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

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.

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

eLife ◽

10.7554/elife.39468 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 20

Author(s):

Jose Arturo Gutierrez-Triana ◽

Tinatini Tavhelidse ◽

Thomas Thumberger ◽

Isabelle Thomas ◽

Beate Wittbrodt ◽

...

Keyword(s):

Genome Editing ◽

Single Copy ◽

Gene Replacement ◽

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.

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

10.1101/358424 ◽

2018 ◽

Cited By ~ 2

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 ◽

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.

Simultaneous precise editing of multiple genes in human cells

Nucleic Acids Research ◽

10.1093/nar/gkz669 ◽

2019 ◽

Vol 47 (19) ◽

pp. e116-e116 ◽

Cited By ~ 17

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 ◽

A Genome ◽

Dependent Protein ◽

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.

Precision genome editing in the CRISPR era

Biochemistry and Cell Biology ◽

10.1139/bcb-2016-0137 ◽

2017 ◽

Vol 95 (2) ◽

pp. 187-201 ◽

Cited By ~ 49

Author(s):

Jayme Salsman ◽

Graham Dellaire

Keyword(s):

Gene Therapy ◽

Genome Editing ◽

Point Mutations ◽

Dna Double Strand Breaks ◽

End Joining ◽

Genetic Changes ◽

New Era ◽

Repair Template ◽

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.

Efficient Disruption and Replacement of an Effector Gene in the Oomycete Phytophthora sojae using CRISPR/Cas9

10.1101/025023 ◽

2015 ◽

Author(s):

Yufeng Fang ◽

Brett M Tyler

Keyword(s):

Gene Replacement ◽

Phytophthora Species ◽

Phytophthora Sojae ◽

Targeted Mutagenesis ◽

End Joining ◽

Soybean Seedlings ◽

Effector Gene ◽

Rxlr Effector ◽

Phytophthora sojae is a pathogenic oomycete that infects soybean seedlings as well as stems and roots of established plants, costing growers $1–2 billion per year. Due to its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. Despite the availability of several genome sequences, the lack of efficient techniques for targeted mutagenesis and gene replacement have long hampered genetic studies of pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR effector gene Avr4/6 as target, we observed that in the absence of a homologous template, the repair of Cas9-induced double-strand breaks (DSBs) in P. sojae was mediated by non-homologous end joining (NHEJ), primarily resulting in short indels. Most mutants were homozygous, presumably due to gene conversion triggered by Cas9-mediated cleavage of non-mutant alleles. When donor DNA was present, homology directed repair (HDR) was observed, which resulted in the replacement of the target gene with the donor DNA. By testing the specific virulence of several NHEJ mutants and HDR -mediated gene replacements on soybeans, we have validated the contribution of Avr4/6 to recognition by soybean R gene loci, Rps4 and Rps6, but also uncovered additional contributions to resistance by these two loci. Our results establish a powerful tool for studying functional genomics in Phytophthora, which provides new avenues for better control of this pathogen.

Genome Editing Tools und ihr Einsatz in der experimentellen Augenheilkunde

Klinische Monatsblätter für Augenheilkunde ◽

10.1055/s-0042-119205 ◽

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 ◽

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.

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

Agricultural Reviews ◽

10.18805/ag.r-2298 ◽

2021 ◽

Author(s):

Varsha Kumari ◽

Priyanka Kumawat ◽

Sharanabasappa Yeri ◽

Shyam Singh Rajput

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Crop Improvement ◽

End Joining ◽

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.

CRISPResso: sequencing analysis toolbox for CRISPR genome editing

10.1101/031203 ◽

2015 ◽

Cited By ~ 4

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 ◽

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.

Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing

International Journal of Molecular Sciences ◽

10.3390/ijms22168571 ◽

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 ◽

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.

Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement

BMC Biology ◽

10.1186/s12915-019-0711-z ◽

2019 ◽

Vol 17 (1) ◽

Cited By ~ 3

Author(s):

Jes Alexander ◽

Gregory M. Findlay ◽

Martin Kircher ◽

Jay Shendure

Keyword(s):

Genome Editing ◽

Cpg Island ◽

Strand Break ◽

End Joining ◽

Exogenous Dna ◽

Cpg Dinucleotides ◽

Epigenome Editing ◽

Abstract Background Recent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double-strand break (DSB), followed by non-homologous end joining (NHEJ)- or homology-directed repair-mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas9) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, with the aim of driving replacement of the DNA sequence intervening the dual cuts via NHEJ. Results In a proof of concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event. Conclusions This study opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.