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CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

Posted by Mary Gearing on Sep 28, 2020 8:00:00 AM

Originally published Mar 3, 2016 and last updated Sep 28, 2020 by Will Arnold.

Although CRISPR systems were first discovered in bacteria, most CRISPR-based genome engineering has taken place in other organisms. In many bacteria, unlike other organisms, CRISPR-induced double stranded breaks are lethal because the non-homologous end-joining (NHEJ) repair pathway is not very robust. In many cases, homology-directed repair does not function effectively either, but scientists have devised means of co-opting phage genetic systems to facilitate homologous recombination in bacteria. These quirks change the way CRISPR-mediated genome engineering functions in bacteria, but have no fear - plasmids from Addgene depositors are making it easier than ever to use CRISPR  in E. coli and other bacterial species. Read on to learn about the tools available for bacteria and some of the applications for which they’ve been used.

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Topics: CRISPR, CRISPR Expression Systems and Delivery Methods

Overcoming the AAV Size Limitation for CRISPR Delivery

Posted by Mary Gearing on Sep 16, 2020 9:00:00 AM

Originally published Jul 14, 2015 and last updated Sep 16, 2020 by Beth Kenkel. 

CRISPR genome editing has quickly become a popular system for in vitro and germline genome editing, but in vivo gene editing approaches have been limited by problems with Cas9 delivery. Adeno-associated viral vectors (AAV) are commonly used for in vivo gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of certain tissues. However, packaging Streptococcus pyogenes (SpCas9) and a gRNA together (~4.2 kb) into an AAV vector is challenging due to its packaging capacity of AAV (~4.7 kb). While this approach has been proven feasible, it leaves little room for additional regulatory elements. Feng Zhang's group previously packaged Cas9 and multiple gRNAs into separate AAV vectors, increasing overall packaging capacity but necessitating purification and co-infection of two AAVs.

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Topics: CRISPR, CRISPR Expression Systems and Delivery Methods

Nanoblades: Tiny CRISPR Ninjas for Genome Editing Difficult Cells

Posted by Beth Kenkel on Sep 26, 2019 8:50:00 AM

CRISPR is a simple and versatile tool for genome engineering, but its utility is dependent on its ability to infiltrate cells. Options for CRISPR delivery include plasmid transfection, RNP electroporation, and viral transduction; but these methods aren’t stealthy enough to gain access to some cells and tissues, such as human induced pluripotent stem cells (hiPSCs). Nanoblades, a new CRISPR delivery method developed by the Ricci Lab and the T. Ohlmann Lab, adds a covert tool to the CRISPR tool box. 

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Topics: CRISPR, CRISPR Expression Systems and Delivery Methods

Mobile-CRISPRi: Bringing CRISPRi to Diverse Bacteria

Posted by Beth Kenkel on Apr 4, 2019 8:53:46 AM

The vast majority of bacteria are undomesticated which limits the tools scientists can use to study them. For example, gene knockdown with CRISPR interference (CRISPRi) has been limited to lab-adapted bacteria because it has been challenging to introduce CRISPRi machinery into diverse bacteria species. Existing protocols can transfer CRISPRi into a single bacterial strain, such as a B. subtilis, or a narrow range of bacterial species, such as the human gut bacteria B. thetaiotaomicron, Mycobacterium, Pseudomonas, and E. coli. However, many non-model bacterial species lack genetic tools.

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Topics: CRISPR, CRISPR Expression Systems and Delivery Methods

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, CRISPR 101, CRISPR Expression Systems and Delivery Methods

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