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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

Multiplex Genome Editing with CRISPR-Cpf1

Posted by Beth Kenkel on May 9, 2017 10:12:15 AM

There’s a new development for CRISPR-Cpf1 genome editing!  A recent paper from Feng Zhang's lab describes how to use Cpf1 for multiplex genome editing.  For a few reasons, Cpf1 is a simplified system for editing multiple targets compared to Cas9.  Read on to learn more about Cpf1 multiplexing.  For an in-depth review of Cpf1, check out this blog post or see Addgene's CRISPR guide page for a review of Cas9.  For a brief comparison of Cpf1 vs. Cas9, see the table below.

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Topics: CRISPR

How to Design Your gRNA for CRISPR Genome Editing

Posted by Guest Blogger on May 3, 2017 11:00:00 AM

This Post was updated on May 3, 2017 with additional information and resources. 

This post was contributed by guest blogger, Addgene Advisory Board member, and Associate Director of the Genetic Perturbation Platform at the Broad Institute, John Doench.

CRISPR technology has made it easier than ever both to engineer specific DNA edits and to perform functional screens to identify genes involved in a phenotype of interest. This blog post will discuss differences between these approaches, as well as provide updates on how best to design gRNAs. You can also find validated gRNAs for your next experiment in Addgene's Validated gRNA Sequence Datatable.

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Topics: Plasmid How To, Genome Engineering, Lab Tips, CRISPR

Plasmids for Endogenous Gene Tagging in Human Cells

Posted by Guest Blogger on Apr 6, 2017 9:02:59 AM

This post was contributed by the gene editing team at the Allen Institute for Cell Science. Learn more by visiting the Allen Cell Explorer at allencell.org and the Allen Institute website at alleninstitute.org.

A classic challenge in cell biology is making sure that what we observe through the microscope represents reality as accurately as possible. This is especially true in the case of protein tagging to elucidate cellular structures. Overexpression methods flood the cell with protein, which can both interfere with a cell’s normal function and result in a ubiquitous background signal that makes it hard to visualize the precise location of the protein or structure of interest.

Endogenous gene tagging is an ideal solution because it allows for tagging and visualization of specific, individual proteins under endogenous regulatory control. But even with the advent of CRISPR/Cas9 technology, inserting large tags into a precise location in the genome is still inefficient, particularly in human cell lines. Furthermore, the quality control necessary to ensure the edited cells are behaving normally can be prohibitively expensive for many labs.

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Topics: Plasmid How To, CRISPR, Techniques

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