scholarly journals Efficient Genome Editing in Bacillus licheniformis Mediated by a Conditional CRISPR/Cas9 System

2020 ◽  
Vol 8 (5) ◽  
pp. 754
Author(s):  
Youran Li ◽  
Hanrong Wang ◽  
Liang Zhang ◽  
Zhongyang Ding ◽  
Sha Xu ◽  
...  
Keyword(s):  
Success Rate ◽  
Genome Editing ◽  
Bacillus Licheniformis ◽  
Genetic Manipulation ◽  
Inducible Promoter ◽  
Bacterial Cells ◽  
Protospacer Adjacent Motif ◽  
Constitutive Overexpression ◽  
Short Palindromic Repeat ◽  
Overexpression System

Bacillus licheniformis is widely used to produce multiple enzymes and chemicals in industrial fermentation. It is also an organism that is hard to genetically manipulate, which is mainly attributed to its extremely low transformation efficiency. The lack of genetic modification technology severely limits its further application. In this study, an all-in-one conditional clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 plasmid was developed for B. licheniformis with the cas9 gene under the control of a xylose-inducible promoter. By means of this design, the expression of the cas9 gene could be repressed without xylose, which significantly improved the transformation ratio from less than 0.1 cfu/μg to 2.42 cfu/μg DNA. Compared with this conditional system, a constitutive overexpression system led to significant growth retardation in bacterial cells. Both the biomass and specific growth rate decreased greatly. After transformation, successful genome editing could be triggered by 0.5% xylose. When the α-amylase gene amyL was used as a genomic target, the efficiencies of its disruption using three different protospacer-adjacent motif (PAM) sequences were 64.3%, 70.9%, and 47.1%, respectively. Moreover, temperature plays a pivotal role in the function of the constructed CRISPR system. The maximum success rate reached 97% at 20 °C, while higher temperatures negatively impacted the function of the system. These results suggested that the design with a cas9 gene under the strict control of a xylose-inducible promoter significantly improved the success rate of genome editing in this host. This work contributes to the development of genetic manipulation and furthers the use of B. licheniformis as an efficient industrial workhorse.

scholarly journals Development of an Efficient Genome Editing Tool in Bacillus licheniformis Using CRISPR-Cas9 Nickase

2018 ◽  
Vol 84 (6) ◽  
Author(s):  
Kaifeng Li ◽  
Dongbo Cai ◽  
Zhangqian Wang ◽  
Zhili He ◽  
Shouwen Chen
Keyword(s):  
Genome Editing ◽  
Bacillus Licheniformis ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Single Gene ◽  
Strain Improvement ◽  
Future Research ◽  
Widespread Application ◽  
Content Type ◽  
Dna Fragment

ABSTRACT Bacillus strains are important industrial bacteria that can produce various biochemical products. However, low transformation efficiencies and a lack of effective genome editing tools have hindered its widespread application. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 techniques have been utilized in many organisms as genome editing tools because of their high efficiency and easy manipulation. In this study, an efficient genome editing method was developed for Bacillus licheniformis using a CRISPR-Cas9 nickase integrated into the genome of B. licheniformis DW2 with overexpression driven by the P43 promoter. The yvmC gene was deleted using the CRISPR-Cas9n technique with homology arms of 1.0 kb as a representative example, and an efficiency of 100% was achieved. In addition, two genes were simultaneously disrupted with an efficiency of 11.6%, and the large DNA fragment bacABC (42.7 kb) was deleted with an efficiency of 79.0%. Furthermore, the heterologous reporter gene aprN , which codes for nattokinase in Bacillus subtilis , was inserted into the chromosome of B. licheniformis with an efficiency of 76.5%. The activity of nattokinase in the DWc9nΔ7/pP43SNT-S sacC strain reached 59.7 fibrinolytic units (FU)/ml, which was 25.7% higher than that of DWc9n/pP43SNT-S sacC . Finally, the engineered strain DWc9nΔ7 (Δ epr Δ wprA Δ mpr Δ aprE Δ vpr Δ bprA Δ bacABC ), with multiple disrupted genes, was constructed using the CRISPR-Cas9n technique. Taken together, we have developed an efficient genome editing tool based on CRISPR-Cas9n in B. licheniformis . This tool could be applied to strain improvement for future research. IMPORTANCE As important industrial bacteria, Bacillus strains have attracted significant attention due to their production of biological products. However, genetic manipulation of these bacteria is difficult. The CRISPR-Cas9 system has been applied to genome editing in some bacteria, and CRISPR-Cas9n was proven to be an efficient and precise tool in previous reports. The significance of our research is the development of an efficient, more precise, and systematic genome editing method for single-gene deletion, multiple-gene disruption, large DNA fragment deletion, and single-gene integration in Bacillus licheniformis via Cas9 nickase. We also applied this method to the genetic engineering of the host strain for protein expression.

scholarly journals CRISPR-Cas12a-Assisted Recombineering in Bacteria

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Mei-Yi Yan ◽  
Hai-Qin Yan ◽  
Gai-Xian Ren ◽  
Ju-Ping Zhao ◽  
Xiao-Peng Guo ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mycobacterium Smegmatis ◽  
Genetic Manipulation ◽  
Antibiotic Resistance Gene ◽  
Point Mutations ◽  
Content Type ◽  
Short Palindromic Repeat ◽  
New Type ◽  
System Point ◽  
Selection Of

ABSTRACT Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms. Here, we developed and optimized a CRISPR-Cas12a-assisted recombineering system to facilitate genetic manipulation in bacteria. Using this system, point mutations, deletions, insertions, and gene replacements can be easily generated on the chromosome or native plasmids in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis. Because CRISPR-Cas12a-assisted recombineering does not require introduction of an antibiotic resistance gene into the chromosome to select for recombinants, it is an efficient approach for generating markerless and scarless mutations in bacteria. IMPORTANCE The CRISPR-Cas9 system has been widely used to facilitate genome editing in many bacteria. CRISPR-Cas12a (Cpf1), a new type of CRISPR-Cas system, allows efficient genome editing in bacteria when combined with recombineering. Cas12a and Cas9 recognize different target sites, which allows for more precise selection of the cleavage target and introduction of the desired mutation. In addition, CRISPR-Cas12a-assisted recombineering can be used for genetic manipulation of plasmids and plasmid curing. Finally, Cas12a-assisted recombineering in the generation of point mutations, deletions, insertions, and replacements in bacteria has been systematically analyzed. Taken together, our findings will guide efficient Cas12a-mediated genome editing in bacteria.

Toxic Effect of 2-ethyl (bicyclo[2.2.1] heptane) on Bacterial Cells

2019 ◽  
Vol 35 (6) ◽  
pp. 67-72 ◽  
Author(s):  
I.V. Manukhov ◽  
L.S. Yaguzhinsky ◽  
M.V. Bermeshev ◽  
M.A. Zisman ◽  
V.G. Pevgov ◽  
...  
Keyword(s):  
Toxic Effect ◽  
Atmospheric Oxygen ◽  
Inducible Promoter ◽  
Oxygen Species ◽  
Bacterial Cells ◽  
Unique Identifier ◽  
E Coli ◽  
Lux Biosensors ◽  
High Level ◽  
Ministry Of Higher Education

Toxic effect of 2-ethylnorbornane (2-ethyl(bicyclo[2.2.1]heptane) (EBH)) on bacteria has been studied using the E. coli pRecA-lux and E. coli pKatG- lux cells as lux-biosensors. It was shown that the addition of EBH to the incubation medium leads to death and growth retardation, high level oxidative stress and DNA damage in E. coli cells. It is assumed that the oxidation of EBH with atmospheric oxygen causes the formation of reactive oxygen species in the medium, which makes a major contribution to the toxicity of this substance. biosensor, luciferase, bioluminescence, inducible promoter, PrecA, PkatG The authors are grateful to Stanislav Filippovich Chalkin for the development of interdisciplinary ties in the scientific community. The work was financially supported by the Ministry of Higher Education and Science of Russia (Project Unique Identifier RFMEFI60417X0181, Agreement No. 14.604.21.0181 of 26.09.2017).

scholarly journals Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuu Asano ◽  
Kensuke Yamashita ◽  
Aoi Hasegawa ◽  
Takanori Ogasawara ◽  
Hoshie Iriki ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dictyostelium Discoideum ◽  
Streptococcus Pyogenes ◽  
Nucleotide Substitution ◽  
Point Mutations ◽  
Targeted Mutagenesis ◽  
Single Amino Acid ◽  
Specific Gene ◽  
Knock Out ◽  
Protospacer Adjacent Motif

AbstractThe powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

scholarly journals 1724. Plasmid-free CRISPR-Cas9 System for Genetic Engineering of Rhizopus delemar

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S632-S632
Author(s):  
Yiyou Gu ◽  
Clara Baldin ◽  
Teklegiorgis Gebremariam ◽  
Abdullah Alqarihi ◽  
Zeinab Mamouei ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Etiologic Agent ◽  
Pcr Analysis ◽  
Free System ◽  
Rhizopus Delemar ◽  
Additional Tool ◽  
Qrt Pcr ◽  
Enzyme Screening ◽  
Serious Infection ◽  
Short Palindromic Repeat

Abstract Background Mucormycosis is a serious infection caused by fungi of the order Mucorales. Rhizopus delemar is the most common etiologic agent of mucormycosis. Pathogenesis studies of mucormycosis have been hampered by poor genetic trackability of the organism, owing to rare chromosomal integration events and multinucleated nature of the cells. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system has been widely used in genetic manipulation through efficient homologous and non-homologous break points in a variety of organisms including R. delemar. However, plasmid-free CRISPR/Cas9 system has not been previously described in the fungus. Here, we introduce a rapid plasmid-free system for inducing orotidine 5’-phosphate decarboxylase (pyrF) gene mutation in R. delemar. Methods Protoplasts of R. delemar 99–880 strain were transformed with 20 nucleotide gRNA targeting the N-terminus of pyrF gene and the Cas9 enzyme. Screening for pyrF auxotrophy was carried out by plating transformed protoplasts on potato dextrose agar (PDA) plates containing 1 mg/mL 5-fluoroorotic acid (5-FOA) and 100 µg/mL uracil. Putative mutant strains were selected for uracil auxotrophy by plating simultaneously on media with or without uracil. pyrF disruption was verified by using PCR and qRT–PCR. Results Approximately100 transformants were generated through plating on 5-FOA plates. Only three transformants did not grow on minimal medium lacking uracil, indicating that they were true pyrF null mutants. PCR analysis showed that these three transformants have undergone nucleotide deletion events within the pyrF gene. The lack of pyrF gene expression was further verified by using qRT–PCR relative to wild-type R. delemar 99–880. Conclusion Similar to the plasmid-based genome manipulation strategy, the plasmid-free CRISPR/Cas9 system can induce gene editing in R. delemar. This rapid and simple approach adds an additional tool in our conquest to understand pathogenesis of mucormycosis. Disclosures All authors: No reported disclosures.

scholarly journals Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1526
Author(s):  
Joanna E. Kowalczyk ◽  
Shreya Saha ◽  
Miia R. Mäkelä
Keyword(s):  
Genome Editing ◽  
Plant Biomass ◽  
Genetic Manipulation ◽  
White Rot ◽  
White Rot Fungus ◽  
Detailed Knowledge ◽  
Wild Type ◽  
Low Frequencies ◽  
Dichomitus Squalens

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.

scholarly journals Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

2021 ◽  
Author(s):  
Soo-Young Yum ◽  
Goo Jang ◽  
Okjae Koo
Keyword(s):  
Genome Editing ◽  
Cytidine Deaminase ◽  
Chromosomal Rearrangements ◽  
Dna Breaks ◽  
Base Editing ◽  
Multiple Loci ◽  
Double Strand Dna Breaks ◽  
Protospacer Adjacent Motif ◽  
Motif Site ◽  
Programmable Nucleases

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3-5 base editing windows, 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

scholarly journals CRISPR base editors: genome editing without double-stranded breaks

2018 ◽  
Vol 475 (11) ◽  
pp. 1955-1964 ◽  
Author(s):  
Ayman Eid ◽  
Sahar Alshareef ◽  
Magdy M. Mahfouz
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Genetic Diseases ◽  
Great Promise ◽  
Cytidine Deaminases ◽  
Strand Breaks ◽  
Base Editing ◽  
Potential Applications ◽  
Short Palindromic Repeat ◽  
Basic Biology

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 adaptive immunity system has been harnessed for genome editing applications across eukaryotic species, but major drawbacks, such as the inefficiency of precise base editing and off-target activities, remain. A catalytically inactive Cas9 variant (dead Cas9, dCas9) has been fused to diverse functional domains for targeting genetic and epigenetic modifications, including base editing, to specific DNA sequences. As base editing does not require the generation of double-strand breaks, dCas9 and Cas9 nickase have been used to target deaminase domains to edit specific loci. Adenine and cytidine deaminases convert their respective nucleotides into other DNA bases, thereby offering many possibilities for DNA editing. Such base-editing enzymes hold great promise for applications in basic biology, trait development in crops, and treatment of genetic diseases. Here, we discuss recent advances in precise gene editing using different platforms as well as their potential applications in basic biology and biotechnology.

scholarly journals Advanced Biological Imaging for Intracellular Micromanipulation: Methods and Applications

2020 ◽  
Vol 10 (20) ◽  
pp. 7308
Author(s):  
Wendi Gao ◽  
Libo Zhao ◽  
Zhuangde Jiang ◽  
Dong Sun
Keyword(s):  
Success Rate ◽  
Genome Editing ◽  
Biomedical Research ◽  
Genetic Diagnosis ◽  
Biological Imaging ◽  
Robotic Systems ◽  
Current Development ◽  
Insufficient Information ◽  
Processing Methods ◽  
Future Extension

Intracellular micromanipulation assisted by robotic systems has valuable applications in biomedical research, such as genetic diagnosis and genome-editing tasks. However, current studies suffer from a low success rate and a large operation damage because of insufficient information on the operation information of targeted specimens. The complexity of the intracellular environment causes difficulties in visualizing manipulation tools and specimens. This review summarizes and analyzes the current development of advanced biological imaging sampling and computational processing methods in intracellular micromanipulation applications. It also discusses the related limitations and future extension, providing an important reference about this field.

New insights of CRISPR technology in human pathogenic fungi

2019 ◽  
Vol 14 (14) ◽  
pp. 1243-1255 ◽  
Author(s):  
Elvira Román ◽  
Daniel Prieto ◽  
Rebeca Alonso-Monge ◽  
Jesús Pla
Keyword(s):  
Mammalian Cells ◽  
Genetic Manipulation ◽  
Pathogenic Fungi ◽  
Biological Research ◽  
Therapeutic Approaches ◽  
New Therapeutic Approaches ◽  
Short Palindromic Repeat ◽  
Future Outcomes ◽  
Near Future ◽  
Genome Manipulation

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems have emerged as a powerful tool for genome manipulation. Class 2 type II CRISPR/ CAS9 is so far the most studied system and has been implemented in many biological systems such as mammalian cells, plants, fungi and bacteria. Fungi are important causes of human diseases worldwide. Genetic manipulation of pathogenic fungi is critical to develop new therapeutic approaches and novel antifungals. We will review here the progress done with CRISPR/ CAS9 systems in human pathogenic fungi, with emphasis in Candida albicans and the main modifications that have improved their usefulness in biological research. We finally discuss possible future outcomes and applications to the developed in a near future.

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