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Some Like it Hot: Thermostable GeoCas9

Posted by Beth Kenkel on Sep 14, 2017 8:40:16 AM

Cas9 is the genome editing tool of choice for a number of model organisms: mammalian cells, yeast, drosophila, plants, worms, zebrafish, frogs, some bacteria; but not thermophilic (high heat loving) bacteria. Until recently the only available Cas9 proteins were isolated from mesophilic (medium heat loving) bacteria, such as Streptococcus pyogene’s SpCas9. These Cas9 proteins don’t work well at high temperatures, so to use them in thermophiles, bacteria must be grown at lower temps. This approach only works for facultative thermophiles (high OR medium heat loving), but not obligate thermophiles. However, the recent discovery of GeoCas9 by the Doudna lab has opened up the field of thermophilic bacteria to CRISPR/Cas9 genome editing.

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Topics: Genome Engineering, CRISPR

3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

Posted by Guest Blogger on Sep 5, 2017 9:58:42 AM

This post was contributed by guest bloggers Dominik Paquet and Dylan Kwart from Ludwig-Maximilians-University in Munich and Marc Tessier-Lavigne’s lab at the Rockefeller University in NYC.

The CRISPR/Cas9 system is a versatile tool for precise gene editing in many organisms and model systems. We have used CRISPR/Cas9 extensively for the purpose of making sequence-specific changes in human induced pluripotent stem cells (iPSCs). The CRISPR/Cas9 com­plex is very efficient at introducing double stranded breaks (DSBs) into genomic DNA in many cell types and often results in biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous end-joining (NHEJ) pathway, leading to nonspecific nucleotide insertions, dele­tions or other mutations, referred to as ‘indels’. While this is convenient for generating gene knockouts, NHEJ repair does not allow introduction of specific sequence changes.

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Topics: CRISPR

Human Germline Editing Using CRISPR

Posted by Mary Gearing on Aug 10, 2017 10:19:54 AM

Note: After this blog was published, a bioRxiv preprint that questions the conclusion of inter-homologue recombination was released. This blog has not been updated in response to this paper.

Any hint of CRISPR editing in human embryos has been met with a storm of media coverage. But the paper published August 2nd in Nature gives us even more to talk about, as it represents another step towards CRISPR germline editing of disease-causing mutations. But how close are we really, and what new questions does this paper bring up? We’ll sift through the paper to understand what Shoukhrat Mitalipov and his colleagues have achieved, and how the field will move forward from this work.

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Topics: CRISPR

Custom CRISPR Screens & the Green Listed Software

Posted by Guest Blogger on Jul 11, 2017 10:30:00 AM

This post was contributed by guest blogger Fredrik Wermeling, leader of a research group at the Centrum for Molecular Medicine (CMM), Department of Medicine, Solna, Karolinska Institutet, in Sweden.

It can be very time consuming to design 5 guide RNAs (gRNAs) targeting each of the 1000 genes you’d like to investigate in your next CRISPR screen. Luckily, the Green Listed software can help you do just this, probably in less than a minute (1).

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Topics: CRISPR

Anti-CRISPRs: Switching Off CRISPR-Cas9

Posted by Beth Kenkel on May 23, 2017 10:30:00 AM

CRISPR-Cas9 technology is constantly evolving. Variants of Cas9 can be used for genome editingactivating gene expression, repressing gene expression, and much more. But there’s one thing that’s been missing: a way to shut off Cas9’s activity after it’s been turned on. The concern is that the longer Cas9 remains active in a cell, the greater chances there are for off-target edits to occur. Although methods to switch on Cas9 activity using light or drugs have been developed, the field lacked an “off-switch” for Cas9.

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Topics: CRISPR

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