scholarly journals Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies.

2019 ◽  
Author(s):  
Rita Sharma ◽  
Yan Liang ◽  
Mi Yeon Lee ◽  
Venkataramana Pidatala ◽  
Jennifer Charlotte Mortimer ◽  
...  
Keyword(s):  
Genome Editing ◽  
Fluorescent Protein ◽  
Transient Transformation ◽  
Reliable System ◽  
High Reproducibility ◽  
Guide Rna ◽  
Expression Construct ◽  
Speed Up ◽  
Transient System ◽  
Green Fluorescent

Abstract Objectives Sorghum is one of the most recalcitrant species for transformation. Considering the time and effort required for stable transformation in sorghum, establishing a transient system to screen the efficiency and full functionality of vector constructs is highly desirable. Results Here, we report an Agrobacterium-mediated transient transformation assay with intact sorghum leaves using green fluorescent protein as marker. It also provides a good monocot alternative to tobacco and protoplast assays with a direct, native and more reliable system for testing single guide RNA (sgRNA) expression construct efficiency. Given the simplicity and ease of transformation, high reproducibility, and ability to test large constructs, this method can be widely adopted to speed up functional genomic and genome editing studies.

scholarly journals Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies

2019 ◽  
Author(s):  
Rita Sharma ◽  
Yan Liang ◽  
Mi Yeon Lee ◽  
Venkataramana R. Pidatala ◽  
Jenny C. Mortimer ◽  
...  
Keyword(s):  
Genome Editing ◽  
Fluorescent Protein ◽  
Transient Transformation ◽  
Reliable System ◽  
High Reproducibility ◽  
Guide Rna ◽  
Expression Construct ◽  
Speed Up ◽  
Transient System ◽  
Green Fluorescent

ABSTRACTObjectivesSorghum is one of the most recalcitrant species for transformation. Considering the time and effort required for stable transformation in sorghum, establishing a transient system to screen the efficiency and full functionality of vector constructs is highly desirable.ResultsHere, we report an Agrobacterium-mediated transient transformation assay with intact sorghum leaves using green fluorescent protein as marker. It also provides a good monocot alternative to tobacco and protoplast assays with a direct, native and more reliable system for testing single guide RNA (sgRNA) expression construct efficiency. Given the simplicity and ease of transformation, high reproducibility, and ability to test large constructs, this method can be widely adopted to speed up functional genomic and genome editing studies.

scholarly journals Hydrodynamics-Based Transplacental Delivery as a Useful Noninvasive Tool for Manipulating Fetal Genome

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1744
Author(s):  
Shingo Nakamura ◽  
Naoko Ando ◽  
Satoshi Watanabe ◽  
Eri Akasaka ◽  
Masayuki Ishihara ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Genome Editing ◽  
Fluorescent Protein ◽  
Guide Rna ◽  
Gene Insertion ◽  
Fetal Toxicity ◽  
A Genome ◽  
Wild Type Female ◽  
Green Fluorescent ◽  
Exogenous Plasmid

We previously demonstrated that the injection of pregnant wild-type female mice (carrying enhanced green fluorescent protein (EGFP)-expressing transgenic fetuses) at embryonic day (E) 12.5 with an all-in-one plasmid conferring the expression of both Cas9 and guide RNA (targeted to the EGFP cDNA) complexed with the gene delivery reagent, resulted in some fetuses exhibiting reduced fluorescence in their hearts and gene insertion/deletion (indel) mutations. In this study, we examined whether the endogenous myosin heavy-chain α (MHCα) gene can be successfully genome-edited by this method in the absence of a gene delivery reagent with potential fetal toxicity. For this, we employed a hydrodynamics-based gene delivery (HGD) system with the aim of ensuring fetal gene delivery rates and biosafety. We also investigated which embryonic stages are suitable for the induction of genome editing in fetuses. Of the three pregnant females injected at E9.5, one had mutated fetuses: all examined fetuses carried exogenous plasmid DNA, and four of 10 (40%) exhibited mosaic indel mutations in MHCα. Gene delivery to fetuses at E12.5 and E15.5 did not cause mutations. Thus, the HGD-based transplacental delivery of a genome editing vector may be able to manipulate the fetal genomes of E9.5 fetuses.

scholarly journals CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yoshihiko Nanasato ◽  
Masafumi Mikami ◽  
Norihiro Futamura ◽  
Masaki Endo ◽  
Mitsuru Nishiguchi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cryptomeria Japonica ◽  
Fluorescent Protein ◽  
Japanese Cedar ◽  
Targeted Mutagenesis ◽  
Embryogenic Tissue ◽  
Breeding Period ◽  
Single Mutation ◽  
Knock Out ◽  
Guide Rna

AbstractCryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

scholarly journals Supramolecular nanosubstrate–mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies

2020 ◽  
Vol 6 (43) ◽  
pp. eabb7107
Author(s):  
Peng Yang ◽  
Shih-Jie Chou ◽  
Jindian Li ◽  
Wenqiao Hui ◽  
Wenfei Liu ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Integration Site ◽  
Genetic Diseases ◽  
Proliferative Potential ◽  
Adeno Associated Virus ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Green Fluorescent ◽  
Virus Integration

Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate–mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)–encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies.

scholarly journals Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in C. elegans

2018 ◽  
Author(s):  
Gregoriy A. Dokshin ◽  
Krishna S. Ghanta ◽  
Katherine M. Piscopo ◽  
Craig C. Mello
Keyword(s):  
Green Fluorescent Protein ◽  
Cost Effectiveness ◽  
Genome Editing ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
C Elegans ◽  
Multiple Loci ◽  
Green Fluorescent ◽  
Fluorescent Protein Tags ◽  
Protein Tags

AbstractCRISPR-based genome editing using ribonucleoprotein (RNP) complexes and synthetic single stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs – such as green fluorescent protein (GFP) tags remains challenging in many systems. Here we describe a streamlined and optimized editing protocol for the nematode C. elegans. We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging all twelve of the Worm-specific Argonaute (WAGO) proteins in C. elegans using ssODN donors. In addition, we describe a novel PCR-based partially single-stranded “hybrid” donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.

scholarly journals CRISPR/Cas9-mediated genome editing in a reef-building coral

2018 ◽  
Vol 115 (20) ◽  
pp. 5235-5240 ◽  
Author(s):  
Phillip A. Cleves ◽  
Marie E. Strader ◽  
Line K. Bay ◽  
John R. Pringle ◽  
Mikhail V. Matz
Keyword(s):  
Genome Editing ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Mutant Gene ◽  
Mutation Induction ◽  
Induced Mutations ◽  
Important Species ◽  
Ribonucleoprotein Complexes ◽  
Genes Encoding ◽  
Green Fluorescent

Reef-building corals are critically important species that are threatened by anthropogenic stresses including climate change. In attempts to understand corals’ responses to stress and other aspects of their biology, numerous genomic and transcriptomic studies have been performed, generating a variety of hypotheses about the roles of particular genes and molecular pathways. However, it has not generally been possible to test these hypotheses rigorously because of the lack of genetic tools for corals. Here, we demonstrate efficient genome editing using the CRISPR/Cas9 system in the coral Acropora millepora. We targeted the genes encoding fibroblast growth factor 1a (FGF1a), green fluorescent protein (GFP), and red fluorescent protein (RFP). After microinjecting CRISPR/Cas9 ribonucleoprotein complexes into fertilized eggs, we detected induced mutations in the targeted genes using changes in restriction-fragment length, Sanger sequencing, and high-throughput Illumina sequencing. We observed mutations in ∼50% of individuals screened, and the proportions of wild-type and various mutant gene copies in these individuals indicated that mutation induction continued for at least several cell cycles after injection. Although multiple paralogous genes encoding green fluorescent proteins are present in A. millepora, appropriate design of the guide RNA allowed us to induce mutations simultaneously in more than one paralog. Because A. millepora larvae can be induced to settle and begin colony formation in the laboratory, CRISPR/Cas9-based gene editing should allow rigorous tests of gene function in both larval and adult corals.

scholarly journals A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

2017 ◽  
Vol 4 (5) ◽  
pp. 170095 ◽  
Author(s):  
Tom Beneke ◽  
Ross Madden ◽  
Laura Makin ◽  
Jessica Valli ◽  
Jack Sunter ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Large Scale ◽  
Leishmania Major ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Gene Knockout ◽  
Guide Rna ◽  
Functional Studies

Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major , Leishmania mexicana and bloodstream form Trypanosoma brucei ; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens.

scholarly journals Gene editing in bacteria via SpCas9/sgRNA ribonucleoprotein complexes

2021 ◽  
Author(s):  
Ruben Dario Arroyo-Olarte ◽  
Ricardo Neftali Bravo-Rodriguez ◽  
Edgar Morales-Rios
Keyword(s):  
Green Fluorescent Protein ◽  
Homologous Recombination ◽  
Gene Editing ◽  
Fluorescent Protein ◽  
High Toxicity ◽  
Guide Rna ◽  
Ribonucleoprotein Complexes ◽  
Gfp Reporter ◽  
Blue Fluorescent Protein ◽  
Green Fluorescent

Gene editing has been revolutionized by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas technology in a variety of organisms. In bacteria, however, CRISPR-Cas still holds many caveats such as high toxicity of Cas9 and off-target editing effects. In this work we develop a system for the incorporation of Cas9/single guide RNA ribonucleoprotein complexes in bacteria and their successful application for gene editing via homologous recombination. Targeting of a green fluorescent protein (GFP) reporter allows for easy verification of gene-edition via conversion to blue-fluorescent protein (BFP), mediated by the well characterized 196T > C (Tyr66His) mutation.

scholarly journals Possible differences in efficiency of guide RNA with different spacer sequence in CRISPR knock-down or knock-out of particular gene

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Gene Expression ◽  
Genome Editing ◽  
Target Gene ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Bacterial Cells ◽  
Fluorescent Intensity ◽  
Guide Rna ◽  
Targeting Efficiency ◽  
Template Dna

Cluster regularly interspersed short palindromic repeats (CRISPR) mediated genome editing has emerged as the dominant technique for modulating the expression of target genes. Specifically, when coupled with different effectors, CRISPR could be utilized to either activate or repress gene expression. Specificity of the CRISPR gene editing method arises from the unique spacer sequence in guide RNA that mediates the specific localization of Cas9 endonuclease to particular stretches of DNA. However, complementary base pairing between the guide RNA and template DNA depends critically on existence of protospacer adjacent motif (PAM) sequence immediately downstream of the spacer sequence. Such three nucleotide PAM sequence could be present at multiple loci in a given gene, which meant that different spacer sequence could be incorporated in guide RNA design to target the same gene. Given that different spacer sequences have different binding affinities to template DNA, differences could exist in the efficiency in which CRISPR-Cas9 could be guided to generate a double strand break in a particular gene locus. Using green fluorescent protein (GFP) reporter gene expressed in recombinant Escherichia coli as experimental system, this study sought to understand if differences in targeting efficiency exist between guide RNA with different spacer sequence that could target the same gene. Fluorescent intensity of cells at the population level would serve as readout of the targeting efficiency. For example, spacer sequence in guide RNA that could better activate the endonuclease activity of Cas9 would result in lower fluorescent intensity of GFP. To check for the effect of expression mode on targeting efficiency of guide RNA, GFP gene would be expressed on a plasmid in E. coli as well as integrated into the genome of the bacterium. Doing so would provide critical information on whether the CRISPR-Cas9 system has differentiated efficacy in generating double strand breaks in genomic versus plasmid DNA. Such information would inform future experimental design involving CRISPR-Cas9 genome editing technology as well as hold implications on how CRISPR evolved as an adaptive immune system in defending bacterial cells against foreign DNA. Given the goal of the study to understand the relative extent in which a target gene would be disrupted by CRISPR-Cas9 guided by different spacer sequence on guide RNA, no repair module for the target gene would be provided. Collectively, multiple occurrence of PAM sequence in a target gene meant that different spacer sequences could be used in CRISPR-Cas9 to downregulate gene expression. Relative efficacies of different spacer sequence in guide RNA in achieving targeted gene inactivation remain poorly understood and constitutes the basis of this study, which hopefully would provide guidance on the selection of specific spacer sequence that would yield the most efficacious disruption of gene expression at the genome and plasmid level.

scholarly journals Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus

2019 ◽  
Vol 116 (32) ◽  
pp. 15774-15783 ◽  
Author(s):  
Claudio Hidalgo-Cantabrana ◽  
Yong Jun Goh ◽  
Meichen Pan ◽  
Rosemary Sanozky-Dawes ◽  
Rodolphe Barrangou
Keyword(s):  
Genome Editing ◽  
Fluorescent Protein ◽  
Stop Codon ◽  
Mucosal Vaccine ◽  
Green Fluorescent Protein Gene ◽  
Type I ◽  
Lactobacillus Crispatus ◽  
Class 1 ◽  
Flexible Genome ◽  
Green Fluorescent

CRISPR-Cas systems are now widely used for genome editing and transcriptional regulation in diverse organisms. The compact and portable nature of class 2 single effector nucleases, such as Cas9 or Cas12, has facilitated directed genome modifications in plants, animals, and microbes. However, most CRISPR-Cas systems belong to the more prevalent class 1 category, which hinges on multiprotein effector complexes. In the present study, we detail how the native type I-E CRISPR-Cas system, with a 5′-AAA-3′ protospacer adjacent motif (PAM) and a 61-nucleotide guide CRISPR RNA (crRNA) can be repurposed for efficient chromosomal targeting and genome editing in Lactobacillus crispatus, an important commensal and beneficial microbe in the vaginal and intestinal tracts. Specifically, we generated diverse mutations encompassing a 643-base pair (bp) deletion (100% efficiency), a stop codon insertion (36%), and a single nucleotide substitution (19%) in the exopolysaccharide priming-glycosyl transferase (p-gtf). Additional genetic targets included a 308-bp deletion (20%) in the prophage DNA packaging Nu1 and a 730-bp insertion of the green fluorescent protein gene downstream of enolase (23%). This approach enables flexible alteration of the formerly genetically recalcitrant species L. crispatus, with potential for probiotic enhancement, biotherapeutic engineering, and mucosal vaccine delivery. These results also provide a framework for repurposing endogenous CRISPR-Cas systems for flexible genome targeting and editing, while expanding the toolbox to include one of the most abundant and diverse systems found in nature.

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