Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification, and Desirable “Clean” Mutant Selection inClostridium beijerinckiias an Example

2016 ◽  
Vol 5 (7) ◽  
pp. 721-732 ◽  
Author(s):  
Yi Wang ◽  
Zhong-Tian Zhang ◽  
Seung-Oh Seo ◽  
Patrick Lynn ◽  
Ting Lu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Bacterial Genome ◽  
Mutant Selection ◽  
Single Nucleotide ◽  
Nucleotide Modification

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

Single Nucleotide Substitutions Effectively Block Cas9 and Allow for Scarless Genome Editing in Caenorhabditis elegans

2021 ◽  
Author(s):  
Jeffrey C Medley ◽  
Shilpa Hebbar ◽  
Joel T Sydzyik ◽  
Anna Y. Zinovyeva
Keyword(s):  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Paternal Genome ◽  
Single Nucleotide ◽  
C Elegans ◽  
Homology Directed Repair ◽  
Maternal Genome ◽  
Guide Sequence

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize off-target sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts post-injection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

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 Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiwei Hu ◽  
Yannan Wang ◽  
Qian Liu ◽  
Yan Qiu ◽  
Zhiyu Zhong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Target Sites ◽  
Guide Sequence ◽  
Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

scholarly journals Reassessment of Viroid RNA Cytosine Methylation Status at the Single Nucleotide Level

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 357
Author(s):  
Francesco Di Serio ◽  
Enza Maria Torchetti ◽  
José-Antonio Daròs ◽  
Beatriz Navarro
Keyword(s):  
Cytosine Methylation ◽  
Methylation Status ◽  
Potato Spindle Tuber Viroid ◽  
Nucleotide Level ◽  
Single Nucleotide ◽  
Protein Coding ◽  
Nucleotide Modification ◽  
Rna Structural Motifs ◽  
Infectious Cycle ◽  
Positive Results

Composed of a few hundreds of nucleotides, viroids are infectious, circular, non-protein coding RNAs able to usurp plant cellular enzymes and molecular machineries to replicate and move in their hosts. Several secondary and tertiary RNA structural motifs have been implicated in the viroid infectious cycle, but whether modified nucleotides, such as 5C-methylcytosine (m5C), also play a role has not been deeply investigated so far. Here, the possible existence of m5C in both RNA polarity strands of potato spindle tuber viroid and avocado sunblotch viroid -which are representative members of the nucleus- and chloroplast-replicating viroids, respectively- has been assessed at single nucleotide level. We show that a standard bisulfite protocol efficiently used for identifying m5C in cellular RNAs may generate false positive results in the case of the highly structured viroid RNAs. Applying a bisulfite conversion protocol specifically adapted to RNAs with high secondary structure, no m5C was identified in both polarity strands of both viroids, indicating that this specific nucleotide modification does not likely play a role in viroid biology.

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 Prediction of synonymous corrections by the BE-FF computational tool expands the targeting scope of base editing

2020 ◽  
Author(s):  
Rabinowitz Roy ◽  
Abadi Shiran ◽  
Almog Shiri ◽  
Offen Daniel
Keyword(s):  
Genome Editing ◽  
Point Mutations ◽  
Nucleotide Variation ◽  
Single Nucleotide Variation ◽  
Computational Tool ◽  
Single Nucleotide ◽  
Base Editing ◽  
A Genome ◽  
Wide Range ◽  
Reference Protein

ABSTRACTBase editing is a genome-editing approach that employs the CRISPR/Cas system to precisely install point mutations within the genome. A cytidine or adenosine deaminase enzyme is fused to a deactivated Cas and converts C to T or A to G, respectively. The diversified repertoire of base editors, varied in their Cas and deaminase proteins, provides a wide range of functionality. However, existing base-editors can only induce transition substitutions in a specified region determined by the base editor, thus, they are incompatible for many point mutations. Here, we present BE-FF (Base Editors Functional Finder), a novel computational tool that identifies suitable base editors to correct the translated sequence erred by a given single nucleotide variation. Even if a perfect correction of the single nucleotide variation is not possible, BE-FF detects synonymous corrections to produce the reference protein. To assess the potential of BE-FF, we analysed a database of human pathogenic point mutations and found suitable base editors for 60.9% of the transition mutations. Importantly, 19.4% of them were made possible only by synonymous corrections. Moreover, we detected 298 cases in which pathogenic mutations caused by transversions were potentially repairable by base editing via synonymous corrections, although it had been thought impractical. The BE-FF tool and the database are available at https://www.danioffenlab.com/be-ff.GRAPHICAL ABSTRACT

scholarly journals Mutant Selection Window and Characterization of Allelic Diversity for Ciprofloxacin-Resistant Mutants of Rhodococcus equi

2010 ◽  
Vol 54 (8) ◽  
pp. 3520-3523 ◽  
Author(s):  
Hidekazu Niwa ◽  
Brent A. Lasker
Keyword(s):  
Amino Acid ◽  
Allelic Diversity ◽  
Dna Gyrase ◽  
Rhodococcus Equi ◽  
Mutant Selection ◽  
Single Nucleotide ◽  
Selection Window ◽  
Mutant Prevention Concentration ◽  
Resistant Mutants

ABSTRACT The mutant prevention concentration (MPC) for ciprofloxacin was determined for two Rhodococcus equi strains. The MPC for both strains was 32 μg/ml, which is above the peak serum concentration of ciprofloxacin obtainable by oral administration in humans. Nine single nucleotide changes corresponding to eight amino acid substitutions in the quinolone resistance-determining regions of DNA gyrase subunits A and B were characterized. Only mutants with amino acid changes in Ser-83 of GyrA were highly resistant (≥64 μg/ml). Our results suggest that ciprofloxacin monotherapy against R. equi infection may result in the emergence of ciprofloxacin-resistant mutants.

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
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