Latest Posts

All Posts

New Optimized Genome-wide CRISPRko, CRISPRi, and CRISPRa Libraries

Posted by Alyssa Cecchetelli on Oct 4, 2018 8:44:18 AM

CRISPR pooled libraries have allowed scientists to easily perform genome-wide screens to effectively and efficiently investigate gene function. CRISPR libraries can be used to knock out, inhibit or activate target genes by combining specific sgRNAs with Cas9 or Cas9 derivatives.

Read More >

Topics: CRISPR, pooled libraries

Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

Posted by Beth Kenkel on Sep 13, 2018 9:55:58 AM

Genome-wide CRISPR/Cas9 screens are a high-throughput systematic approach for identifying genes involved in a biological process. These screens provide an alternative to genome-wide RNAi screens, which although highly effective, are affected by low on-target efficacy, non-specific toxicity, and off-target effects. The flaws of RNAi screens are well characterized and strategies exist to control for these faults. However, it’s still unclear if similar pitfalls exist for CRISPR screens and how best to design these screens to controls for flaws. Recently the Bassik Lab at Stanford developed a new genome-wide CRISPR knockout screen to analyze the following unanswered questions about CRISPR screen design.

Read More >

Topics: CRISPR

CRISPR 101: Ribonucleoprotein (RNP) delivery

Posted by Andrew Hempstead on Sep 6, 2018 8:02:59 AM

CRISPR has greatly enhanced the ability of scientists to make genomic alterations, bringing about a revolution in genome engineering, with new techniques rapidly being developed. Performing a CRISPR experiment requires delivery of, at minimum, two components: the Cas9 protein and a guide RNA (gRNA) targeting your genomic site of interest. This is commonly performed by transfecting cells with a plasmid, such as PX459, which encodes Cas9 and contains a site for inserting a custom gRNA.  While this methodology has proven to be incredibly valuable to scientists, there are some potential complications that must be considered when using this method:

  1.     Cells must be amenable to transfection or viral transduction
  2.     Appropriate promoters must be chosen for both Cas9 and gRNA expression  
  3.     Plasmid DNA may be incorporated into the genome
  4.     Off-target effects can occur due to prolonged Cas9 expression
  5.     The requirement for Cas9 transcription and translation delays editing
Read More >

Topics: CRISPR 101, CRISPR, Genome Engineering

Finding nucleic acids with SHERLOCK and DETECTR

Posted by Alyssa Cecchetelli on Aug 30, 2018 8:28:06 AM

Sensitive and specific nucleic acid detection is crucial for clinical diagnostics, genotyping, and biotechnological advancements. Current 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. Recently scientists have utilized CRISPR-Cas9 protein variants, Cas13, and Cas12a, to develop simple, portable, and inexpensive platforms to reliably detect nucleic acids at the atomolar level.

Read More >

Topics: CRISPR, Genome Engineering, Plasmid Technology

Hassle-free 96-well format epitope tagging using Cas9 ribonucleoprotein

Posted by Guest Blogger on Jun 28, 2018 11:01:10 AM

This post was contributed by guest blogger Pooran Dewari, a postdoc in Steve Pollard’s lab at the MRC Centre for Regenerative Medicine (CRM), Edinburgh.

Most commercial antibodies do not work in pull-down assays: Epitope tagging provides a solution

Proteins - the workhorses of the cell – never work alone in the cellular milieu. It is, therefore, critical to understand how proteins interact with one another (or with DNA) to perform diverse biochemical tasks in the cell. One of the most popular approaches to study protein interactions is the pull-down assay, wherein a protein of interest can be captured along with its associated partners. Common pull-down assays include immunoprecipitation mass spectrometry (IP/MS) and chromatin immunoprecipitation (ChIP). In IP/MS, a target protein is first immunoprecipitated - along with its associated protein complexes - from the cell-lysate using antibodies against the target protein. The captured protein complexes are then analysed by mass spectrometry to identify the interacting proteins. Similarly, in ChIP-seq assays, chromatin fragments that are bound by a protein of interest are pulled-down and later coupled to high-throughput sequencing to identify genome-wide binding patterns of the target protein.

Read More >

Topics: CRISPR, Techniques

Click here to subscribe to the Addgene Blog
 
Subscribe

 

Recent Posts