in sdiA significantly affects S.enterica biofilm formation, cell adhesion and invasion. However, sdiA mutation did not affect bacterial intracellular survival. Moreover, in vivo bacterial pathogenesis was markedly lowered in S.enterica ΔsdiA in comparison with the wild-type strain. Significantly, double-mutant sdiA and ssaV attenuated the S. enterica virulence and in vivo pathogenesis. Moreover, mutations in selected genes increased Salmonella susceptibility to tested antibiotics, as revealed by determining the MICs and MBICs of these antibiotics. Altogether, current results clearly highlight the importance of the CRISPR-Cas9 system as a bacterial genome editing tool and the valuable role of SdiA in S.enterica virulence. The present findings extend the understanding of virulence regulation and host pathogenesis of Salmonellaenterica.

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