Purified Cas9 Protein for Insect Genome Modification with CRISPRs

The interest in CRISPR/Cas9 genome-editing and -modification technologies is quite extensive and there has been a flood of data dealing with optimizing efficiency among other things.

Injecting Insect Embryos

Injecting Insect Embryos

Drosophila melanogaster

Drosophila melanogaster

 

 

 

 

 

 

 

In insect systems there are a number of options by which the components of the CRISPR/Cas9 system can be delivered to germ cells: Cas9 mRNA injections (and gRNA), Cas9 transgene expression in germ cells, transgene expression of gRNAs to name a few.

Lee et al (2014) report on yet another approach – injection of purified Cas9 protein and the necessary RNA components. Lee et al (2014) report on their efforts to mutagenize Drosophila melanogaster using ebony (e) as a target, which has been used in other optimization efforts in D. melanogaster so comparisons are more meaningful.

Lee et al (2014) find that germ-line mutation transmission rates ranged from 2.6% – 20%. In addition they found that the percentage of mutants arising from mutant-producing G1 offspring ranged from 1.7% – 25%.

Cas9 + gRNA

Cas9 + gRNA

So while this approach certainly can be effective and Cas9 protein is available commercially, the performance, at least in D. melanogaster, was not as good as when Cas9 mRNA was used as a source of Cas9 or when Cas9 was expressed in the germ-line from an integrated transgene. Direct Cas9 protein injection did outperform plasmid DNA containing a Cas9 transcription unit under the regulatory control of a strong promoter and transiently expressed following injection into embryos.

It is worth noting that the authors used T7 endonuclease I (T7E1) to look for mutations. T7E1 is a heteroduplex-sensitive endonuclease and works very much like the Surveyor system.

 

Lee J-S, Kwak S-J, Kim J, Kim A-K, Noh HM, Kim J-S, Yu K (2014) RNA-Guided Genome Editing in Drosophila with the Purified Cas9 Protein. G3: Genes|Genomes|Genetics 4: 1291-1295
doi:10.1534/g3.114.012179

 

 

Recent IGTRCN posts dealing with this topic:

Expanding the CRISPR/Cas Tool Box for Drosophila Engineering

 sgRNA Design & Target Specificity

Vasa Cas9 Experession Yields Good Mutation Rates in Drosophila

Germ-line Expression of Cas9 Improves Knock-in Rates

CRISPR/Cas9 in Drosophila – Other Insects not far behind

 

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1 Comment

  1. Great results. Do you think a concentration of 4000 ng/ul for the Cas9 is optimal? Would a higher concentration be even more effective? Our company is currently only selling 4000 ng/ul but we’re
    considering higher concentration if they would be useful.
    oceaniabio.com

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