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

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

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 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 CRISPR/Cas9-based genome editing for simultaneous interference with gene expression and protein stability

2017 ◽  
Vol 45 (20) ◽  
pp. e171-e171 ◽  
Author(s):  
Virginia Martínez ◽  
Ida Lauritsen ◽  
Tonja Hobel ◽  
Songyuan Li ◽  
Alex Toftgaard Nielsen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Stability ◽  
Genome Editing

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

scholarly journals Importance of the Q/N-rich Segment for Protein Stability and Activity of Endogenous Mouse TDP-43

2021 ◽  
Author(s):  
Toshiya Sato ◽  
Kanako Oda ◽  
Seiko Sakai ◽  
Rika Kato ◽  
Saori Yamamori ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Subcellular Localization ◽  
Protein Stability ◽  
Genome Editing ◽  
Nuclear Protein ◽  
Dna Binding Protein ◽  
Mouse Development ◽  
The Stability ◽  
Lateral Sclerosis ◽  
Mouse Lines

Abstract TAR DNA-binding protein 43 kDa (TDP-43), a nuclear protein, plays an important role in the molecular pathogenesis of amyotrophic lateral sclerosis (ALS). TDP-43 aggregation and translocation out of the nucleus are crucial factors in ALS. TDP-43 aggregation results from its resistance to degradation, to which the long-disordered C-terminal region (CTR) is thought to contribute. The CTR has two Gly, aromatic, and Ser-rich (GaroS) segments and an amyloidogenic core divided into a hydrophobic patch and a Gln/Asn (Q/N)-rich segment. Although TDP-43 lacking the CTR is known to be unstable, as observed in knock-in mice, it is unclear which of these segments contributes to the stability of TDP-43. Here, we generated 12 mouse lines lacking the various sub-regions of CTR by genome editing and compared the protein stability, activity, and subcellular localization of TDP-43. We demonstrated the functional diversity of the four segments of CTR, finding that the presence of Q/N-rich segment greatly restored the protein stability and activity of TDP-43. In addition, we found that the second GaroS deletion did not affect protein stability and mouse development.

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