scholarly journals PNA–NLS conjugates as single-molecular activators of target sites in double-stranded DNA for site-selective scission

2013 ◽  
Vol 11 (32) ◽  
pp. 5233 ◽  
Author(s):  
Yuichiro Aiba ◽  
Yuya Hamano ◽  
Wataru Kameshima ◽  
Yasuyuki Araki ◽  
Takehiko Wada ◽  
...  
Keyword(s):  
Double Stranded Dna ◽  
Target Sites ◽  
Site Selective

scholarly journals Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiwei Hu ◽  
Yannan Wang ◽  
Qian Liu ◽  
Yan Qiu ◽  
Zhiyu Zhong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Target Sites ◽  
Guide Sequence ◽  
Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

Activation of Double-stranded DNA by One pcPNA Strand for Its Site-selective Scission with CeIV/EDTA

2007 ◽  
Vol 36 (6) ◽  
pp. 780-781 ◽  
Author(s):  
Yuichiro Aiba ◽  
Yoji Yamamoto ◽  
Makoto Komiyama
Keyword(s):  
Double Stranded Dna ◽  
Site Selective

Artificial restriction DNA cutter for site-selective scission of double-stranded DNA with tunable scission site and specificity

2008 ◽  
Vol 3 (4) ◽  
pp. 655-662 ◽  
Author(s):  
Makoto Komiyama ◽  
Yuichiro Aiba ◽  
Yoji Yamamoto ◽  
Jun Sumaoka
Keyword(s):  
Double Stranded Dna ◽  
Artificial Restriction ◽  
Site Selective

scholarly journals Tandem paired nicking promotes precise genome editing with scarce interference by p53

2019 ◽  
Author(s):  
Toshinori Hyodo ◽  
Md Lutfur Rahman ◽  
Sivasundaram Karnan ◽  
Takuji Ito ◽  
Atsushi Toyoda ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Dna Recombination ◽  
Genetic Changes ◽  
Double Stranded Dna ◽  
Cas9 Nuclease ◽  
Target Sites ◽  
P53 Activation ◽  
Additional Nucleotide

SummaryTargeted knock-in mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites. A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knock-in. Here, we demonstrate that tandem paired nicking, a method for targeted knock-in involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knock-in largely equivalent to that of the Cas9 nuclease-based approach. Tandem paired nicking seems to accomplish targeted knock-in via DNA recombination analogous to Holliday’s model, and creates intended genetic changes in the genome without introducing additional nucleotide changes such as silent mutations. Targeted knock-in through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering.

scholarly journals An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Oguz Kanca ◽  
Jonathan Zirin ◽  
Jorge Garcia-Marques ◽  
Shannon Marie Knight ◽  
Donghui Yang-Zhou ◽  
...  
Keyword(s):  
S2 Cells ◽  
Single Stranded Dna ◽  
Double Stranded Dna ◽  
Target Sites ◽  
Multiple Uses

We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018a). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.

scholarly journals Rapid site-selective hydrolysis of double-stranded DNA by use of Ce(IV)/EDTA and PNA bearing phosphate group

2005 ◽  
Vol 49 (1) ◽  
pp. 277-278 ◽  
Author(s):  
Yuichiro Aiba ◽  
Masao Mori ◽  
Yoji Yamamoto ◽  
Makoto Komiyama
Keyword(s):  
Phosphate Group ◽  
Selective Hydrolysis ◽  
Double Stranded Dna ◽  
Site Selective ◽  
Hydrolysis Of

Site-Selective Binding of Nanoparticles to Double-Stranded DNA via Peptide Nucleic Acid “Invasion”

ACS Nano ◽  
2011 ◽  
Vol 5 (4) ◽  
pp. 2467-2474 ◽  
Author(s):  
Andrea L. Stadler ◽  
Dazhi Sun ◽  
Mathew M. Maye ◽  
Daniel van der Lelie ◽  
Oleg Gang
Keyword(s):  
Nucleic Acid ◽  
Peptide Nucleic Acid ◽  
Selective Binding ◽  
Double Stranded Dna ◽  
Site Selective
Sign in / Sign up

Export Citation Format

Share Document