scholarly journals Direct Delivery of Cas9-sgRNA Ribonucleoproteins into Cells Using a Nanoneedle Array

2019 ◽  
Vol 9 (5) ◽  
pp. 965 ◽  
Author(s):  
Ayana Yamagishi ◽  
Daisuke Matsumoto ◽  
Yoshio Kato ◽  
Yuki Honda ◽  
Mone Morikawa ◽  
...  
Keyword(s):  
Aspect Ratio ◽  
Genome Editing ◽  
High Throughput ◽  
Mammalian Cells ◽  
Non Invasive ◽  
Nanoneedle Array ◽  
Novel Method ◽  
Direct Delivery ◽  
Target Effects ◽  
2D Array

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a powerful and widely used tool for genome editing. Recently, it was reported that direct delivery of Cas9-sgRNA ribonucleoproteins (RNPs) reduced off-target effects. Therefore, non-invasive, high-throughput methods are needed for direct delivery of RNPs into cells. Here, we report a novel method for direct delivery of RNPs into cells using a nanostructure with a high-aspect-ratio and uniform nanoneedles. This nanostructure is composed of tens of thousands of nanoneedles laid across a 2D array. Through insertion of the nanoneedle array previously adsorbed with Cas9-sgRNA, it was possible to deliver RNPs directly into mammalian cells for genome editing.

scholarly journals Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity

2020 ◽  
Vol 6 (6) ◽  
pp. eaay0187 ◽  
Author(s):  
Sabine Aschenbrenner ◽  
Stefan M. Kallenberger ◽  
Mareike D. Hoffmann ◽  
Adrian Huck ◽  
Roland Eils ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Genome Editing ◽  
Mammalian Cells ◽  
Viral Vectors ◽  
Transient Transfection ◽  
Target Specificity ◽  
Guide Rnas ◽  
Modeling And Experiments ◽  
Fine Tune ◽  
Target Effects

The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a combination of mathematical modeling and experiments, we first determined how CRISPR-Cas9 activity profiles relate to Cas9 specificity. We then used artificially weakened anti-CRISPR (Acr) proteins either coexpressed with or directly fused to Cas9 to fine-tune its activity toward selected levels, thereby achieving an effective kinetic insulation of ON- and OFF-target editing events. We demonstrate highly specific genome editing in mammalian cells using diverse single-guide RNAs prone to potent OFF-targeting. Last, we show that our strategy is compatible with different modes of delivery, including transient transfection and adeno-associated viral vectors. Together, we provide a highly versatile approach to reduce CRISPR-Cas OFF-target effects via kinetic insulation.

scholarly journals Efficient multiplex genome editing using CRISPR-Mb3Cas12a in mice

2019 ◽  
Author(s):  
Zhuqing Wang ◽  
Yue Wang ◽  
Shawn Wang ◽  
Andrew J Gorzalski ◽  
Hayden McSwiggin ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Editing Efficiency ◽  
Protospacer Adjacent Motif ◽  
Summary Statement ◽  
Strict Requirement ◽  
Moraxella Bovoculi ◽  
Target Effects

AbstractDespite many advantages over Cas9, Cas12a has not been widely used in genome editing in mammalian cells largely due to its strict requirement of the TTTV protospacer adjacent motif (PAM) sequence. Here, we report that Mb3Cas12a (Moraxella bovoculi AAX11_00205) could edit the genome in murine zygotes independent of TTTV PAM sequences and with minimal on-target mutations and close to 100% editing efficiency when crRNAs of 23nt spacers were used.Summary statementCRISPR-Mb3Cas12a can target a broader range of sequences in murine zygotes compared to AsCas12a and LbCas12a, and has lower on-target effects than Cas9 and high overall knock-in efficiency.

Bee Markers: A Novel Method for Non-Invasive Short Term Marking of Bats

2020 ◽  
Vol 21 (2) ◽  
pp. 465 ◽  
Author(s):  
Lucinda Kirkpatrick ◽  
Grzegorz Apoznański ◽  
Luc De Bruyn ◽  
Ralf Gyselings ◽  
Tomasz Kokurewicz
Keyword(s):  
Short Term ◽  
Non Invasive ◽  
Novel Method

scholarly journals Arrays of high-aspect ratio microchannels for high-throughput isolation of circulating tumor cells (CTCs)

2013 ◽  
Vol 20 (10-11) ◽  
pp. 1815-1825 ◽  
Author(s):  
Mateusz L. Hupert ◽  
Joshua M. Jackson ◽  
Hong Wang ◽  
Małgorzata A. Witek ◽  
Joyce Kamande ◽  
...  
Keyword(s):  
Aspect Ratio ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
High Throughput ◽  
High Aspect Ratio
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