scholarly journals Assessing the efficacy of CRISPR/Cas9 genome editing in the wheat pathogen Parastagonspora nodorum

2020 ◽  
Author(s):  
Haseena Khan ◽  
Megan C McDonald ◽  
Simon J Willams ◽  
Peter Solomon
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Pathogenic Fungus ◽  
Point Mutations ◽  
Dna Breaks ◽  
Gene Manipulation ◽  
Homology Directed Repair ◽  
Rnp Complex ◽  
Parastagonospora Nodorum

Abstract Background: The genome-editing tool CRISPR/Cas9 has revolutionized gene manipulation by providing an efficient method to generate targeted mutations. This technique deploys the Cas9 endonuclease and a guide RNA (gRNA) which interact to form a Cas9-gRNA complex that initiates gene editing through the introduction of double stranded DNA breaks. We tested the efficacy of the CRISPR/Cas9 approach as a means of facilitating a variety of reverse genetic approaches in the wheat pathogenic fungus Parastagonospora nodorum . Results: Parastagonospora nodorum protoplasts were transformed with the Cas9 protein and gRNA in the form of a preassembled ribonuclear protein (RNP) complex targeting the Tox3 effector gene. Subsequent screening of the P. nodorum transformants revealed 100% editing of those mutants screened. We further tested the efficacy of RNP complex when co-transformed with a Tox3 -Homology Directed Repair cassette harbouring 1 kb of homologous flanking DNA. Subsequent screening of resulting transformants demonstrated homologous recombination efficiencies exceeding 70%. A further transformation with a Tox3 -Homology Directed Repair cassette harbouring a selectable marker with 50 bp micro-homology flanks was also achieved 25% homologous recombination efficiency. The success of these homology directed repair approaches demonstrate that CRISPR/Cas9 is amenable to other in vivo DNA manipulation approaches such as the insertion of DNA and generating point mutations. Conclusion: These data highlight the significant potential that CRISPR/Cas9 has in expediting gene transgene-free knockouts in Parastagonospora nodorum and also in facilitating other gene manipulation approaches. Access to these tools will significantly decrease the time required to assess the requirement of gene for disease and to undertake functional studies to determine its role.

scholarly journals Assessing the efficacy of CRISPR/Cas9 genome editing in the wheat pathogen Parastagonspora nodorum

2020 ◽  
Author(s):  
Haseena Khan ◽  
Megan C McDonald ◽  
Simon J Willams ◽  
Peter Solomon
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Pathogenic Fungus ◽  
Point Mutations ◽  
Dna Breaks ◽  
Gene Manipulation ◽  
Homology Directed Repair ◽  
Rnp Complex ◽  
Parastagonospora Nodorum

Abstract Background: The genome-editing tool CRISPR/Cas9 has revolutionized gene manipulation by providing an efficient method to generate targeted mutations. This technique deploys the Cas9 endonuclease and a guide RNA (sgRNA) which interact to form a Cas9-sgRNA complex that initiates gene editing through the introduction of double stranded DNA breaks. We tested the efficacy of the CRISPR/Cas9 approach as a means of facilitating a variety of reverse genetic approaches in the wheat pathogenic fungus Parastagonospora nodorum. Results: Parastagonospora nodorum protoplasts were transformed with the Cas9 protein and sgRNA in the form of a preassembled ribonuclear protein (RNP) complex targeting the Tox3 effector gene. Subsequent screening of the P. nodorum transformants revealed 100% editing of those mutants screened. We further tested the efficacy of RNP complex when co-transformed with a Tox3-Homology Directed Repair cassette harbouring 1 kb of homologous flanking DNA. Subsequent screening of resulting transformants demonstrated homologous recombination efficiencies exceeding 70%. A further transformation with a Tox3-Homology Directed Repair cassette harbouring a selectable marker with 50 bp micro-homology flanks was also achieved with 25% homologous recombination efficiency. The success of these homology directed repair approaches demonstrate that CRISPR/Cas9 is amenable to other in vivo DNA manipulation approaches such as the insertion of DNA and generating point mutations. Conclusion: These data highlight the significant potential that CRISPR/Cas9 has in expediting transgene-free gene knockouts in Parastagonospora nodorum and also in facilitating other gene manipulation approaches. Access to these tools will significantly decrease the time required to assess the requirement of gene for disease and to undertake functional studies to determine its role.

scholarly journals Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing

2018 ◽  
Vol 115 (31) ◽  
pp. E7379-E7388 ◽  
Author(s):  
Laura J. Smith ◽  
Jason Wright ◽  
Gabriella Clark ◽  
Taihra Ul-Hasan ◽  
Xiangyang Jin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Homologous Recombination ◽  
Genome Editing ◽  
High Efficiency ◽  
Dna Breaks ◽  
Hematopoietic Stem ◽  
A Genome ◽  
Component Approach ◽  
Il2rg Gene

The precise correction of genetic mutations at the nucleotide level is an attractive permanent therapeutic strategy for human disease. However, despite significant progress, challenges to efficient and accurate genome editing persist. Here, we report a genome editing platform based upon a class of hematopoietic stem cell (HSC)-derived clade F adeno-associated virus (AAV), which does not require prior nuclease-mediated DNA breaks and functions exclusively through BRCA2-dependent homologous recombination. Genome editing is guided by complementary homology arms and is highly accurate and seamless, with no evidence of on-target mutations, including insertion/deletions or inclusion of AAV inverted terminal repeats. Efficient genome editing was demonstrated at different loci within the human genome, including a safe harbor locus, AAVS1, and the therapeutically relevant IL2RG gene, and at the murine Rosa26 locus. HSC-derived AAV vector (AAVHSC)-mediated genome editing was robust in primary human cells, including CD34+cells, adult liver, hepatic endothelial cells, and myocytes. Importantly, high-efficiency gene editing was achieved in vivo upon a single i.v. injection of AAVHSC editing vectors in mice. Thus, clade F AAV-mediated genome editing represents a promising, highly efficient, precise, single-component approach that enables the development of therapeutic in vivo genome editing for the treatment of a multitude of human gene-based diseases.

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 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 Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species

2020 ◽  
Vol 48 (10) ◽  
pp. e57-e57
Author(s):  
Xiaozhen He ◽  
Wenfeng Chen ◽  
Zhen Liu ◽  
Guirong Yu ◽  
Youbang Chen ◽  
...  
Keyword(s):  
Genome Editing ◽  
Human Peripheral Blood ◽  
Point Mutations ◽  
Peripheral Blood Lymphocytes ◽  
Dna Double Strand Breaks ◽  
Site Specific ◽  
Low Efficiency ◽  
Multiple Species ◽  
Balancing Efficiency

Abstract Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and ‘cleaner’ knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.

scholarly journals Regulation of class switch recombination and somatic mutation by AID phosphorylation

2008 ◽  
Vol 205 (11) ◽  
pp. 2585-2594 ◽  
Author(s):  
Kevin M. McBride ◽  
Anna Gazumyan ◽  
Eileen M. Woo ◽  
Tanja A. Schwickert ◽  
Brian T. Chait ◽  
...  
Keyword(s):  
Somatic Mutation ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Point Mutations ◽  
Class Switch Recombination ◽  
Variable Region ◽  
Dna Breaks ◽  
Class Switching ◽  
Class Switch

Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates somatic mutation and class switch recombination in B lymphocytes by introducing uracil:guanine mismatches into DNA. Repair pathways process these mismatches to produce point mutations in the Ig variable region or double-stranded DNA breaks in the switch region DNA. However, AID can also produce off-target DNA damage, including mutations in oncogenes. Therefore, stringent regulation of AID is required for maintaining genomic stability during maturation of the antibody response. It has been proposed that AID phosphorylation at serine 38 (S38) regulates its activity, but this has not been tested in vivo. Using a combination of mass spectrometry and immunochemical approaches, we found that in addition to S38, AID is also phosphorylated at position threonine 140 (T140). Mutation of either S38 or T140 to alanine does not impact catalytic activity, but interferes with class switching and somatic hypermutation in vivo. This effect is particularly pronounced in haploinsufficient mice where AID levels are limited. Although S38 is equally important for both processes, T140 phosphorylation preferentially affects somatic mutation, suggesting that posttranslational modification might contribute to the choice between hypermutation and class switching.

scholarly journals Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Raed Ibraheim ◽  
Phillip W. L. Tai ◽  
Aamir Mir ◽  
Nida Javeed ◽  
Jiaming Wang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Control Measure ◽  
Type I ◽  
Adeno Associated Virus ◽  
Sequencing Platform ◽  
Homology Directed Repair ◽  
Safety Risks ◽  
Quality Control Measure ◽  
Hereditary Tyrosinemia

AbstractAdeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.

scholarly journals Precision genome editing in the CRISPR era

2017 ◽  
Vol 95 (2) ◽  
pp. 187-201 ◽  
Author(s):  
Jayme Salsman ◽  
Graham Dellaire
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Point Mutations ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Genetic Changes ◽  
New Era ◽  
Homology Directed Repair ◽  
Repair Template ◽  
Non Homologous End Joining

With the introduction of precision genome editing using CRISPR–Cas9 technology, we have entered a new era of genetic engineering and gene therapy. With RNA-guided endonucleases, such as Cas9, it is possible to engineer DNA double strand breaks (DSB) at specific genomic loci. DSB repair by the error-prone non-homologous end-joining (NHEJ) pathway can disrupt a target gene by generating insertions and deletions. Alternatively, Cas9-mediated DSBs can be repaired by homology-directed repair (HDR) using an homologous DNA repair template, thus allowing precise gene editing by incorporating genetic changes into the repair template. HDR can introduce gene sequences for protein epitope tags, delete genes, make point mutations, or alter enhancer and promoter activities. In anticipation of adapting this technology for gene therapy in human somatic cells, much focus has been placed on increasing the fidelity of CRISPR–Cas9 and increasing HDR efficiency to improve precision genome editing. In this review, we will discuss applications of CRISPR technology for gene inactivation and genome editing with a focus on approaches to enhancing CRISPR–Cas9-mediated HDR for the generation of cell and animal models, and conclude with a discussion of recent advances and challenges towards the application of this technology for gene therapy in humans.

scholarly journals Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break

2019 ◽  
Author(s):  
Geoffrey L. Rogers ◽  
Hsu-Yu Chen ◽  
Heidy Morales ◽  
Paula M. Cannon
Keyword(s):  
Homologous Recombination ◽  
Progenitor Cells ◽  
Genome Editing ◽  
High Efficiency ◽  
Zinc Finger Nucleases ◽  
Dna Breaks ◽  
Adeno Associated Virus ◽  
Hematopoietic Stem ◽  
Double Stranded Dna ◽  
Dna Break

AbstractAdeno-associated virus (AAV) vectors are frequently used as donor templates for genome editing by homologous recombination. Although modification rates are typically under 1%, they are greatly enhanced by targeted double-stranded DNA breaks (DSBs). A recent report described clade F AAVs mediating high-efficiency homologous recombination-based editing in the absence of DSBs. The clade F vectors included AAV9 and a series isolated from human hematopoietic stem/progenitor cells (HSPCs). We evaluated these vectors by packaging homology donors into AAV9 and an AAVHSC capsid and examining their ability to insert GFP at the CCR5 or AAVS1 loci in human HSPCs and cell lines. As a control we used AAV6, which effectively edits HSPCs, but only when combined with a targeted DSB. Each AAV vector promoted GFP insertion in the presence of matched CCR5 or AAVS1 zinc finger nucleases (ZFNs), but none supported detectable editing in the absence of the nucleases. Rates of editing with ZFNs correlated with transduction efficiencies for each vector, implying no differences in the ability of donor sequences delivered by the different vectors to direct genome editing. Our results therefore do not support that clade F AAVs can perform high efficiency genome editing in the absence of a DSB.

scholarly journals Retron Editing for Precise Genome Editing without Exogenous Donor DNA in Human Cells

2021 ◽  
Author(s):  
Xiangfeng Kong ◽  
Zikang Wang ◽  
Yingsi Zhou ◽  
Xing Wang ◽  
Linyu Shi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Human Cells ◽  
Exogenous Dna ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Coding Rna ◽  
Bacterial Genetic ◽  
Low Efficiency

CRISPR-Cas9 mediated seamless genome editing can be achieved by incorporating donor DNA into the CRISPR-Cas9 target loci via homology-directed repair (HDR), albeit with relative low efficiency due to the inefficient delivery of exogenous DNA. Retrons are bacterial genetic element composed of a non-coding RNA (ncRNA) and reverse transcriptase (RT). Retrons coupled with CRISPR-Cas9 have been shown to enhance precise genome editing via HDR in yeast through fusing guide RNA (gRNA) to the 3′ end of retron ncRNA, producing multicopy single-stranded DNA (msDNA) covalently tethered to gRNA. Here, we further engineered retrons by fusing Cas9 with E.coli RT from different clades and joining gRNA at the 5′ end of retron ncRNA, and found that retron editing can achieve precise genome editing efficiently in human cells. By co- expression of Cas9-RT fusions and retron-ncRNA gRNA (rgRNA) in HEK293T cells, we demonstrated the rates of retron editing at endogenous genomic loci was up to 10 %. We expect our retron editing system could aid in advancing the ex vivo and in vivo therapeutic applications of retron.

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