CRISPR-Cas9 mediated “Mutagenic Chain Reaction”

Bill Reid, Ph.D. Postdoctoral Researcher, Institute for Bioscience and Biotechnology Research, University of Maryland College Park

Bill Reid, Ph.D. Postdoctoral Researcher, Institute for Bioscience and Biotechnology Research, University of Maryland College Park
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A recent paper in Science by Gantz and Bier (2015) investigated the potential of utilizing the CRISPR-Cas9 system as, essentially, a programmable homing endonuclease to generate homozygous loss-of-function (LOF) mutants.

Advances in genome editing have allowed for the directed generation of specific LOF mutants in insects, however much downstream work is still needed in order to achieve a homozygous line. In the model insect, Drosophila melanogaster, the generation of a homozygous LOF mutant can be achieved through genetic crosses with available multiple balancer lines, while in other insect systems, homozygosity is achieved through brute force crossing and genotyping.

Generalized homing mechanisms for mobile group I introns

Generalized homing mechanisms for mobile group I introns. Other homing endonucleases (hundreds) have similar consequences to genomes harboring them. Rapid gene conversion or as Gantz and Bier might say, a ‘mutagenic chain reaction’

As a proof of concept, Gantz and Bier generated yellow-mutants in D. melanogaster through the use of CRISPR-Cas9 mediated genome editing and included a homologous repair (HR) donor containing a vasa-Cas9 U6:3-yl-sgRNA cassette. Upon incorporation of the donor, the LOF mutants became yMCR mutants, which were LOF yellow mutants that were also capable of expressing the vasa-promoter driven Cas9 nuclease and the U6:3 driven yellow-targeting sgRNA.

The yMCR mutants effectively perpetuated the initial CRISPR-Cas9 mediated genome editing event- serving as both the Cas9/sgRNA source, as well as the HR template, which Gantz and Bier termed the “mutagenic chain reaction”.

This system proved to be highly efficient at generating and perpetuating the generation of LOF mutants, resulting in yellow offspring even in the F1 outcross to wild-type flies.

While the concept of homing endonucleases to drive genetic change in insect populations is not new, the potential versatility of using the CRISPR-Cas9 system represents an accessible, and very powerful, means of doing so.

MCR

A schematic representation of Gantz and Bier’s synthetic homing endonuclease system.

 

As the authors write in their penultimate sentence,

“a dialogue on this [mutagenic chain reaction] topic should become an immediate high priority issue”.

 

Gantz, V. M., Bier, E., 2015  The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations. Science: doi: 10.1126/science.aaa5945.

Bohannon, J., 2015  Biologists devise invasion plan for mutations. Science 347:  1300doi: 10.1126/science.347.6228.1300.

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1 Comment

  1. Mutagenic Chain Reaction – does not look more dangerous that Wolbachia or P-elemnts that spread
    in Drosophila melanogaster population during last 100 years

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