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 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.

CRISPR/Cas9: a historical and chemical biology perspective of targeted genome engineering

2016 ◽  
Vol 45 (24) ◽  
pp. 6666-6684 ◽  
Author(s):  
Amrita Singh ◽  
Debojyoti Chakraborty ◽  
Souvik Maiti
Keyword(s):  
Genome Editing ◽  
Chemical Biology ◽  
Genome Engineering ◽  
A Genome

The development and adaptation of CRISPR–Cas9 as a genome editing tool and chemical biology approaches for modulating its activity.

scholarly journals Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Kyunghun Min ◽  
Yuichi Ichikawa ◽  
Carol A. Woolford ◽  
Aaron P. Mitchell
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Genetic Analysis ◽  
Genome Editing ◽  
Drug Targets ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Content Type ◽  
Biological Features ◽  
Link Type

ABSTRACT The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation. Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C. albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C. albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation.

scholarly journals Mechanistic Insights into theCis-andTrans-acting Deoxyribonuclease Activities of Cas12a

2018 ◽  
Author(s):  
Daan C. Swarts ◽  
Martin Jinek
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Cleavage Product ◽  
List Type ◽  
Francisella Novicida ◽  
Ssdna Binding ◽  
Double Stranded Dna ◽  
Target Dna ◽  
Dna Strands ◽  
Dna Strand

HIGHLIGHTSTarget ssDNA binding allosterically induces unblocking of the RuvC active sitePAM binding facilitates unwinding of dsDNA targetsNon-target DNA strand cleavage is prerequisite for target DNA strand cleavageAfter DNA cleavage, Cas12a releases the PAM-distal DNA productSUMMARYCRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA incisand non-target single stranded DNAs (ssDNA) intrans.To elucidate the molecular basis for both deoxyribonuclease cleavage modes, we performed structural and biochemical studies onFrancisella novicidaCas12a. We show how crRNA-target DNA strand hybridization conformationally activates Cas12a, triggering itstrans-acting, non-specific, single-stranded deoxyribonuclease activity. In turn,cis-cleavage of double-stranded DNA targets is a result of PAM-dependent DNA duplex unwinding and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent,trans-active state. Together, these results provide a revised model for the molecular mechanism of Cas12a enzymes that explains theircis- andtrans-acting deoxyribonuclease activities, and additionally contribute to improving Cas12a-based genome editing.

scholarly journals Tandem paired nicking promotes precise genome editing with scarce interference by p53

2019 ◽  
Author(s):  
Toshinori Hyodo ◽  
Md Lutfur Rahman ◽  
Sivasundaram Karnan ◽  
Takuji Ito ◽  
Atsushi Toyoda ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Dna Recombination ◽  
Genetic Changes ◽  
Double Stranded Dna ◽  
Cas9 Nuclease ◽  
Target Sites ◽  
P53 Activation ◽  
Additional Nucleotide

SummaryTargeted knock-in mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites. A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knock-in. Here, we demonstrate that tandem paired nicking, a method for targeted knock-in involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knock-in largely equivalent to that of the Cas9 nuclease-based approach. Tandem paired nicking seems to accomplish targeted knock-in via DNA recombination analogous to Holliday’s model, and creates intended genetic changes in the genome without introducing additional nucleotide changes such as silent mutations. Targeted knock-in through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering.

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 Highly efficient generation of bacteria leaf blight resistance and transgene-free rice using genome editing and multiplexed selection system

2020 ◽  
Author(s):  
Kun Yu ◽  
Zhiqiang Liu ◽  
Huaping Gui ◽  
Lizhao Geng ◽  
Juan Wei ◽  
...  
Keyword(s):  
Protein Binding ◽  
Genome Editing ◽  
Leaf Blight ◽  
Genomic Research ◽  
Blight Resistance ◽  
Selection System ◽  
Transcription Activator ◽  
Efficient Generation ◽  
A Genome ◽  
Selection Of

Abstract Background Rice leaf blight is a worldwide devastating disease caused by bacteria Xanthomonas oryzae pv. Oryzae (Xoo). The UPT (up-regulated by transcription activator-like 1 effector) box in promoter region of the rice Xa13 gene played a key role in Xoo pathogenicity. Mutation of key bacterial protein binding site in UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistant to bacterial.Highly efficient generation and selection transgene-free, edited plants helpful to shorten and simple the gene editing breeding process. Selective elimination of transgenic pollen of E0 plants can enrich proportion of E1 transgene-free offspring and expression of the color mark gene in seeds makes the selection of E2 plants is very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacteria leaf blight resistance and transgene-free rice plants.Results We introduced site specific mutations into the UPT box using CRISPR/Cas12a technology to hamper TAL (Transcription-Activator Like effectors) protein binding and gene activation, and generated genome edited rice with improved bacteria blight resistance. Transgenic pollens of E0 plants were eliminated by pollen specific expression of α-amylase gene Zmaa1, the proportion of transgene-free plants were enriched from 25% to 50% in single T-DNA insertion events in E1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced 50% cost and achieved up to 98.64% of accuracy for selection of transgene-free edited plants. Conclusion We demonstrated core nucleotide deletion in the UPT box of Xa13 promoter conferred resistance to rice blight and selection of transgene-free plants were boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding.

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 CRISPR/Cas12a mediated genome engineering in photosynthetic bacteria

2020 ◽  
Author(s):  
Yang Zhang ◽  
Jifeng Yuan
Keyword(s):  
Genome Editing ◽  
Transcriptional Repression ◽  
Gene Editing ◽  
Photosynthetic Bacteria ◽  
Genome Engineering ◽  
Biotechnological Applications ◽  
End Joining ◽  
Editing Efficiency ◽  
Francisella Novicida ◽  
Non Homologous End Joining

ABSTRACTPurple non-sulfur photosynthetic bacteria (PNSB) such as R. capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. In this study, we sought to develop the class II RNA-guided CRISPR/Cas12a system from Francisella novicida for both genome editing and gene down-regulation in R. capsulatus. About 90% editing efficiency was achieved by using CRISPR/Cas12a driven by a strong promoter Ppuc when targeting ccoO or nifH gene. When both genes were simultaneously targeted, the multiplex gene editing efficiency reached >63%. In addition, CRISPR interference using deactivated Cas12a was also evaluated using reporter genes gfp and lacZ, and the repression efficiency reached >80%. In summary, our work represents the first report to develop CRISPR/Cas12a mediated genome editing/transcriptional repression in R. capsulatus, which would greatly accelerate PNSB-related researches.IMPORTANCEPurple non-sulfur photosynthetic bacteria (PNSB) such as R. capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. However, lack of efficient gene editing tools remains a main obstacle for progressing in PNSB-related researches. Here, we developed CRISPR/Cas12a for genome editing via the non-homologous end joining (NHEJ) repair machinery in R. capsulatus. In addition, DNase-deactivated Cas12a was found to simultaneously suppress multiple targeted genes. Taken together, our work offers a new set of tools for efficient genome engineering in PNSB such as R. capsulatus.

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