Rapid and marker-free gene replacement in citric acid-producing Aspergillus tubingensis (A. niger) WU-2223L by the CRISPR/Cas9 system-based genome editing technique using DNA fragments encoding sgRNAs

2021 ◽  
Author(s):  
Isato Yoshioka ◽  
Kohtaro Kirimura
Keyword(s):  
Citric Acid ◽  
Genome Editing ◽  
Gene Replacement ◽  
Dna Fragments ◽  
Aspergillus Tubingensis ◽  
Marker Free ◽  
Free Gene

scholarly journals Efficient marker free CRISPR/Cas9 genome editing for functional analysis of gene families in filamentous fungi

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Tim M. van Leeuwe ◽  
Mark Arentshorst ◽  
Tim Ernst ◽  
Ebru Alazi ◽  
Peter J. Punt ◽  
...  
Keyword(s):  
Genome Editing ◽  
Filamentous Fungi ◽  
Marker Gene ◽  
Gene Families ◽  
Selection Marker ◽  
Dna Fragments ◽  
Additional Advantage ◽  
Multiple Gene ◽  
Large Gene ◽  
Marker Free

Abstract Background CRISPR/Cas9 mediated genome editing has expedited the way of constructing multiple gene alterations in filamentous fungi, whereas traditional methods are time-consuming and can be of mutagenic nature. These developments allow the study of large gene families that contain putatively redundant genes, such as the seven-membered family of crh-genes encoding putative glucan–chitin crosslinking enzymes involved in cell wall biosynthesis. Results Here, we present a CRISPR/Cas9 system for Aspergillus niger using a non-integrative plasmid, containing a selection marker, a Cas9 and a sgRNA expression cassette. Combined with selection marker free knockout repair DNA fragments, a set of the seven single knockout strains was obtained through homology directed repair (HDR) with an average efficiency of 90%. Cas9–sgRNA plasmids could effectively be cured by removing selection pressure, allowing the use of the same selection marker in successive transformations. Moreover, we show that either two or even three separate Cas9–sgRNA plasmids combined with marker-free knockout repair DNA fragments can be used in a single transformation to obtain double or triple knockouts with 89% and 38% efficiency, respectively. By employing this technique, a seven-membered crh-gene family knockout strain was acquired in a few rounds of transformation; three times faster than integrative selection marker (pyrG) recycling transformations. An additional advantage of the use of marker-free gene editing is that negative effects of selection marker gene expression are evaded, as we observed in the case of disrupting virtually silent crh family members. Conclusions Our findings advocate the use of CRISPR/Cas9 to create multiple gene deletions in both a fast and reliable way, while simultaneously omitting possible locus-dependent-side-effects of poor auxotrophic marker expression.

scholarly journals Using the Endogenous CRISPR-Cas System of Heliobacterium modesticaldum To Delete the Photochemical Reaction Center Core Subunit Gene

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Patricia L. Baker ◽  
Gregory S. Orf ◽  
Kimberly Kevershan ◽  
Michael E. Pyne ◽  
Taner Bicer ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Reaction Center ◽  
Photochemical Reaction ◽  
Genetic Locus ◽  
Gene Replacement ◽  
Type I ◽  
Content Type ◽  
Heliobacterium Modesticaldum

ABSTRACT In Heliobacterium modesticaldum, as in many Firmicutes, deleting genes by homologous recombination using standard techniques has been extremely difficult. The cells tend to integrate the introduced plasmid into the chromosome by a single recombination event rather than perform the double recombination required to replace the targeted locus. Transformation with a vector containing only a homologous recombination template for replacement of the photochemical reaction center gene pshA produced colonies with multiple genotypes, rather than a clean gene replacement. To address this issue, we required an additional means of selection to force a clean gene replacement. In this study, we report the genetic structure of the type I-A and I-E CRISPR-Cas systems from H. modesticaldum, as well as methods to leverage the type I-A system for genome editing. In silico analysis of the CRISPR spacers revealed a potential consensus protospacer adjacent motif (PAM) required for Cas3 recognition, which was then tested using an in vivo interference assay. Introduction of a homologous recombination plasmid that carried a miniature CRISPR array targeting sequences in pshA (downstream of a naturally occurring PAM sequence) produced nonphototrophic transformants with clean replacements of the pshA gene with ∼80% efficiency. Mutants were confirmed by PCR, sequencing, optical spectroscopy, and growth characteristics. This methodology should be applicable to any genetic locus in the H. modesticaldum genome. IMPORTANCE The heliobacteria are the only phototrophic members of the largely Gram-positive phylum Firmicutes, which contains medically and industrially important members, such as Clostridium difficile and Clostridium acetobutylicum. Heliobacteria are of interest in the study of photosynthesis because their photosynthetic system is unique and the simplest known. Since their discovery in the early 1980s, work on the heliobacteria has been hindered by the lack of a genetic transformation system. The problem of introducing foreign DNA into these bacteria has been recently rectified by our group; however, issues still remained for efficient genome editing. The significance of this work is that we have characterized the endogenous type I CRISPR-Cas system in the heliobacteria and leveraged it to assist in genome editing. Using the CRISPR-Cas system allowed us to isolate transformants with precise replacement of the pshA gene encoding the main subunit of the photochemical reaction center.

scholarly journals Nontransgenic Marker-Free Gene Disruption by an Episomal CRISPR System in the Oleaginous Microalga, Nannochloropsis oceanica CCMP1779

2018 ◽  
Vol 7 (4) ◽  
pp. 962-968 ◽  
Author(s):  
Eric Poliner ◽  
Tomomi Takeuchi ◽  
Zhi-Yan Du ◽  
Christoph Benning ◽  
Eva M. Farré
Keyword(s):  
Gene Disruption ◽  
Nannochloropsis Oceanica ◽  
Crispr System ◽  
Marker Free ◽  
Free Gene

Human iPSC modeling reveals mutation-specific responses to gene therapy in Best disease

2019 ◽  
Author(s):  
Divya Sinha ◽  
Benjamin Steyer ◽  
Pawan K. Shahi ◽  
Katherine Mueller ◽  
Rasa Valiauga ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Gene Replacement ◽  
Patient Specific ◽  
Genomic Alteration ◽  
Inherited Disorders ◽  
Induced Pluripotent ◽  
Best Disease

AbstractDominantly inherited disorders are not typically considered therapeutic candidates for gene augmentation. Here, we utilized patient-specific induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in iPSC-RPE models of recessive bestrophinopathy and dominant Best disease caused by two different ion binding domain mutations. A dominant Best disease iPSC-RPE model that did not respond to gene augmentation showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then tested gene editing in all three dominant Best disease iPSC-RPE models, which produced premature stop codons exclusively within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of RPE transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some dominant Best disease patients and that non-responders are candidates for alternate approaches such as genome editing. However, testing genome editing strategies for on-target efficiency and off-target events using patient-matched iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected patients.SignificanceDominantly inherited disorders pose distinct challenges for gene therapies, particularly in the face of extreme mutational diversity. We tested whether a broad gene replacement strategy could reverse the cellular phenotype of Best disease, a dominant blinding condition that targets retinal pigment epithelium (RPE). Using RPE generated from patient-specific induced pluripotent stem cells (iPSCs), we show that gene replacement functionally overcomes some, but not all, of the tested mutations. In comparison, all dominant Best disease models tested were phenotypically corrected after mutation-specific genome editing, although one off-target genomic alteration was discovered. Our results support a two-tiered approach to gene therapy for Best disease, guided by safety and efficacy testing in iPSC-RPE models to maximize personal and public health value.

scholarly journals Marker-free coselection for CRISPR-driven genome editing in human cells

2017 ◽  
Vol 14 (6) ◽  
pp. 615-620 ◽  
Author(s):  
Daniel Agudelo ◽  
Alexis Duringer ◽  
Lusiné Bozoyan ◽  
Caroline C Huard ◽  
Sophie Carter ◽  
...  
Keyword(s):  
Genome Editing ◽  
Human Cells ◽  
Marker Free

scholarly journals Enhancement of homology-directed repair with chromatin donor templates in cells

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Grisel Cruz-Becerra ◽  
James T Kadonaga
Keyword(s):  
Genome Editing ◽  
Dna Fragments ◽  
Natural Form ◽  
Naked Dna ◽  
Homology Directed Repair ◽  
Coding Regions ◽  
Wide Range ◽  
Low Efficiency ◽  
Donor Template ◽  
In Cells

A key challenge in precise genome editing is the low efficiency of homology-directed repair (HDR). Here we describe a strategy for increasing the efficiency of HDR in cells by using a chromatin donor template instead of a naked DNA donor template. The use of chromatin, which is the natural form of DNA in the nucleus, increases the frequency of HDR-edited clones as well as homozygous editing. In addition, transfection of chromatin results in negligible cytotoxicity. These findings suggest that a chromatin donor template should be useful for a wide range of HDR applications such as the precise insertion or replacement of DNA fragments that contain the coding regions of genes.

scholarly journals Efficient Targeted Gene Insertion in Maize using Agrobacterium-Mediated Delivery

2020 ◽  
Author(s):  
Sergei Svitashev ◽  
Dave Peterson ◽  
Pierjuigi Barone ◽  
Brian Lenderts ◽  
Chris Schwartz ◽  
...  
Keyword(s):  
Genome Editing ◽  
Strand Break ◽  
Plant Genome ◽  
Specific Gene ◽  
Crop Species ◽  
Gene Insertion ◽  
Maize Transformation ◽  
Dna Double Strand Break ◽  
Genomic Location ◽  
Marker Free

Abstract CRISPR-Cas is a powerful DNA double strand break technology with wide-ranging applications in plant genome modification. However, the efficiency of genome editing depends on various factors including plant genetic transformation processes and types of modifications desired. Agrobacterium infection is the preferred method of transformation and delivery of editing components into the plant cell. While this method has been successfully used to generate gene knock-outs in multiple crops, precise nucleotide replacement and especially gene insertion into a pre-defined genomic location remain highly challenging. Here we report an efficient, heritable, selectable marker-free site-specific gene insertion in maize using Agrobacterium-mediated delivery. Advancements in maize transformation and new vector design enabled targeted insertion with frequencies as high as 8–10%. Importantly, these advancements allowed not only an improvement of the frequency but also of the quality of generated events. These results further enable the application of genome editing for trait product development in a wide variety of crop species amenable to Agrobacterium-mediated transformation.

scholarly journals A novel and effective Cre/lox-based genetic tool for repeated, targeted and markerless integration in Yarrowia lipolytica

2020 ◽  
Author(s):  
Qinghua Zhou ◽  
Liangcheng Jiao ◽  
Wenjuan Li ◽  
Zhiming Hu ◽  
Yunchong Li ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Protein Production ◽  
Genome Engineering ◽  
Selection Marker ◽  
The Novel ◽  
Genetic Tool ◽  
Recombination System ◽  
Marker Free ◽  
Free Gene ◽  
Gene Integration

AbstractThe unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless gene integration based on Cre/lox sitespecific recombination system. The developed tool required only a single selection marker and could completely excise all of the unnecessary sequences. A total of three plasmids were created and seven rounds of marker-free gene integration were examined in Y. lipolytica. All the integration efficiencies remained above 90%, and analysis of protein production and growth characteristics of the engineered strains confirmed that genome modification via the novel genetic tool was feasible. Further work also confirmed the genetic tool was effective for integration of other genes, loci, and strains. Thus, this study significantly promotes the application of Cre/lox system and presents a powerful tool for genome engineering in Y. lipolytica.

scholarly journals Use of Bacteriophage λ Recombination Functions To Promote Gene Replacement in Escherichia coli

1998 ◽  
Vol 180 (8) ◽  
pp. 2063-2071 ◽  
Author(s):  
Kenan C. Murphy
Keyword(s):  
Escherichia Coli ◽  
Gene Replacement ◽  
Dna Fragments ◽  
Bacteriophage Λ ◽  
E Coli ◽  
Linear Dna ◽  
Dna Fragment ◽  
Elevated Rate

ABSTRACT Replacement of Escherichia coli’s RecBCD function with phage λ’s Red function generates a strain whose chromosome recombines with short linear DNA fragments at a greatly elevated rate. The rate is at least 70-fold higher than that exhibited by arecBC sbcBC or recD strain. The value of the system is highlighted by gene replacement with a PCR-generated DNA fragment. The ΔrecBCD::P lac -red kanreplacement allele can be P1 transduced to other E. colistrains, making the hyper-Rec phenotype easily transferable.

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