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Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

Posted by Alyssa Cecchetelli on May 26, 2020 9:15:00 AM

If you’re using CRISPR to make a genome edit, how do you know if your CRISPR experiment was successful in your organism or cell type? You can use DNA sequencing or other molecular cloning techniques to determine CRISPR/sgRNA efficiency of an experiment and confirm the correct edit was made to the genome without any off target effects. However, these methods can be labor intensive and quite time consuming.  

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Topics: CRISPR, Other CRISPR Tools

A new generation of adenine base editors improves editing in primary human cells

Posted by Susanna Bachle on May 7, 2020 9:15:00 AM

Adenine base editors (ABE) mediate A•T-to-G•C base changes (Figure 1), but it can be challenging to make these base changes, especially in primary human cells. Now, scientists at Beam Therapeutics have found a way to improve editing in primary human cells (Gaudelli et al., 2020).

One of the widely used base editing systems, ABE7.10 (and the starting point for a new generation of ABEs), consists of 3 components: 

  • a deaminase (TadA, originally from E.coli, named TadA7.10 in ABE7.10) 
  • a catalytically impaired Cas protein (dCas or Cas nickase) 
  • a guide RNA that targets the complex of TadA and dCas to the genomic DNA of interest 
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Topics: CRISPR, Base Editing

SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas

Posted by Guest Blogger on May 5, 2020 9:15:00 AM

This post was contributed by Shravanti Suresh from Iowa State University.

Since its appearance, SARS-CoV-2 has spread to almost every part of the world manifesting as a full-fledged pandemic. Containing the spread of this virus has become an utmost priority for countries around the world and to do so, the WHO recommends one strategy: testing, tracking, and social distancing.

With countries like South Korea, one of the earlier epicenters of the outbreak ultimately flattening the curve, it has become evident that widespread testing is crucial in controlling this pandemic. Currently, the CDC uses RT-qPCR tests to diagnose COVID-19 and some serological tests to determine past exposure. However, the limited availability of reagents and equipment and the long turnaround times, have led researchers to turn to other technologies like CRISPR.

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Topics: CRISPR, Cas Proteins, COVID-19

Finding nucleic acids with SHERLOCK and DETECTR

Posted by Alyssa Cecchetelli on Apr 16, 2020 9:00:09 AM

Originally published Aug 30, 2018 and last updated April 16, 2020.

Sensitive and specific nucleic acid detection is crucial for clinical diagnostics, genotyping, and biotechnological advancements. Many methods of nucleic acid detection however, either lack the sensitivity or the specificity to detect nucleic acids at low concentrations and/or are too expensive, time-consuming, and complex to use outside of standard laboratories. In the case of the COVID-19 pandemic, qPCR can be used to diagnose the presence of SARS-CoV-2 RNA, but inadequate access to reagents and equipment has become a bottleneck.

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Topics: CRISPR, Cas Proteins, Other CRISPR Tools, COVID-19

Expanding the Targeting Scope and Editing Efficiency of Adenine Base Editors

Posted by Susanna Bachle on Mar 17, 2020 9:32:55 AM

David Liu’s lab created the first base editor in 2016 (Komor et al., 2016) and since then has been trying to expand their precision editing capabilities. Base editors make specific DNA base changes and consist of a catalytically impaired Cas protein (dCas or Cas nickase) fused to a DNA-modifying enzyme, in this case a deaminase. Base changes from C•G-to-T•A are mediated by cytosine base editors (CBEs) and base changes from A•T-to-G•C are mediated by adenine base editors (ABEs). How does this work? Through molecular biology teamwork. The guide RNA (gRNA) specifies the editing target site on the DNA, the Cas domain directs the modifying enzyme to the target site, and the deaminase induces the DNA base change without a DNA double-strand break. But base editors aren’t perfect. They may be slow, can only target certain sites, or make only a subset of base substitutions. 

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Topics: CRISPR, Base Editing

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