scholarly journals A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs

2021 ◽  
Vol 3 ◽  
Author(s):  
Wenzhi Jiang ◽  
Jenifer Bush ◽  
Jen Sheen
Keyword(s):  
Genome Editing ◽  
Technology Development ◽  
End Joining ◽  
Genomic Changes ◽  
Homology Directed Repair ◽  
Efficient Gene ◽  
Gfp Reporter ◽  
Gfp Reporter Gene ◽  
Development Evaluation ◽  
Diverse Plant Species

The ultimate goal of technology development in genome editing is to enable precisely targeted genomic changes in any cells or organisms. Here we describe protoplast systems for precise and efficient DNA sequence changes with preassembled Cas9 ribonucleoprotein (RNP) complexes in Arabidopsis thaliana, Nicotiana benthamiana, Brassica rapa, and Camelina sativa. Cas9 RNP-mediated gene disruption with dual gRNAs could reach ∼90% indels in Arabidopsis protoplasts. To facilitate facile testing of any Cas9 RNP designs, we developed two GFP reporter genes, which led to sensitive detection of nonhomologous end joining (NHEJ) and homology-directed repair (HDR), with editing efficiency up to 85 and 50%, respectively. When co-transfected with an optimal single-stranded oligodeoxynucleotide (ssODN) donor, precise editing of the AtALS gene via HDR reached 7% by RNPs. Significantly, precise mutagenesis mediated by preassembled primer editor (PE) RNPs led to 50% GFP reporter gene recovery in protoplasts and up to 4.6% editing frequency for the specific AtPDS mutation in the genome. The rapid, versatile and efficient gene editing by CRISPR RNP variants in protoplasts provides a valuable platform for development, evaluation and optimization of new designs and tools in gene and genomic manipulation and is applicable in diverse plant species.

scholarly journals Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

2018 ◽  
Author(s):  
Wannaporn Ittiprasert ◽  
Victoria H. Mann ◽  
Shannon E. Karinshak ◽  
Avril Coghlan ◽  
Gabriel Rinaldi ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Genome Editing ◽  
Human Macrophage ◽  
Wild Type ◽  
End Joining ◽  
Chromosomal Breakage ◽  
Knock Out ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

AbstractCRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 (ω1) ofSchistosoma mansonias a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5’-and 3’-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed ∼4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding ω1 were reduced >80% and lysates of ω1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of ω1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

scholarly journals Highly efficient homology-directed repair using transient CRISPR/Cpf1-geminiviral replicon in tomato

2019 ◽  
Author(s):  
Tien Van Vu ◽  
Velu Sivankalyani ◽  
Eun-Jung Kim ◽  
Duong Thi Hai Doan ◽  
Mil Thi Tran ◽  
...  
Keyword(s):  
Genome Editing ◽  
De Novo ◽  
Phenotypic Selection ◽  
Culture Conditions ◽  
Nonhomologous End Joining ◽  
Physical Culture ◽  
End Joining ◽  
Dark Cycle ◽  
Homology Directed Repair ◽  
Error Prone Repair

ABSTRACTGenome editing via the homology-directed repair (HDR) pathway in somatic plant cells is very inefficient compared to error-prone repair by nonhomologous end joining (NHEJ). Here, we increased HDR-based genome editing efficiency approximately 3-fold compared to a Cas9-based single-replicon system via the use of de novo multi-replicon systems equipped with CRISPR/LbCpf1 in tomato and obtained replicon-free but stable HDR alleles. The efficiency of CRISPR/LbCpf1-based HDR was significantly modulated by physical culture conditions such as temperature and light. Ten days of incubation at 31°C under a light/dark cycle after Agrobacterium-mediated transformation resulted in the best performance among the tested conditions. Furthermore, we developed our single-replicon system into a multi-replicon system that effectively increased HDR efficiency. Although this approach is still challenging, we showed the feasibility of HDR-based genome editing of a salt-tolerant SlHKT1;2 allele without genomic integration of antibiotic markers or any phenotypic selection. Self-pollinated offspring plants carrying the HKT1;2 HDR allele showed stable inheritance and germination tolerance in the presence of 100 mM NaCl. Our work may pave the way for transgene-free editing of alleles of interest in asexually as well as sexually reproducing plants.

scholarly journals Simultaneous precise editing of multiple genes in human cells

2019 ◽  
Vol 47 (19) ◽  
pp. e116-e116 ◽  
Author(s):  
Stephan Riesenberg ◽  
Manjusha Chintalapati ◽  
Dominik Macak ◽  
Philipp Kanis ◽  
Tomislav Maricic ◽  
...  
Keyword(s):  
Genome Editing ◽  
Kinase Inhibitor ◽  
Double Strand Breaks ◽  
End Joining ◽  
Nucleotide Substitutions ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
A Genome ◽  
Dependent Protein ◽  
Non Homologous End Joining

Abstract When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.

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 Efficient single-copy HDR by 5’ modified long dsDNA donors

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Jose Arturo Gutierrez-Triana ◽  
Tinatini Tavhelidse ◽  
Thomas Thumberger ◽  
Isabelle Thomas ◽  
Beate Wittbrodt ◽  
...  
Keyword(s):  
Genome Editing ◽  
Single Copy ◽  
Gene Replacement ◽  
End Joining ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

CRISPR/Cas9 efficiently induces targeted mutations via non-homologous-end-joining but for genome editing, precise, homology-directed repair (HDR) of endogenous DNA stretches is a prerequisite. To favor HDR, many approaches interfere with the repair machinery or manipulate Cas9 itself. Using Medaka we show that the modification of 5’ ends of long dsDNA donors strongly enhances HDR, favors efficient single-copy integration by retaining a monomeric donor conformation thus facilitating successful gene replacement or tagging.

scholarly journals CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Wen-Wei Zhang ◽  
Greg Matlashewski
Keyword(s):  
Visceral Leishmaniasis ◽  
Genome Editing ◽  
Cleavage Site ◽  
Leishmania Donovani ◽  
Expression Vectors ◽  
Drug Selection ◽  
End Joining ◽  
Content Type ◽  
Homology Directed Repair ◽  
Cas9 Nuclease

ABSTRACT The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani. IMPORTANCE Leishmania donovani is the causative agent of fatal visceral leishmaniasis. To understand Leishmania infection and pathogenesis and identify new drug targets for control of leishmaniasis, more-efficient ways to manipulate this parasite genome are required. In this study, we have implemented CRISPR-Cas9 genome-editing technology in L. donovani. Both single- and dual-gRNA expression vectors were developed using a strong RNA polymerase I promoter and ribozymes. With this system, it was possible to generate loss-of-function insertion and deletion mutations and introduce drug selection markers and the GFP sequence precisely into the L. donovani genome. These methods greatly improved the ability to manipulate this parasite genome and will help pave the way for high-throughput functional analysis of Leishmania genes. This study further revealed that double-stranded DNA breaks created by CRISPR-Cas9 were repaired by the homology-directed repair (HDR) pathway and microhomology-mediated end joining (MMEJ) in Leishmania.

Genome Editing Tools und ihr Einsatz in der experimentellen Augenheilkunde

2017 ◽  
Vol 234 (03) ◽  
pp. 329-334
Author(s):  
M. Yanik ◽  
W. Wende ◽  
K. Stieger
Keyword(s):  
Genome Editing ◽  
Neurodegenerative Erkrankungen ◽  
Transcription Activator ◽  
End Joining ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

ZusammenfassungNeue molekularbiologische Werkzeuge revolutionieren zurzeit die Genomchirurgie (genome editing) mit weitreichendem Einfluss auch auf die experimentelle Augenheilkunde. Neben den bereits etablierten Systemen wie den Zinkfingernukleasen (ZFN) oder Transcription-activator-like-Effector-Nukleasen (TALEN) sind es insbesondere die CRISPR-/Cas-Systeme (CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated), die überraschend einfach einen gezielten und präzisen Schnitt im Genom lebender Zellen ermöglichen. Dieser DNA-Doppelstrangbruch wird in der Zelle mittels NHEJ (non-homologous end joining) oder HDR (homology directed repair) repariert und kann ausgenutzt werden, um ein defektes Gen zu deaktivieren oder mithilfe einer korrekten Gensequenz zu reparieren. Die Genome-Editing-Technologie eröffnet damit bisher ungeahnte Möglichkeiten in der Grundlagenforschung, Biotechnologie, biomedizinischen Forschung bis hin zu ersten klinischen Anwendungen. Neurodegenerative Erkrankungen der Netzhaut stehen dabei aufgrund der guten Zugänglichkeit und des Immunprivilegs des Auges mit im Fokus des Interesses von Forschern und Firmen.

scholarly journals CRISPR-Cas9: A Genome Editing Tool in Crop Plants: A Review

2021 ◽  
Author(s):  
Varsha Kumari ◽  
Priyanka Kumawat ◽  
Sharanabasappa Yeri ◽  
Shyam Singh Rajput
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Crop Improvement ◽  
End Joining ◽  
Homology Directed Repair ◽  
Ligase Iv ◽  
A Genome ◽  
Target Dna ◽  
Non Homologous End Joining ◽  
Dna Strand

Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease 9 (CRISPR-Cas9) system is a rapid technology for gene editing. CRISPR-Cas9 is an RNA guided gene editing tool where Cas9 acts as endonuclease by cutting the target DNA strand. Double Stranded Breaks (DBS) can be repaired by non-homologous end joining (NHEJ) and homology-directed repair (HDR). The NHEJ employs DNA ligase IV to rejoin the broken ends which cause insertion or deletion mutations, whereas HDR repairs the DSBs based on a homologous complementary template and results in perfect repair of broken ends. CRISPR-Cas9 impart diverse advantageous features in contrast with the conventional methods. In this review article, we have discussed CRISPR-Cas9 based genome editing along with its mechanism of action and role in crop improvement.

scholarly journals Efficient single-copy HDR by 5’ modified long dsDNA donors

2018 ◽  
Author(s):  
Jose Arturo Gutierrez-Triana ◽  
Tinatini Tavhelidse ◽  
Thomas Thumberger ◽  
Isabelle Thomas ◽  
Beate Wittbrodt ◽  
...  
Keyword(s):  
Genome Editing ◽  
Single Copy ◽  
Gene Replacement ◽  
End Joining ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

ABSTRACTCRISPR/Cas9 efficiently induces targeted mutations via non-homologous-end-joining but for genome editing, precise, homology-directed repair (HDR) of endogenous DNA stretches is a prerequisite. To favor HDR, many approaches interfere with the repair machinery or manipulate Cas9 itself. Using Medaka we show that the modification of 5’ ends of long dsDNA donors strongly enhances HDR, favors efficient single-copy integration by retaining a monomeric donor conformation thus facilitating successful gene replacement or tagging.

scholarly journals CRISPResso: sequencing analysis toolbox for CRISPR genome editing

2015 ◽  
Author(s):  
Luca Pinello ◽  
Matthew C Canver ◽  
Megan D Hoban ◽  
Stuart H Orkin ◽  
Donald B Kohn ◽  
...  
Keyword(s):  
Genome Editing ◽  
Comprehensive Evaluation ◽  
Targeted Mutagenesis ◽  
Sequencing Analysis ◽  
End Joining ◽  
Sequencing Errors ◽  
Homology Directed Repair ◽  
Experimental Variation ◽  
Non Homologous End Joining ◽  
Sequence Quality

Recent progress in genome editing technologies, in particular the CRISPR-Cas9 system, has provided new opportunities to investigate the biological functions of genomic sequences by targeted mutagenesis. Double strand breaks (DSBs) resulting from site-specific Cas9 cleavage can be resolved by endogenous DNA repair pathways such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). Deep sequencing of amplified genomic regions allows for quantitative and sensitive detection of targeted mutations. However, no standard analytic tool to date has been developed to systematically enumerate and visualize these events, and to solve challenging issues such as amplification or sequencing errors, experimental variation in sequence quality, ambiguous alignment of variable length indels, and difficulty in deconvoluting mixed HDR/NHEJ outcomes. To address these issues we developed CRISPResso, a robust computational pipeline that enables accurate quantification and visualization of CRISPR-Cas9 outcomes as well as comprehensive evaluation of effects on coding sequences, noncoding elements and selected off-target sites.

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