scholarly journals High Homology-Directed Repair Using Mitosis Phase and Nucleus Localizing Signal

2020 ◽  
Vol 21 (11) ◽  
pp. 3747
Author(s):  
Jeong Pil Han ◽  
Yoo Jin Chang ◽  
Dong Woo Song ◽  
Beom Seok Choi ◽  
Ok Jae Koo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
High Efficiency ◽  
End Joining ◽  
Homology Directed Repair ◽  
Cas9 Protein ◽  
Localization Signal ◽  
Non Homologous End Joining ◽  
The Relationship ◽  
Donor Template

In homology-directed repair, mediated knock-in single-stranded oligodeoxynucleotides (ssODNs) can be used as a homologous template and present high efficiency, but there is still a need to improve efficiency. Previous studies have mainly focused on controlling double-stranded break size, ssODN stability, and the DNA repair cycle. Nevertheless, there is a lack of research on the correlation between the cell cycle and single-strand template repair (SSTR) efficiency. Here, we investigated the relationship between cell cycle and SSTR efficiency. We found higher SSTR efficiency during mitosis, especially in the metaphase and anaphase. A Cas9 protein with a nuclear localization signal (NLS) readily migrated to the nucleus; however, the nuclear envelope inhibited the nuclear import of many nucleotide templates. This seemed to result in non-homologous end joining (NHEJ) before the arrival of the homologous template. Thus, we assessed whether NLS-tagged ssODNs and free NLS peptides could circumvent problems posed by the nuclear envelope. NLS-tagging ssODNs enhanced SSTR and indel efficiency by 4-fold compared to the control. Our results suggest the following: (1) mitosis is the optimal phase for SSTR, (2) the donor template needs to be delivered to the nucleus before nuclease delivery, and (3) NLS-tagging ssODNs improve SSTR efficiency, especially high in mitosis.

scholarly journals Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mollie S. Schubert ◽  
Bernice Thommandru ◽  
Jessica Woodley ◽  
Rolf Turk ◽  
Shuqi Yan ◽  
...  
Keyword(s):  
Design Parameters ◽  
Optimized Design ◽  
End Joining ◽  
Exogenous Dna ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Mammalian Cell Lines ◽  
Non Homologous End Joining ◽  
Dna Template ◽  
Donor Template

AbstractCRISPR–Cas proteins are RNA-guided nucleases used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing an exogenous DNA template during repair allows for the intentional, precise incorporation of a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less accurate NHEJ-mediated repair. Here, we describe comprehensive design considerations and optimized methods for highly efficient HDR using single-stranded oligodeoxynucleotide (ssODN) donor templates for several CRISPR–Cas systems including S.p. Cas9, S.p. Cas9 D10A nickase, and A.s. Cas12a delivered as ribonucleoprotein (RNP) complexes. Features relating to guide RNA selection, donor strand preference, and incorporation of blocking mutations in the donor template to prevent re-cleavage were investigated and were implemented in a novel online tool for HDR donor template design. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: https://www.idtdna.com/HDR

scholarly journals Improved methods and optimized design for CRISPR Cas9 and Cas12a homology-directed repair

2021 ◽  
Author(s):  
Mollie S Schubert ◽  
Bernice Thommandru ◽  
Jessica Woodley ◽  
Rolf Turk ◽  
Shuqi Yan ◽  
...  
Keyword(s):  
Optimized Design ◽  
End Joining ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Mammalian Cell Lines ◽  
Non Homologous End Joining ◽  
Dna Template ◽  
Donor Template

CRISPR-Cas proteins are used to introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing a DNA template during repair allows for precise introduction of a desired mutation via the HDR pathway. However, rates of repair by HDR are often slow compared to the more rapid but less accurate NHEJ-mediated repair. Here, we describe comprehensive design considerations and optimized methods for highly efficient HDR using single-stranded oligodeoxynucleotide (ssODN) donor templates for several CRISPR-Cas systems including S.p. Cas9, S.p. Cas9 D10A nickase, and A.s. Cas12a delivered as ribonucleoprotein complexes with synthetic guide RNAs. Features relating to guide RNA selection, donor strand preference, and incorporation of blocking mutations in the donor template to prevent re-cleavage were investigated and were implemented in a novel online tool for HDR donor template design. Additionally, we employ chemically modified HDR donor templates in combination with a small molecule to boost HDR efficiency up to 10-fold. These findings allow for high frequencies of precise repair utilizing HDR in multiple mammalian cell lines. Tool availability: www.idtdna.com/HDR

scholarly journals A CyclinB2-Cas9 fusion promotes the homology-directed repair of double-strand breaks

2019 ◽  
Author(s):  
Manuel M. Vicente ◽  
Afonso Mendes ◽  
Margarida Cruz ◽  
José R. Vicente ◽  
Vasco M. Barreto
Keyword(s):  
Cell Cycle ◽  
Strand Break ◽  
Relative Increase ◽  
End Joining ◽  
New Era ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Dna Lesion ◽  
Non Homologous End Joining ◽  
Cycle Dependent

ABSTRACTThe discovery of clustered regularly interspaced palindromic repeats (CRISPR), a defense system against viruses found in bacteria, launched a new era in gene targeting. The key feature of this technique is the guiding of the endonuclease Cas9 by single guide RNAs (sgRNA) to specific sequences, where a DNA lesion is introduced to trigger DNA repair. The CRISPR/Cas9 system may be extremely relevant for gene therapy, but the technique needs improvement to become a safe and fully effective tool. The Cas9-induced double-strand break (DSB) is repaired by one of two pathways, the error-prone Non-homologous end joining (NHEJ) or the high-fidelity Homology Direct Repair (HDR). Shifting the repair of the DSB to HDR is challenging, given the efficiency of NHEJ. Here we describe an engineered protein approach to increase knock-in efficiency by promoting the relative increase in Cas9 activity in G2, the phase of the cell cycle where HDR is more active. Cas9 was fused to the degradation domain of proteins known to be degraded in G1. The activity of two chimeric proteins, Geminin-Cas9 and CyclinB2-Cas9, is demonstrated, as well as their cell-cycle-dependent degradation. The chimeras shifted the repair of the DSBs to the HDR repair pathway compared to the commonly used Cas9. The application of cell cycle specific degradation tags could pave the way for more efficient and secure gene editing applications of the CRISPR/Cas9 system.

scholarly journals Gene targeting facilitated by engineered sequence-specific nucleases and its potential for applications in crop improvement

2021 ◽  
Author(s):  
Daisuke Miki ◽  
Rui Wang ◽  
Jing Li ◽  
Dali Kong ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Agronomic Traits ◽  
Higher Plants ◽  
Crop Improvement ◽  
Strand Break ◽  
End Joining ◽  
Homology Directed Repair ◽  
Target Dna ◽  
Targeted Gene Editing ◽  
Non Homologous End Joining

Abstract Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world’s population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining (NHEJ), and are only rarely repaired via error-free homology-directed repair (HDR) if an appropriate donor template is provided. Gene targeting (GT), i.e., the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.

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.

Editing livestock genomes with site-specific nucleases

2014 ◽  
Vol 26 (1) ◽  
pp. 74 ◽  
Author(s):  
Daniel F. Carlson ◽  
Wenfang Tan ◽  
Perry B. Hackett ◽  
Scott C. Fahrenkrug
Keyword(s):  
Genetic Alterations ◽  
Large Animal ◽  
Dna Breaks ◽  
Transcription Activator ◽  
End Joining ◽  
Site Specific ◽  
Homology Directed Repair ◽  
Double Strand Dna Breaks ◽  
Non Homologous End Joining ◽  
Inactive Genes

Over the past 5 years there has been a major transformation in our ability to precisely manipulate the genomes of animals. Efficiencies of introducing precise genetic alterations in large animal genomes have improved 100 000-fold due to a succession of site-specific nucleases that introduce double-strand DNA breaks with a specificity of 10–9. Herein we describe our applications of site-specific nucleases, especially transcription activator-like effector nucleases, to engineer specific alterations in the genomes of pigs and cows. We can introduce variable changes mediated by non-homologous end joining of DNA breaks to inactive genes. Alternatively, using homology-directed repair, we have introduced specific changes that support either precise alterations in a gene’s encoded polypeptide, elimination of the gene or replacement by another unrelated DNA sequence. Depending on the gene and the mutation, we can achieve 10%–50% effective rates of precise mutations. Applications of the new precision genetics are extensive. Livestock now can be engineered with selected phenotypes that will augment their value and adaption to variable ecosystems. In addition, animals can be engineered to specifically mimic human diseases and disorders, which will accelerate the production of reliable drugs and devices. Moreover, animals can be engineered to become better providers of biomaterials used in the medical treatment of diseases and disorders.

Homologous Recombination, Non-Homologous End-Joining and Cell Cycle: Genomes Angels

2004 ◽  
Vol 5 (1) ◽  
pp. 49-58
Author(s):  
Delacote F. ◽  
Guirouilh-Barbat J. ◽  
Lambert S. ◽  
B. Lopez
Keyword(s):  
Cell Cycle ◽  
Homologous Recombination ◽  
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.

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