scholarly journals Multiplex genomic edition in Ashbya gossypii using CRISPR-Cpf1

2019 ◽  
Author(s):  
Alberto Jiménez ◽  
Birgit Hoff ◽  
José Luis Revuelta
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Marker Genes ◽  
Target Sequence ◽  
Ashbya Gossypii ◽  
Nucleotide Substitutions ◽  
Large Deletions ◽  
Auxotrophic Marker ◽  
Selection Of

AbstractThe CRISPR/Cas technologies constitute an essential tool for rapid genome engineering of many organisms, including fungi. The CRISPR/Cas9 system adapted for the industrial fungus Ashbya gossypii enables the efficient genome editing for the introduction of deletions, insertions and nucleotide substitutions. However, the Cas9 system is constrained to the existence of an specific 5’-NGG-3’ PAM sequence in the target site.Here we present a new CRISPR/Cas system for A. gossypii that expands the molecular toolbox available for microbial engineering of this fungus. The use of Cpf1 nuclease from Lachnospiraceae bacterium allows to employ a T-rich PAM sequence (5’-TTTN-3’) and facilitates the implementation of a multiplexing CRISPR/Cpf1 system adapted for A. gossypii. The system has been validated for the introduction of large deletions into five different auxotrophic marker genes (HIS3, ADE2, TRP1, LEU2 and URA3). The use of both crRNA and dDNA arrays in a multi-CRISPR/Cpf1 system was demonstrated to be an efficient strategy for multiplex gene deletion of up to four genes using a single multi-CRISPR/Cpf1 plasmid. Our results also suggest that the selection of the target sequence may significantly affect to the edition efficiency of the system.

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 The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato

2019 ◽  
Author(s):  
Florian Veillet ◽  
Laura Chauvin ◽  
Marie-Paule Kermarrec ◽  
François Sevestre ◽  
Mathilde Merrer ◽  
...  
Keyword(s):  
Solanum Tuberosum ◽  
Genome Editing ◽  
Genome Engineering ◽  
Basic Research ◽  
Loss Of Function ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Discrete Variation ◽  
Tetraploid Potato ◽  
Base Editing

AbstractGenome editing has recently become a method of choice for basic research and functional genomics, and holds great potential for molecular plant breeding applications. The powerful CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to generate knockout mutants. Recently, the development of base editors has broadened the scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knockout the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective screening strategy based on high-resolution melting analysis followed by direct Sanger sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but unwanted insertions of plasmid DNA were also observed. We successfully created tetra-allelic mutants with impaired amylose biosynthesis, confirming the loss-of-function of the StGBSSI protein. The second main objective of this work was to demonstrate the proof of concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete variation in the amino acid sequence and generating a loss-of-function allele. The successful application of base editing in the tetraploid potato opens up new avenues for genome engineering in this species.Key MessageThe StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base editing strategies, leading to plants with impaired amylose biosynthesis.

scholarly journals A portable CRISPR-Cas9N system for flexible genome engineering in Lactobacillus acidophilus, Lactobacillus gasseri and Lactobacillus paracasei.

2021 ◽  
Author(s):  
Yong Jun Goh ◽  
Rodolphe Barrangou
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Genome Editing ◽  
Lactobacillus Acidophilus ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Lactobacillus Paracasei ◽  
Universal System ◽  
Lactobacillus Gasseri ◽  
Health Promoting

Diverse Lactobacillus strains are widely used as probiotic cultures in the dairy and dietary supplements industries, and specific strains such as Lactobacillus acidophilus NCFM have been engineered for the development of biotherapeutics. To expand the Lactobacillus manipulation toolbox with enhanced efficiency and ease, we present here a CRISPR-SpyCas9D10A nickase (Cas9N)-based system for programmable engineering of L. acidophilus NCFM, a model probiotic bacterium. Successful single-plasmid delivery system was achieved with the engineered pLbCas9N vector harboring cas9N under the regulation of a Lactobacillus promoter and a cloning region for customized sgRNA and editing template. The functionality of the pLbCas9N system was validated in NCFM with targeted chromosomal deletions ranging between 300 bp and 1.9 kb at various loci (rafE, lacS and ltaS), yielding 35-100% mutant recovery rates. Genome analysis of the mutants confirmed precision and specificity of the pLbCas9N system. To showcase the versatility of this system, we also inserted a mCherry fluorescent protein gene downstream of the pgm gene to create a polycistronic transcript. The pLbCas9N system was further deployed in other species to generate concurrent single base substitution and gene deletion in Lactobacillus gasseri ATCC 33323, and an in-frame gene deletion in Lactobacillus paracasei Lpc-37, highlighting the portability of the system in phylogenetically distant Lactobacillus species, where its targeting activity was not interfered by endogenous CRISPR-Cas systems. Collectively, these editing outcomes illustrate the robustness and versatility of the pLbCas9N system for genome manipulations in diverse lactobacilli, and open new avenues for the engineering of health-promoting lactic acid bacteria. Importance This work describes the development of a broad-host range CRISPR-based editing system for genome manipulations in three Lactobacillus species, which belong to lactic acid bacteria (LAB) commonly known for their long history of use in food fermentations and as indigenous members of healthy microbiota, and their emerging roles in human and animal commercial health-promoting applications.  We exploited the established CRISPR-SpyCas9 nickase for flexible and precise genome editing applications in Lactobacillus acidophilus, and further demonstrated the efficacy of this universal system in two distantly related Lactobacillus species.  This versatile Cas9-based system facilitates genome engineering compared to conventional gene replacement systems, and represents a valuable gene editing modality in species that do not possess native CRISPR-Cas systems.  Overall, this portable tool contributes to expanding the genome editing toolbox of LAB for studying their health-promoting mechanisms and engineering of these beneficial microbes as next-generation vaccines and designer probiotics.

scholarly journals Establishment and application of a CRISPR–Cas12a assisted genome-editing system in Zymomonas mobilis

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Wei Shen ◽  
Jun Zhang ◽  
Binan Geng ◽  
Mengyue Qiu ◽  
Mimi Hu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Zymomonas Mobilis ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Strain Improvement ◽  
Genomic Research ◽  
Francisella Novicida ◽  
A Genome

Abstract Background Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. Results Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR–Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR–Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR–Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. Conclusions This study applied CRISPR–Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR–Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

scholarly journals Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

2016 ◽  
Vol 82 (17) ◽  
pp. 5421-5427 ◽  
Author(s):  
Josef Altenbuchner
Keyword(s):  
Bacillus Subtilis ◽  
Genome Editing ◽  
Genome Engineering ◽  
Shuttle Vector ◽  
Temperature Sensitive ◽  
Target Sequence ◽  
Target Specificity ◽  
Content Type ◽  
A Genome ◽  
Single Plasmid

ABSTRACTThe clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system fromStreptococcus pyogeneshas recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing ofBacillus subtilis. The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication forEscherichia coli, the temperature-sensitive replication origin of plasmid pE194tsforB. subtilis, and a kanamycin resistance gene working in both organisms. For genome editing, it carries thecas9gene under the control of theB. subtilismannose-inducible promoter PmanPand a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. This sgRNA guides the Cas9 nuclease to its target. The 20-nucleotide spacer sequence at the 5′ end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site. Repair of the DSBs and the required modification of the genome are achieved by adding homology templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homology templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in theB. subtilischromosome and by repair of thetrpC2mutation ofB. subtilis168.IMPORTANCEIn prokaryotes, most methods used for scarless genome engineering are based on selection-counterselection systems. The disadvantages are often the lack of a suitable counterselection marker, the toxicity of the compounds needed for counterselection, and the requirement of certain mutations in the target strain. CRISPR-Cas systems were recently developed as important tools for genome editing. The single-plasmid system constructed for the genome editing ofB. subtilisovercomes the problems of counterselection methods. It allows deletions and introduction of point mutations. It is easy to handle and very efficient, and it may be adapted for use in other firmicutes.

scholarly journals One-plasmid based CRISPR-Cas9 editing for gene deletion in Lactococcus lactis

2021 ◽  
Author(s):  
José Miguel Miquelão Santos ◽  
Gabriel António Amaro Monteiro ◽  
Duarte Miguel de França Teixeira dos Prazeres ◽  
Sofia de Oliveira Dias Duarte
Keyword(s):  
Genome Editing ◽  
Lactococcus Lactis ◽  
Plasmid Dna ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Deletion Mutants ◽  
Cell Factories ◽  
Yield And Quality ◽  
Delivery Vehicles

Abstract Lactococcus lactis strains are promising cell factories and delivery vehicles of plasmid DNA and recombinant protein for therapeutic applications. However, the limited yields of recombinant molecules obtained with these bacteria limits their wide applicability. Genome engineering of this host may solve the problem. However, the current genome editing toolbox available for L. lactis is either too laborious or incapable of large edits, limiting the scope of strain editing experiments. In this work, the basis for a one-plasmid CRISPR-Cas9 based genome editing plasmid was developed and tested. The new plasmid (pTCas9dO) adapted from the pKCcas9dO plasmid was used to delete 657 bp of the lactococcal nuclease nth of L. lactis subsp. lactis LMG19460, with the aim of improving yield and quality of plasmid DNA replicated in this strain. Although deletion mutants were successfully generated, plasmid curing was unsuccessful. Thus, further modifications are required before the plasmid is truly applicable for genome editing experiments. Unexpectedly, the generated deletion mutants generated a roughly 40% decrease in plasmid yield alongside with a decrease in the quality of produced pDNA.

scholarly journals Inducible Plasmid Self-Destruction (IPSD) assisted genome engineering in lactobacilli and bifidobacteria

2019 ◽  
Author(s):  
Fanglei Zuo ◽  
Zhu Zeng ◽  
Lennart Hammarström ◽  
Harold Marcotte
Keyword(s):  
Synthetic Biology ◽  
Homologous Recombination ◽  
Genetic Engineering ◽  
Genome Editing ◽  
Genome Engineering ◽  
Bacterial Species ◽  
Knock Out ◽  
Recombination System ◽  
A Genome ◽  
Selection Of

ABSTRACTGenome engineering is essential for application of synthetic biology in probiotics including lactobacilli and bifidobacteria. Several homologous recombination system-based mutagenesis tools have been developed for these bacteria but still, have many limitations in different species or strains. Here we developed a genome engineering method based on an inducible self-destruction plasmid delivering homologous DNA into bacteria. Excision of the replicon by induced recombinase facilitates selection of homologous recombination events. This new genome editing tool called Inducible Plasmid Self-Destruction (IPSD) was successfully used to perform gene knock-out and knock-in in lactobacilli and bifidobacteria. Due to its simplicity and universality, the IPSD strategy may provide a general approach for genetic engineering of various bacterial species.

scholarly journals Universal toxin-based selection for precise genome engineering in human cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Songyuan Li ◽  
Nina Akrap ◽  
Silvia Cerboni ◽  
Michelle J. Porritt ◽  
Sandra Wimberger ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Restriction Enzymes ◽  
Genetically Engineered ◽  
Human Cells ◽  
Selection Strategy ◽  
Nucleotide Substitutions ◽  
Base Editing ◽  
Selection For

AbstractProkaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems.

SELECTION OF PREPARENTAL LINES OF PLYMOUTH ROCK CHICKEN USING MARKER GENES K AND k

2018 ◽  
Vol 53 (6) ◽  
pp. 1162-1168
Author(s):  
D.N. Efimov ◽  
◽  
Zh.V. Emanuylova ◽  
E.V. Zhuravleva ◽  
A.V. Egorova ◽  
...  
Keyword(s):  
Marker Genes ◽  
Selection Of

scholarly journals Organelle Genetics in Plants

2021 ◽  
Vol 22 (4) ◽  
pp. 2104
Author(s):  
Pedro Robles ◽  
Víctor Quesada
Keyword(s):  
Rna Editing ◽  
Posttranscriptional Regulation ◽  
Genome Engineering ◽  
Plant Mitochondria ◽  
Plastid Dna ◽  
Chloroplast Genomes ◽  
Wide Range ◽  
Organelle Genetics ◽  
Selection Of

Eleven published articles (4 reviews, 7 research papers) are collected in the Special Issue entitled “Organelle Genetics in Plants.” This selection of papers covers a wide range of topics related to chloroplasts and plant mitochondria research: (i) organellar gene expression (OGE) and, more specifically, chloroplast RNA editing in soybean, mitochondria RNA editing, and intron splicing in soybean during nodulation, as well as the study of the roles of transcriptional and posttranscriptional regulation of OGE in plant adaptation to environmental stress; (ii) analysis of the nuclear integrants of mitochondrial DNA (NUMTs) or plastid DNA (NUPTs); (iii) sequencing and characterization of mitochondrial and chloroplast genomes; (iv) recent advances in plastid genome engineering. Here we summarize the main findings of these works, which represent the latest research on the genetics, genomics, and biotechnology of chloroplasts and mitochondria.

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