A recent paper by Xue et al (2014) has described a novel use of the Gal4 system in conjunction with CRISPR/Cas9 to induce tissue specific gene silencing that may prove be an alternative to more established approaches that utilize RNA interference.
The technique has been dubbed ‘CMCM’ or CRISPR/Cas9-mediated conditional mutagenesis. As suggested in their paper, one drawback of RNAi for conditional knockdown is that it is often not 100% efficient in silencing gene expression and also that not all tissues perform RNAi equally efficiently (examples given include less susceptibility of testes and ovaries).
Xue et al (2014) hypothesised that since CRISP/Cas9 system can show very high efficiency of targeted gene mutagenesis, maybe it could be also used to analyse gene knockdown phenotypes by mutation of the target genomic DNA in single generations rather than aiming directly at mRNA.
The Gal4 system, used to regulate conditional tissue specific gene expression, has been a workhorse for Drosophila genetic analysis since its development in the early 1990s. Its extreme utility in the fruitfly has tempted other hardy souls to adopt the Gal4 system to their favoured species, which now encompasses a number of the more tractable insects, including different mosquito species, silkworms, and flour beetles. This new CMCM approach may provide an alternative means for tissue specific gene knockdown in these emerging models.
The CMCM system devised involves 3 sets of crossed transgenic lines. One set expressing UAS-controlled Cas9, a DNA cleaving nuclease, a second set ubiquitously expressing guide RNAs that direct Cas9 for sequence specific cleavage, and a driver line that expresses Gal4 that, in this instance, will direct expression of Cas9 from the UAS cassette in defined tissue/s. The upshot of these crosses should lead to tissue specific mutagenesis of the target gene in progeny carrying all three genetic components.
To examine the relative utility of CMCM versus RNAi and known null mutations, Xue et al developed lines that ubiquitously expressed guide RNAs targeted to 6 different genes, each marked with RFP. Cas9 expression was placed under the control of UAS, marked with GFP. RFP/GFP progeny were then crossed to a number of different red eye marked driver lines, expressing Gal4 in the testes, ovaries, wings or eyes.
To summarise their results, CMCM silencing phenotypes were at least comparable to RNAi based approaches in all six gene targets, and was clearly superior when analysing testes specific phentoypes. The effects were shown to be specific by observing morphological changes only in the expected tissues in Notch mutants using wing and eye specific driver lines. This was also backed up by genomic DNA sequencing of isolated tissues from these mutants.
Phenotype severity could be increased by including multiple gRNA targets in one construct, as well as by keeping flies at 28ºC (known to increase Gal4 activity). The work also demonstrated that alternative ubiquitous promoters had different efficiencies; indicating that some degree of optimisation may be necessary to apply the system to other organisms, as well as suggesting that for ideal comparison, the gRNAs lines should be created by site-specific phiC31-mediated integration.
Overall, the paper suggests that CMCM is a viable approach to gene silencing.
In the Drosophila world, the ready availability of RNAi lines targeting most genes makes it likely that, in the near future, genome wide screens will still be RNAi based. The authors suggest though that CMCM will provide an independent method of validation for putative loss of function candidates. It also provides an alternative silencing method for genes or tissues in which RNAi is inefficient.
On this note, a recent blog (Oct14) featured the use of “CRISPR/Cas9 for RNA Cleavage” and it would be interesting to adopt this novel RNA targeting methodology to the Gal4 system and compare its efficiency with CMCM.
In species beyond the vinegar fly, where Gal4 based genetic modification strategies are being developed, CMCM may provide a suitable alternative to RNAi for those hard to reach places.
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