scholarly journals Repair of a Site-Specific DNA Cleavage: Old-School Lessons for Cas9-Mediated Gene Editing

2017 ◽  
Vol 13 (2) ◽  
pp. 397-405 ◽  
Author(s):  
Danielle N. Gallagher ◽  
James E. Haber
Keyword(s):  
Dna Cleavage ◽  
Gene Editing ◽  
Site Specific ◽  
A Site

DNA synthesis errors associated with double-strand-break repair.

Genetics ◽  
1995 ◽  
Vol 140 (3) ◽  
pp. 965-972 ◽  
Author(s):  
J N Strathern ◽  
B K Shafer ◽  
C B McGill
Keyword(s):  
Dna Synthesis ◽  
Dna Cleavage ◽  
Genome Duplication ◽  
Inducible Promoter ◽  
Strand Break ◽  
Site Specific ◽  
Large Populations ◽  
Dna Break ◽  
A Site ◽  
Ho Gene

Abstract Repair of a site-specific double-strand DNA break (DSB) resulted in increased reversion frequency for a nearby allele. Site-specific DSBs were introduced into the genome of Saccharomyces cerevisiae by the endonuclease encoded by the HO gene. Expression of the HO gene from a galactose-inducible promoter allowed efficient DNA cleavage at a single site in large populations of cells. To determine whether the DNA synthesis associated with repair of DSBs has a higher error rate than that associated with genome duplication, HO-induced DSBs were generated 0.3 kb from revertible alleles of trp1. The reversion rate of the trp1 alleles was approximately 100-fold higher among cells that had experienced an HO cut than among uninduced cells. The reverted allele was found predominantly on the chromosome that experienced the DNA cleavage.

scholarly journals RNA-CLAMP Enables Photo-activated Control of CRISPR-Cas9 Gene Editing by Site-specific Intramolecular Cross-linking of the sgRNA

2021 ◽  
Author(s):  
Dongyang Zhang ◽  
Shuaijiang Jin ◽  
Luping Liu ◽  
Ember Tota ◽  
Zijie Li ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Mammalian Cells ◽  
Gene Editing ◽  
Cross Linking ◽  
Stimuli Responsive ◽  
Spatiotemporal Resolution ◽  
Site Specific ◽  
Guide Rna ◽  
Dna Cleavage Activity ◽  
Versatile Tool

AbstractHere we introduce RNA-CLAMP, a technology which enables site-specific and enzymatic cross-linking (clamping) of two selected stem loops within an RNA of interest. Intramolecular clamping of the RNA can disrupt normal RNA function, whereas subsequent photo-cleavage of the crosslinker restores activity. We applied the RNA-CLAMP technique to the single guide RNA of the CRISPR-Cas9 gene editing system. By clamping two stem loops of the single-guide RNA (sgRNA) with a photo-cleavable cross-linker, gene editing was completely silenced. Visible light irradiation cleaved the crosslinker and restored gene editing with high spatiotemporal resolution. Furthermore, by designing two photo-cleavable linkers which are responsive to different wavelength of lights, we achieved multiplexed photo-activation of gene editing in mammalian cells. Notably, although the Cas9-sgRNA RNP is not capable of DNA cleavage activity upon clamping, it maintained the capability to bind to the target DNA. The RNA-CLAMP enabled photo-activated CRISPR-Cas9 gene editing platform offers clean background, free choice of activation wavelength and multiplexing capability. We believe that this technology to precisely and rapidly control gene editing will serve as a versatile tool in the future development of stimuli responsive gene editing technologies. Beyond gene editing, RNA-CLAMP provides a site-specific tool for manipulating the internal structure of functional RNAs.

scholarly journals Genome Editing With Targeted Deaminases

2016 ◽  
Author(s):  
Luhan Yang ◽  
Adrian W. Briggs ◽  
Wei Leong Chew ◽  
Prashant Mali ◽  
Marc Guell ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Zinc Finger Nucleases ◽  
Human Stem Cells ◽  
Site Specific ◽  
Cytidine Deaminases ◽  
Genetic Modifications ◽  
Reduced Toxicity ◽  
A Site ◽  
Dna Template

Precise genetic modifications are essential for biomedical research and gene therapy. Yet, traditional homology-directed genome editing is limited by the requirements for DNA cleavage, donor DNA template and the endogenous DNA break-repair machinery. Here we present programmable cytidine deaminases that enable site-specific cytidine to thymidine (C-to-T) genomic edits without the need for DNA cleavage. Our targeted deaminases are efficient and specific in Escherichia coli, converting a genomic C-to-T with 13% efficiency and 95% accuracy. Edited cells do not harbor unintended genomic abnormalities. These novel enzymes also function in human cells, leading to a site-specific C-to-T transition in 2.5% of cells with reduced toxicity compared with zinc-finger nucleases. Targeted deaminases therefore represent a platform for safer and effective genome editing in prokaryotes and eukaryotes, especially in systems where DSBs are toxic, such as human stem cells and repetitive elements targeting.

scholarly journals Kinetics of Nuclear Uptake and Site-Specific DNA Cleavage during CRISPR-Directed Gene Editing in Solid Tumor Cells

2020 ◽  
Vol 18 (6) ◽  
pp. 891-902 ◽  
Author(s):  
Kelly Banas ◽  
Natalia Rivera-Torres ◽  
Pawel Bialk ◽  
Byung-Chun Yoo ◽  
Eric B. Kmiec
Keyword(s):  
Tumor Cells ◽  
Solid Tumor ◽  
Dna Cleavage ◽  
Gene Editing ◽  
Site Specific ◽  
Nuclear Uptake ◽  
Kinetics Of

scholarly journals Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

1989 ◽  
Vol 9 (4) ◽  
pp. 1507-1512 ◽  
Author(s):  
H Zhu ◽  
H Conrad-Webb ◽  
X S Liao ◽  
P S Perlman ◽  
R A Butow
Keyword(s):  
Genetic Analysis ◽  
Rna Processing ◽  
Fold Increase ◽  
Functional Expression ◽  
Rrna Gene ◽  
Yeast Mitochondria ◽  
Wild Type ◽  
Site Specific ◽  
Var1 Gene ◽  
A Site

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.

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