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CRISPR-Cas14: a family of small DNA-targeting enzymes enabling high-fidelity SNP genotyping

Posted by Benoit Giquel on Nov 29, 2018 8:53:18 AM

Before being adapted by scientists to edit the genome of virtually any organisms on this planet, CRISPR-Cas systems were merely adaptive immune systems that provide bacteria protection against infectious agents. Several enzymes behind this immunity have already been discovered and studied but it is only the tip of the iceberg as it has been predicted that many others are still unknown.

In this quest to discover new and maybe more efficient Cas systems, Jennifer Doudna’s lab analyzed metagenomic datasets to try to determine whether simpler and maybe smaller Cas systems could exist in nature.

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Topics: CRISPR

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.

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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.

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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
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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.

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Topics: CRISPR, Genome Engineering, Plasmid Technology

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