Expanding the CRISPR/Cas Tool Box for Drosophila Engineering

Mr. Kasim George, Graduate Student, Molecular and Cell Biology Program, University of Maryland College Park

Mr. Kasim George, Graduate Student, Molecular and Cell Biology Program, University of Maryland College Park

The ability to introduce mutations in the genome of organisms has allowed us to gather information on numerous biological mechanisms.

Drosophila melanogaster By Mr. Checker via Wikimedia Commons

Drosophila melanogaster
 via Wikimedia Commons

However, there are significant drawbacks in the traditional tools used for mutagenesis – lack of specificity that gives rise to unwanted mutations, and low efficiency, making the process labor intensive…UNTIL NOW?

The type II clustered regular interspersed short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, that uses the RNA guided endonuclease Cas9 to introduce double strand breaks in DNA has emerged as an extraordinarily powerful method for genome modification in various organisms.

Cas9 + gRNA

Cas9 + gRNA

Several groups have reported on successful genome editing of Drosophila melanogaster using the CRISPR/Cas system. However, the rates of mutagenesis reported have varied.

Differences in methods used to introduce or express Cas9 and guide-RNA (gRNA) into appropriate cells are thought to contribute significantly to the observed variance in the efficiency of mutagenesis.

Port et al. (2014) report on a set of systematically evaluated transgenic Cas9- and gRNA-expressing lines used to develop what the researchers call an optimized CRISPR/Cas ‘tool box’ for Drosophila genome engineering.

So what’s in this CRISPR/Cas tool box?

The researchers have equipped it with Cas9-expressing lines with ubiquitous, or germ-line restricted Cas9 activity, and gRNA-expressing lines that differential expression gRNA using different promoters. They show that somatic expression of Cas9 and gRNA from integrated transgenes results in extremely high bi-allelic mutations. So high that the system can be used for revealing loss-of-function phenotypes in somatic cells. In one of their experiments they target the ebony locus and somatic mutagenesis is so extensive that the flies have essentially an ebony phenotype.

Bi-allelic somatic mutation of ebony using different Cas9 expression strategies.  from Figure 1 of Port et al (2014)

Bi-allelic somatic mutagenesis of ebony using different Cas9 expression strategies. from Figure 1 of Port et al (2014)

One of their most interesting finding was that germline expression of Cas9 using the nos promote and gRNA  expression from a transgene resulted in between 25 – 100% of the resulting progeny carrying a mutation. Surprisingly, Cas9 regulated with the vasa promoter, showed a significant amount of somatic activity, as result somewhat different from some recent results of others who were also trying to optimize the CRISPR system in flies.

Finally, not only were knock-out efficiencies high but so too were knock-in efficiencies; both with oligonucleotides and long double stranded donor templates

The tool box appears to have all the ‘bells and whistles’ and their findings have demonstrated high fidelity of CRISPR/Cas in Drosophila. All that is left is to demonstrate is high target specificity which will require genome wide searches for induced insertions or deletions. So far the specificity appears ‘CRISPY’ as no non-specific phenotypes were observed.

The strategies for using the CRISPR system described by Port et al. (2014) could be applied to other insects in which transgenic technologies and germline-specific promoters are available.

Fillip Port, Hui-Min Chen, Tzumin Lee, and Simon L. Bullock
Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila PNAS 2014 ; published ahead of print July 7, 2014, doi:10.1073/pnas.1405500111

Port et al. (2014) is an Open Access publication.


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