scholarly journals Highly efficient homology-directed repair using Cas9 protein in Ceratitis capitata

2018 ◽  
Author(s):  
Roswitha A. Aumann ◽  
Marc F. Schetelig ◽  
Irina Häecker
Keyword(s):  
Control Systems ◽  
Genome Editing ◽  
Pest Control ◽  
Genetically Modified Organisms ◽  
Ceratitis Capitata ◽  
Fluorescent Protein ◽  
Great Promise ◽  
Homology Directed Repair ◽  
Highly Efficient ◽  
Ribonucleoprotein Complexes

AbstractBackgroundThe Mediterranean fruit fly Ceratitis capitata is a highly polyphagous and invasive insect pest, causing vast economical damage in horticultural systems. A currently used control strategy is the sterile insect technique (SIT) that reduces pest populations through infertile matings with mass-released, sterilized insects. Transgenic approaches hold great promise to improve key aspects of a successful SIT program. However, there is strict or even prohibitive legislation regarding the release of genetically modified organisms (GMO), while novel CRISPR-Cas technologies might allow to develop genetically enhanced strains for SIT programs classified as non-transgenic.ResultsHere we describe highly efficient homology-directed repair genome editing in C. capitata by injecting pre-assembled CRISPR-Cas9 ribonucleoprotein complexes using different guide RNAs and a short single-stranded oligodeoxynucleotide donor to convert an enhanced green fluorescent protein in C. capitata into a blue fluorescent protein. Six out of seven fertile and individually backcrossed G0 individuals generated 57-90% knock-in rate within their total offspring and 70-96% knock-in rate within their phenotypically mutant offspring.ConclusionConsidering the possibility that CRISPR-induced alterations in organisms could be classified as a non-GMO in the US and Europe, our approach to homology-directed repair genome editing can be used to genetically improve strains for pest control systems like SIT without the need to struggle with GMO directives. Furthermore, it can be used to recreate and use mutations, found in classical mutagenesis screens, for pest control systems.

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 5' modifications improve potency and efficacy of DNA donors for precision genome editing

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Krishna S Ghanta ◽  
Zexiang Chen ◽  
Aamir Mir ◽  
Gregoriy A Dokshin ◽  
Pranathi M Krishnamurthy ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Triethylene Glycol ◽  
Great Promise ◽  
Precise Form ◽  
Homology Directed Repair ◽  
Genomic Location ◽  
Cultured Human Cells ◽  
Repair Template ◽  
Template Dna

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5′-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

scholarly journals 5′ Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome Editing

2018 ◽  
Author(s):  
Krishna S. Ghanta ◽  
Gregoriy A. Dokshin ◽  
Aamir Mir ◽  
Pranathi Meda Krishnamurthy ◽  
Hassan Gneid ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Genetic Basis ◽  
Cell Types ◽  
Great Promise ◽  
Precise Form ◽  
Homology Directed Repair ◽  
Genomic Location ◽  
Repair Template ◽  
Template Dna

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach that directly addresses the underlying genetic basis of disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit the number of donor molecules available to the HDR machinery, thus limiting HDR efficacy. Here, we explore modifications to both double-stranded and single-stranded repair template DNAs and describe simple 5′ end modifications that consistently and dramatically increase donor potency and HDR efficacy across cell types and species.

scholarly journals Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing

2021 ◽  
Vol 22 (16) ◽  
pp. 8571
Author(s):  
Christopher E. Denes ◽  
Alexander J. Cole ◽  
Yagiz Alp Aksoy ◽  
Geng Li ◽  
G. Gregory Neely ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Nuclear Dna ◽  
Great Promise ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
Small Molecule Modulators

Modification of the human genome has immense potential for preventing or treating disease. Modern genome editing techniques based on CRISPR/Cas9 show great promise for altering disease-relevant genes. The efficacy of precision editing at CRISPR/Cas9-induced double-strand breaks is dependent on the relative activities of nuclear DNA repair pathways, including the homology-directed repair and error-prone non-homologous end-joining pathways. The competition between multiple DNA repair pathways generates mosaic and/or therapeutically undesirable editing outcomes. Importantly, genetic models have validated key DNA repair pathways as druggable targets for increasing editing efficacy. In this review, we highlight approaches that can be used to achieve the desired genome modification, including the latest progress using small molecule modulators and engineered CRISPR/Cas proteins to enhance precision editing.

scholarly journals Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Angela Meccariello ◽  
Simona Maria Monti ◽  
Alessandra Romanelli ◽  
Rita Colonna ◽  
Pasquale Primo ◽  
...  
Keyword(s):  
Gene Disruption ◽  
Ceratitis Capitata ◽  
Agricultural Pest ◽  
Highly Efficient ◽  
Ribonucleoprotein Complexes ◽  
Free Gene

scholarly journals Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks

2017 ◽  
Vol 114 (50) ◽  
pp. E10745-E10754 ◽  
Author(s):  
Alexandre Paix ◽  
Andrew Folkmann ◽  
Daniel H. Goldman ◽  
Heather Kulaga ◽  
Michael J. Grzelak ◽  
...  
Keyword(s):  
Genome Editing ◽  
Rational Design ◽  
Genome Engineering ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Template Switching ◽  
Dna Breaks ◽  
Homology Directed Repair ◽  
Switching Characteristics ◽  
Strand Annealing

The RNA-guided DNA endonuclease Cas9 has emerged as a powerful tool for genome engineering. Cas9 creates targeted double-stranded breaks (DSBs) in the genome. Knockin of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double stranded) engage in a high-efficiency HDR mechanism that requires only ∼35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1,000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand annealing. Our findings enable rational design of synthetic donor DNAs for efficient genome editing.

scholarly journals Genome Editing in Bacteria: CRISPR-Cas and Beyond

2021 ◽  
Vol 9 (4) ◽  
pp. 844
Author(s):  
Ruben D. Arroyo-Olarte ◽  
Ricardo Bravo Rodríguez ◽  
Edgar Morales-Ríos
Keyword(s):  
Genome Editing ◽  
Fluorescent Protein ◽  
Bacterial Genome ◽  
Guide Rnas ◽  
Comprehensive View ◽  
Ribonucleoprotein Complexes ◽  
Eukaryotic Organisms ◽  
Multi Step Methods ◽  
Cas Proteins

Genome editing in bacteria encompasses a wide array of laborious and multi-step methods such as suicide plasmids. The discovery and applications of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas based technologies have revolutionized genome editing in eukaryotic organisms due to its simplicity and programmability. Nevertheless, this system has not been as widely favored for bacterial genome editing. In this review, we summarize the main approaches and difficulties associated with CRISPR-Cas-mediated genome editing in bacteria and present some alternatives to circumvent these issues, including CRISPR nickases, Cas12a, base editors, CRISPR-associated transposases, prime-editing, endogenous CRISPR systems, and the use of pre-made ribonucleoprotein complexes of Cas proteins and guide RNAs. Finally, we also address fluorescent-protein-based methods to evaluate the efficacy of CRISPR-based systems for genome editing in bacteria. CRISPR-Cas still holds promise as a generalized genome-editing tool in bacteria and is developing further optimization for an expanded application in these organisms. This review provides a rarely offered comprehensive view of genome editing. It also aims to familiarize the microbiology community with an ever-growing genome-editing toolbox for bacteria.

scholarly journals Reprogramming acetogenic bacteria with CRISPR-targeted base editing via deamination

2020 ◽  
Author(s):  
Peng-Fei Xia ◽  
Isabella Casini ◽  
Sarah Schulz ◽  
Christian-Marco Klask ◽  
Largus T. Angenent ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Genetically Modified Organisms ◽  
Cytidine Deaminase ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Clostridium Ljungdahlii ◽  
Acetogenic Bacteria ◽  
A Genome ◽  
Nucleotide Resolution

AbstractAcetogenic bacteria are rising in popularity as chassis microbes in biotechnology due to their capability of converting inorganic one-carbon (C1) gases to organic chemicals. To fully uncover the potential of acetogenic bacteria, synthetic-biology tools are imperative to either engineer designed functions or to interrogate the physiology. Here, we report a genome-editing tool at a one-nucleotide resolution, namely base editing, for acetogenic bacteria based on CRISPR-targeted deamination. This tool combines nuclease deactivated Cas9 with activation-induced cytidine deaminase to enable cytosine-to-thymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which are generally the bottlenecks for applying conventional CRISPR-Cas systems in bacteria. We designed and validated a modularized base-editing tool in the model acetogenic bacterium Clostridium ljungdahlii. The editing principles were investigated, and an in-silico analysis revealed the capability of base editing across the genome. Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux towards improved acetate production. This resulted in engineered C. ljungdahlii strains with the desired phenotypes and stable genotypes. Our base-editing tool promotes the application and research in acetogenic bacteria and provides a blueprint to upgrade CRISPR-Cas-based genome editing in bacteria in general.SignificanceAcetogenic bacteria metabolize one-carbon (C1) gases, such as industrial waste gases, to produce fuels and commodity chemicals. However, the lack of efficient gene-manipulation approaches hampers faster progress in the application of acetogenic bacteria in biotechnology. We developed a CRISPR-targeted base-editing tool at a one-nucleotide resolution for acetogenic bacteria. Our tool illustrates great potential in engineering other A-T-rich bacteria and links designed single-nucleotide variations with biotechnology. It provides unique advantages for engineering industrially relevant bacteria without creating genetically modified organisms (GMOs) under the legislation of many countries. This base-editing tool provides an example for adapting CRISPR-Cas systems in bacteria, especially those that are highly sensitive to heterologously expressed Cas proteins and have limited ability of receiving foreign DNA.

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