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Choose a strategy

posted on 22 October 2013 by Fillip Port and Simon Bullock

modified on 4 April 2014 and 13 April 2015 by Fillip

What is the best strategy for CRISPR/Cas-mediated genome editing in Drosophila?

There is no general answer to this question as each strategy has certain advantages and disadvantages. For successful genome engineering you have to supply Cas9 and gRNA(s) to the cells of interest (most often germ cells). If you intend to make a precise change by harnessing homologous repair you also need to supply a donor construct. In principle these components can be delivered via injection of plasmid DNA or in vitro transcribed RNA into embryos or by combining transgenes stably integrated into the genome. Which method to choose will depend on the nature of your experiment.

Supplying all components by injection

This is the most flexible approach, but we and others have found that it is also the one that has the lowest efficiency. You can inject either plasmids or in-vitro transcribed RNA for Cas9 and gRNA(s). The disadvantage of RNA is that it requires extra work and reagents and is less stable if send to injection companies. Furthermore, we think that injection of optimised plasmids (such as U6:3-gRNAs) can result in similar gene targeting efficiency as RNA injections. This is why we no longer use RNA in our experiments. Peter Duchek and colleagues have recently described a bi-cistronic plasmid containing cas9 and gRNA and which reportedly results in improved targeting efficiencies (Gokcezade et al. 2014). However, accidental integration of such a plasmid would create an autonomous gene drive and present a biosafety risk and we therefore recommend not using such plasmids until the risk has been systematically assessed.

One advantage of the injection approach is that you can inject into any fly line you want, which can be a benefit if you aim to generate mutations in complex genetic backgrounds (e.g. stocks with FRT chromosomes, additional mutations or transgenes encoding fluorescent proteins). However, such chromosomes can also be crossed to transgenic cas9 lines, followed by gRNA injection into heterozygous embryos.

Injecting gRNA(s) into cas9 transgenic flies

With this approach you introduce gRNA(s) by injection of DNA into embryos that have a transgenic source of Cas9. We and others have found that transgenic Cas9 increases the overall efficiency and robustness of CRISPR/Cas experiments. Different transgenic cas9 strains are available, which differ in expression pattern and activity. Transgenes are available on different chromosomes for additional flexibility. Which line is best suited for your experiment depends on your target. For example, targeting of essential genes benefit from germline specific expression of cas9, whereas for non-essential genes you might want the most active line. Once you have modified your target, you will need to remove the Cas9 transgene. Usually you will have to cross your mutation several times to generate a stock, so this rarely presents any extra work. 

Fully transgenic CRISPR/Cas

At first this approach seems less desirable, as one has to generate a transgenic fly line for every gRNA of interest, a process that usually takes 1-2 months. These gRNA transgenics are then crossed to a transgenic cas9 line. However, fully transgenic CRISPR/Cas has a number of unique properties that make it worth considering this approach. The main advantage is that this protocol gives the highest efficiency. This is mainly due to the fact that gene targeting in cas9 gRNA expressing flies is highly reproducible. Whereas injection of CRISPR/Cas components usually results in a significant fraction of flies that do not transmit any mutations (often around 50%), essentially all transgenic CRISPR/Cas flies pass on mutant alleles to their progeny. Furthermore, with the optimal cas9 source mutagenesis approaches 100% (both alleles in all cells) with the majority of gRNAs. This can drastically cut down the time you have to spend on screening for flies which contain the desired mutation. The high efficiency of transgenic CRISPR/Cas can be problematic when targeting essential genes, but usage of germline restricted cas9 lines and lower efficiency U6:2-gRNA constructs can often avoid lethality.

Another point that is worth considering is that gRNA transgenes can be used for applications beyond the generation of mutant fly lines. For example, crossing gRNA transgenes to our act-cas9 lines directly reveals the null-mutant phenotype in the majority of cases. We also have shown that transgenic CRISPR/Cas generates genetic mosaics and you can use your gRNA transgenes to perform a mosaic analysis of your gene of interest. Furthermore, in combination with the Gal4/UAS system transgenic CRISPR/Cas can be used for tissue specific mutagenesis. These are examples how gRNA transgenes can be useful to study a gene of interest in more detail. 

Donor constructs for homology directed repair

Delivery of donor constructs by injection is the most convenient approach. Kelly Beumer and Dana Carroll have extensively tested which donor constructs support homology directed repair of ZFN and TALEN induced double strand breaks and these findings are very likely to be applicable for the CRISPR/Cas system (paper).

The circumstances of your experiment should determine what strategy you follow. Whereas injection based approaches are good for speed and flexibility, the use of transgenics increases efficiency and consistency, which in turn can cut down the time and effort you spend screening for your mutation.

At the moment we feel that for most experiments which aim to produce a few genome engineered fly lines the introduction of gRNAs by microinjection of DNA into cas9 transgenic embryos presents the best compromise of speed and efficiency. As an alternative approach, CRISPR with independent transgenes has the advantage to be able to reveal mutant phenotypes without the need to establish homozygous lines. The superior efficiency of this system is also advantageous when generating many mutant lines in parallel.



Acknowledgements: We would like to thank Kelly Beumer and Dana Carroll for insightful discussions.