On 2015 Apr 22, Fillip Port commented:

I would like to add that inadvertent integration of a bi-cistronic plasmid, such as the one reported here, into the gRNA target site would create an autonomous gene drive. Genomic integration in the absence of homology arms is expected to be a rare event, but has been reported before. I would therefore suggest to take such a possibility into account. Further reading: PMID:25035423; http://biorxiv.org/content/early/2015/04/01/017384; http://www.sciencemag.org/content/early/2015/03/18/science.aaa5945.abstract.

On 2014 Oct 29, Fillip Port commented:

The following comment was initially posted on the technology blog igtrcn.org

As is the case for many other organisms, CRISPR has rapidly become the method of choice for targeted genome modification in insects. Much of the method development has been taking place in the model organism Drosophila melanogaster, where, in little over a year, 17 publications have demonstrated various ways to harness the CRISPR system for fly genome editing.

However, this flurry of papers has not resulted in a consensus about which protocol is best suited to modify the fly genome, not least because each method comes with certain strengths and weaknesses. CRISPR components – i.e. the Cas9 endonuclease and short gRNAs - can be delivered into fly embryos by microinjection of either plasmid DNA, in-vitro transcribed RNA or purified protein. Microinjection is the most rapid way to introduce CRISPR components into flies and is independent of genetic background, but often suffers from a high degree of variability that leads to overall reduced efficiency. In contrast, transgenic CRISPR in which both Cas9 and gRNAs are expressed from previously integrated transgenes results in reproducible gene targeting at very high rates, but generating the transgenes in the first place is a time consuming process that makes this approach rather slow. Since all gene targeting experiments require Cas9, a popular compromise is to inject gRNA into cas9 expressing transgenic embryos. This approach benefits from good success rates due to the reliable source of Cas9, and is as rapid as microinjection of both components once a lab has acquired one of the publically available cas9 lines.

Now Peter Duchek and colleagues (Gokcezade et al. 2014) present a new protocol for microinjection-based CRISPR in Drosophila melanogaster. The method uses microinjection of DNA plasmids, but rather than injecting a mix of two plasmids encoding Cas9 and gRNA as was done previously, they combine both components on a single plasmid. This small change proves an effective one. In their paper Gokcezade et al. present data from targeting five genes and achieve efficiencies of around 10% mutant offspring when monitoring random InDel mutations and around 5% for precise genome modifications by homology directed repair.

Efficiencies of that range start to make it practical to screen for mutations by PCR based assays, a requirement for scarless genome engineering without the use of visible markers. If such high efficiencies can be routinely achieved on more target genes and in different laboratories then the method presented by Duchek and colleagues will present an attractive alternative to gRNA injections into transgenic cas9 embryos. This is particularly good news for researchers working on insects where transgenic cas9 strains are not available or for those who want to modify a specific genetic background of D. melanogaster. However, in another recent paper Frank Schnorrer and colleagues use the bi-cistronic plasmid presented in Gokcezade et al. to knock-in a RFP selection cassette into the Drosophila genome and achieve efficiencies that are substantially lower than the ones reported in Gokcezade et al. (Zhang et al., 2014, G3, PMID:25324299). This might be because of the larger size of the integration cassette, different target genes and gRNAs or differences in the microinjection procedure.

Like with all other CRISPR protocols proposed today, it will be interesting to see how the bi-cistronic cas9/gRNA plasmid fares in the hands of other insect genome engineers. To aid this Gokcezade et al. have made their plasmids available from the non-profit repository Addgene (Plasmid number 59984 and 59985).

On 2014 Oct 29, Fillip Port commented:

The following comment was initially posted on the technology blog igtrcn.org

As is the case for many other organisms, CRISPR has rapidly become the method of choice for targeted genome modification in insects. Much of the method development has been taking place in the model organism Drosophila melanogaster, where, in little over a year, 17 publications have demonstrated various ways to harness the CRISPR system for fly genome editing.

However, this flurry of papers has not resulted in a consensus about which protocol is best suited to modify the fly genome, not least because each method comes with certain strengths and weaknesses. CRISPR components – i.e. the Cas9 endonuclease and short gRNAs - can be delivered into fly embryos by microinjection of either plasmid DNA, in-vitro transcribed RNA or purified protein. Microinjection is the most rapid way to introduce CRISPR components into flies and is independent of genetic background, but often suffers from a high degree of variability that leads to overall reduced efficiency. In contrast, transgenic CRISPR in which both Cas9 and gRNAs are expressed from previously integrated transgenes results in reproducible gene targeting at very high rates, but generating the transgenes in the first place is a time consuming process that makes this approach rather slow. Since all gene targeting experiments require Cas9, a popular compromise is to inject gRNA into cas9 expressing transgenic embryos. This approach benefits from good success rates due to the reliable source of Cas9, and is as rapid as microinjection of both components once a lab has acquired one of the publically available cas9 lines.

Now Peter Duchek and colleagues (Gokcezade et al. 2014) present a new protocol for microinjection-based CRISPR in Drosophila melanogaster. The method uses microinjection of DNA plasmids, but rather than injecting a mix of two plasmids encoding Cas9 and gRNA as was done previously, they combine both components on a single plasmid. This small change proves an effective one. In their paper Gokcezade et al. present data from targeting five genes and achieve efficiencies of around 10% mutant offspring when monitoring random InDel mutations and around 5% for precise genome modifications by homology directed repair.

Efficiencies of that range start to make it practical to screen for mutations by PCR based assays, a requirement for scarless genome engineering without the use of visible markers. If such high efficiencies can be routinely achieved on more target genes and in different laboratories then the method presented by Duchek and colleagues will present an attractive alternative to gRNA injections into transgenic cas9 embryos. This is particularly good news for researchers working on insects where transgenic cas9 strains are not available or for those who want to modify a specific genetic background of D. melanogaster. However, in another recent paper Frank Schnorrer and colleagues use the bi-cistronic plasmid presented in Gokcezade et al. to knock-in a RFP selection cassette into the Drosophila genome and achieve efficiencies that are substantially lower than the ones reported in Gokcezade et al. (Zhang et al., 2014, G3, PMID:25324299). This might be because of the larger size of the integration cassette, different target genes and gRNAs or differences in the microinjection procedure.

Like with all other CRISPR protocols proposed today, it will be interesting to see how the bi-cistronic cas9/gRNA plasmid fares in the hands of other insect genome engineers. To aid this Gokcezade et al. have made their plasmids available from the non-profit repository Addgene (Plasmid number 59984 and 59985).