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 CRISPR/Cas12a mediated genome engineering in photosynthetic bacteria

2020 ◽  
Author(s):  
Yang Zhang ◽  
Jifeng Yuan
Keyword(s):  
Genome Editing ◽  
Transcriptional Repression ◽  
Gene Editing ◽  
Photosynthetic Bacteria ◽  
Genome Engineering ◽  
Biotechnological Applications ◽  
End Joining ◽  
Editing Efficiency ◽  
Francisella Novicida ◽  
Non Homologous End Joining

ABSTRACTPurple non-sulfur photosynthetic bacteria (PNSB) such as R. capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. In this study, we sought to develop the class II RNA-guided CRISPR/Cas12a system from Francisella novicida for both genome editing and gene down-regulation in R. capsulatus. About 90% editing efficiency was achieved by using CRISPR/Cas12a driven by a strong promoter Ppuc when targeting ccoO or nifH gene. When both genes were simultaneously targeted, the multiplex gene editing efficiency reached >63%. In addition, CRISPR interference using deactivated Cas12a was also evaluated using reporter genes gfp and lacZ, and the repression efficiency reached >80%. In summary, our work represents the first report to develop CRISPR/Cas12a mediated genome editing/transcriptional repression in R. capsulatus, which would greatly accelerate PNSB-related researches.IMPORTANCEPurple non-sulfur photosynthetic bacteria (PNSB) such as R. capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. However, lack of efficient gene editing tools remains a main obstacle for progressing in PNSB-related researches. Here, we developed CRISPR/Cas12a for genome editing via the non-homologous end joining (NHEJ) repair machinery in R. capsulatus. In addition, DNase-deactivated Cas12a was found to simultaneously suppress multiple targeted genes. Taken together, our work offers a new set of tools for efficient genome engineering in PNSB such as R. capsulatus.

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 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 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 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 A CRISPR/Cas9 Method Facilitates Efficient Oligo-Mediated Gene Editing in Debaryomyces Hansenii

2021 ◽  
Author(s):  
Tomas Strucko ◽  
Niklas L Andersen ◽  
Mikkel R Mahler ◽  
José L Martínez ◽  
Uffe H Mortensen
Keyword(s):  
Gene Editing ◽  
Point Mutations ◽  
Debaryomyces Hansenii ◽  
Cell Factory ◽  
Full Potential ◽  
Dna Breaks ◽  
End Joining ◽  
Dna Oligonucleotides ◽  
Non Homologous End Joining ◽  
Basic And Applied Research

Abstract The halophilic and osmotolerant yeast Debaryomyces hansenii has a high potential for cell factory applications due to its resistance to harsh environmental factors and compatibility with a wide substrate range. However, currently available genetic techniques does not allow the full potential of D. hansenii as a cell factory to be harnessed. Moreover, most of the currently available tools rely on the use of auxotrophic markers that are not suitable in wild-type prototrophic strains. In addition, the preferred non-homologous end-joining (NHEJ) DNA damage repair mechanism pose further challenges when precise gene targeting is required. In this study, we present a novel plasmid based CRISPRCUG/Cas9 method for easy and efficient gene editing of the prototrophic strains of D. hansenii. Our toolset design is based on a dominant marker and facilitates quick assembly of the vectors expressing Cas9 and single or multiple sgRNAs that provides possibility for multiplex gene engineering even in prototrophic strains. Moreover, we have constructed an NHEJ deficient D. hansenii that enable our CRISPRCUG/Cas9 tools to support highly efficient introduction of point mutations and single/double gene deletions. Importantly, we also demonstrate that 90-nt single stranded DNA oligonucleotides are sufficient to direct repair of DNA breaks induced by sgRNA-Cas9 resulting in precise edits reaching 100% efficiencies. In conclusion, tools developed in this study will greatly advance basic and applied research in D. hansenii. In addition, we envision that our tools can be rapidly adapted for gene editing of other non-conventional yeast species including the ones belonging to the CUG clade.

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