scholarly journals Genome Editing in Caenorhabditis briggsae using the CRISPR/Cas9 System

2015 ◽  
Author(s):  
Elizabeth Culp ◽  
Cory Richman ◽  
Devika Sharanya ◽  
Bhagwati Gupta
Keyword(s):  
Genome Editing ◽  
Comparative Studies ◽  
Somatic Mutations ◽  
Sister Species ◽  
Efficient Technique ◽  
Caenorhabditis Briggsae ◽  
End Joining ◽  
C Elegans ◽  
Non Homologous End Joining

The CRISPR/Cas9 system is an efficient technique for generating targeted alterations in an organism's genome. Here we describe a methodology for using the CRISPR/Cas9 system to generate mutations via non-homologous end joining in the nematode Caenorhabditis briggsae, a sister species of C. elegans. Evidence for somatic mutations and off-target mutations are also reported. The use of the CRISPR/Cas9 system in C. briggsae will greatly facilitate comparative studies to C. elegans.

scholarly journals Genome editing in the nematode Caenorhabditis briggsae using the CRISPR/Cas9 system

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Elizabeth Culp ◽  
Cory Richman ◽  
Devika Sharanya ◽  
Nikita Jhaveri ◽  
Wouter van den Berg ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Gene Function ◽  
Somatic Mutations ◽  
Caenorhabditis Briggsae ◽  
Loss Of Function ◽  
End Joining ◽  
C Elegans ◽  
Conserved Genes ◽  
Foreign Dna ◽  
Non Homologous End Joining

Abstract The CRISPR/Cas system has recently emerged as a powerful tool to engineer the genome of an organism. The system is adopted from bacteria where it confers immunity against invading foreign DNA. This work reports the first successful use of the CRISPR/Cas system in Caenorhabditis briggsae (a cousin of the well-known nematode C. elegans), to generate mutations via non-homologous end joining. We recovered deletion alleles of several conserved genes by microinjecting plasmids that express Cas9 endonuclease and an engineered CRISPR RNA corresponding to the DNA sequence to be cleaved. Evidence for somatic mutations and off-target mutations are also reported. Our approach allows for the generation of loss-of-function mutations in C. briggsae genes thereby facilitating a comparative study of gene function.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

scholarly journals Use of single guided Cas9 nickase to facilitate precise and efficient genome editing in human iPSCs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pan P. Li ◽  
Russell L. Margolis
Keyword(s):  
Genome Editing ◽  
Disease Modeling ◽  
Neurodegenerative Disorder ◽  
Cag Repeat ◽  
Spinocerebellar Ataxia Type ◽  
End Joining ◽  
Human Ipscs ◽  
Non Homologous End Joining ◽  
Transient Overexpression ◽  
Donor Template

AbstractCas9 nucleases permit rapid and efficient generation of gene-edited cell lines. However, in typical protocols, mutations are intentionally introduced into the donor template to avoid the cleavage of donor template or re-cleavage of the successfully edited allele, compromising the fidelity of the isogenic lines generated. In addition, the double-stranded breaks (DSBs) used for editing can introduce undesirable “on-target” indels within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address these problems, we present an optimized protocol for precise genome editing in human iPSCs that employs (1) single guided Cas9 nickase to generate single-stranded breaks (SSBs), (2) transient overexpression of BCL-XL to enhance survival post electroporation, and (3) the PiggyBac transposon system for seamless removal of dual selection markers. We have used this method to modify the length of the CAG repeat contained in exon 7 of PPP2R2B. When longer than 43 triplets, this repeat causes the neurodegenerative disorder spinocerebellar ataxia type 12 (SCA12); our goal was to seamlessly introduce the SCA12 mutation into a human control iPSC line. With our protocol, ~ 15% of iPSC clones selected had the desired gene editing without “on target” indels or off-target changes, and without the deliberate introduction of mutations via the donor template. This method will allow for the precise and efficient editing of human iPSCs for disease modeling and other purposes.

scholarly journals Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing

2018 ◽  
Vol 19 (1) ◽  
Author(s):  
Tao Guo ◽  
Yi-Li Feng ◽  
Jing-Jing Xiao ◽  
Qian Liu ◽  
Xiu-Na Sun ◽  
...  
Keyword(s):  
Genome Editing ◽  
End Joining ◽  
Non Homologous End Joining

scholarly journals Genome-scale CRISPR screens are efficient in non-homologous end-joining deficient cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joana Ferreira da Silva ◽  
Sejla Salic ◽  
Marc Wiedner ◽  
Paul Datlinger ◽  
Patrick Essletzbichler ◽  
...  
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Knock Out ◽  
Genetic Ablation ◽  
Alternative End Joining ◽  
Non Homologous End Joining ◽  
Genome Scale

Abstract The mutagenic repair of Cas9 generated breaks is thought to predominantly rely on non-homologous end-joining (NHEJ), leading to insertions and deletions within DNA that culminate in gene knock-out (KO). In this study, by taking focused as well as genome-wide approaches, we show that this pathway is dispensable for the repair of such lesions. Genetic ablation of NHEJ is fully compensated for by alternative end joining (alt-EJ), in a POLQ-dependent manner, resulting in a distinct repair signature with larger deletions that may be exploited for large-scale genome editing. Moreover, we show that cells deficient for both NHEJ and alt-EJ were still able to repair CRISPR-mediated DNA double-strand breaks, highlighting how little is yet known about the mechanisms of CRISPR-based genome editing.

A CRISPR-Cpf1-Assisted Non-Homologous End Joining Genome Editing System ofMycobacterium smegmatis

2018 ◽  
Vol 13 (9) ◽  
pp. 1700588 ◽  
Author(s):  
Bingbing Sun ◽  
Junjie Yang ◽  
Sheng Yang ◽  
Richard D. Ye ◽  
Daijie Chen ◽  
...  
Keyword(s):  
Genome Editing ◽  
End Joining ◽  
Non Homologous End Joining

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 Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

2021 ◽  
Author(s):  
Marcos Fernando Basso ◽  
Karoline Estefani Duarte ◽  
Thais Ribeiro Santiago ◽  
Wagner Rodrigo de Souza ◽  
Bruno de Oliveira Garcia ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Gene Knockout ◽  
Setaria Viridis ◽  
End Joining ◽  
Non Homologous End Joining

scholarly journals Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases

2021 ◽  
Author(s):  
Eleonora I. Ioannidi ◽  
Matthew T. N. Yarnall ◽  
Cian Schmitt-Ulms ◽  
Rohan N. Krajeski ◽  
Justin Lim ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
High Efficiency ◽  
End Joining ◽  
Differentiated Cells ◽  
Dividing Cells ◽  
Gene Integration ◽  
Fluorescent Tagging ◽  
Non Homologous End Joining ◽  
Drag And Drop

Programmable and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double-strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. We present Programmable Addition via Site-specific Targeting Elements, or PASTE, which achieves efficient and versatile gene integration at diverse loci by directing insertion with a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. Without generating double stranded breaks, we demonstrate integration of sequences as large as ~36 kb with rates between 10-50% at multiple genomic loci across three human cell lines, primary T cells, and quiescent non-dividing primary human hepatocytes. To further improve PASTE, we discover thousands of novel serine integrases and cognate attachment sites from metagenomes and engineer active orthologs for high-efficiency integration using PASTE. We apply PASTE to fluorescent tagging of proteins, integration of therapeutically relevant genes, and production and secretion of transgenes. Leveraging the orthogonality of serine integrases, we engineer PASTE for multiplexed gene integration, simultaneously integrating three different genes at three genomic loci. PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in non-dividing cells and fewer detectable off-target events. For therapeutic applications, PASTE can be delivered as mRNA with synthetically modified guides to programmably direct insertion of DNA templates carried by AAV or adenoviral vectors. PASTE expands the capabilities of genome editing via drag-and-drop gene integration, offering a platform with wide applicability for research, cell engineering, and gene therapy.

Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

2020 ◽  
Author(s):  
Ruben Schep ◽  
Eva K. Brinkman ◽  
Christ Leemans ◽  
Xabier Vergara ◽  
Ben Morris ◽  
...  
Keyword(s):  
Genome Editing ◽  
Strand Break ◽  
Double Strand Break ◽  
Repair Pathway ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
The Impact

AbstractDNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is particularly important for Cas9-mediated genome editing, where the outcome critically depends on the pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance, and guidance for the design of Cas9-mediated genome editing experiments.

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