“Reverse genetics is currently booming with the establishment of TALEN- and CRISPR-mediated genome engineering”
Xu Zhang, Wouter Koolhaas, Frank Schnorrer (2014)
Indeed it is booming but that does not mean that reverse genetics using CRISPRs or TALENS is easy.
There have been many publications exploring strategies that yield good efficiencies of both knock-out and knock-ins using CRISPR/Cas9. Zhang et al (2014) describe an approach which, while designed and implemented in Drosophila melanogaster, might be used in some other insect systems that have or almost have all of the requisite bits and pieces.
Using CRISPR/Cas9 to create knock-in mutants is an exciting and powerful option. But homology-directed-repair, which is required for knocking in a sequence is not a highly frequent event. Furthermore, as described nicely by Zhang et al (2014) knocking out genes with no visible phenotype requires considerable effort to screen, using PCR, for individuals that have a mutant genotype. Maintaining insects with cryptic mutations can also be challenging.
With that in mind Xu and colleagues developed a two-step process which involves creating a knock-in mutation with a sequence expressing a common visible genetic marker (DsRed). So, while the frequency of knock-ins was only a few percent (the percent of the number of fertile germlines yielding at least one knock-out event), they were easy to detect, making screening much easier and faster.
In addition to the DsRed marker gene, the sequence used as the target for HDR contained two phC31 att P sites. These sites flanked the marker gene and any other sequences in the original knock-in target.
With this knock-in Zhang et al (2014) could exchange the sequences flanked by the attP sites with any sequence they wanted using Recombination Mediated Cassette Exchange (RMCE). PhiC31 mediated RMCE in Drosophila is pretty efficient and RMCE has been demonstrated in some other insects as well. Certainly there is no reason to suspect that RMCE using phiC31 or any other site-specific recombination system would not work in other insects although efficiencies might vary.
Following RMCE the resulting modified gene will contain DNA sequence ‘scars’ which are the recombined attachment sites but if these scars are strategically located in intronic sequences they can be inconsequential.
Xu and colleagues provide a couple of good examples of how they used this strategy and a fairly comprehensive description of their workflow.
So, these investigators have devised a scheme that instead of relying on one low-frequency, difficult-to-find mutation (ie. cryptic knock-ins), they have spread the work out and managed the ‘frequency problems’. They manage the low-frequency knock-in phase by using a visible marker and then finish the genome modification using the more efficient method of RCME.
This approach is not only clever but possibly applicable now to other insects such as Bombyx, Aedes, Anopheles, certain tephritid fruitflies, and Tribolium whose genetic tool boxes now include CRISPR/Cas9 and possibly RMCE.
Zhang X, Koolhaas W, Schnorrer F (2014) A versatile two-step CRISPR- and RMCE-based strategy for efficient genome engineering in Drosophila. bioRxiv first posted online August 11, 2014
Access the most recent version at doi: http://dx.doi.org/10.1101/007864
Recent Related Posts on Technology Topics.