standard Dominant Maternal Effects Caused by CRISPR/Cas9

Expressing Cas9 and guide RNAs from transgenes integrated into the genome of organisms typically leads to higher rates of mutagenesis of the target gene compared to when those components are provided transiently by microinjection, for example. Lin and Potter describe a phenomenon they refer to as non-Mendelian dominant maternal effects that are caused by transgenic Cas9 and guide RNAs under some circumstances.

maternal-effect

Maternal effects manifest themselves in progeny having phenotypes that are dependent on the genotype of the mother and not their genotype. Here all progeny arising from m/+ females have + phenotype while all progeny arising from m/m female have a mutant phenotype. This image is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license.

Lin and Potter, in an earlier publication, described a gene editing technique in Drosophila that involved the expression of gRNAs from integrated transgene as well as the expression of Cas9 from a transgene and the presence of a HDR donor construct as a transgene. They call their method the Homology Assisted CRISPR Knock-in (HACK) method and it is used to convert existing GAL4 lines of D. melanogaster, including enhancer traps, into functional QF2 expressing lines

In working with that system they made an interesting observation that is the basis of their current manuscript.

They observed mutagenesis of progeny that contained none of the Cas9 components – neither the Cas9 nor the gRNA transgene.

Nurse cells contributing RNA and proteins to the developing oocyte. By Imp-GFP_localisation_in_the_Drosophila_oocyte.png: Kristin L. M. Boylan, Sarah Mische, Mingang Li, Guillermo Marqués, Xavier Morin, William Chia, Thomas S. Haysderivative work: Celefin (talk) – Imp-GFP_localisation_in_the_Drosophila_oocyte.png, from Boylan KLM, Mische S, Li M, Marqués G, Morin X, et al. (2008) Motility Screen Identifies Drosophila IGF-II mRNA-Binding Protein—Zipcode-Binding Protein Acting in Oogenesis and Synaptogenesis. PLoS Genet 4(2): e36. doi:10.1371/journal.pgen.0040036 [1], CC BY 2.5, https://commons.wikimedia.org/w/index.php?curid=7197966

The HACK system of Lin and Potter relies on Cas9 expression from a Act5C:Cas9 transgene and gRNA expression from U6:gRNA.  The promoters in both cases are ubiquitously expressed.

In the current publication Lin and Potter only observed the exceptional pattern of mutagenesis in progeny when Act5C:Cas9 and U6:sgRNA were present in the maternal genome even though they were absent from the genome of progeny carrying mutations in the target gene.  The reciprocal situation in which these transgenes were in the paternal genome did not result in mutations in progeny in the absence of the Cas9 and gRNA transgenes.   This pattern of inheritance was reminiscent of a classic maternal effect.

The ubiquitous expression of both Cas9 and gRNA in females results in oocytes loaded with Cas9 and gRNA even in the absence of chromosomes containing the transgenes responsible for expressing these components.

Drosophila melanogaster

Drosophila melanogaster

The frequency of mutagenesis in the absence of the editing transgenes was 87%.  When the chromosomes containing the editing components were present the frequency of mutagenesis was upwards of 98%.

Lin and Potter found the a continuous supply of gRNA as opposed to Cas9 was more critical in mutagenizing the target.  Apparently gRNAs are less stable than the Cas9 protein.

Lin and Potter’s observations are interesting because if suggests that efforts to use Cas9 based gene drive systems might be most effective if they are present in males.  The presence of Cas9 and gRNA in females are expected to result in mutations (NHEJ) in the target in a significant proportion of progeny that do not inherit the gene drive-containing chromosomes, resulting in drive-resistant alleles of the target locus.

Non-Mendelian Dominant Maternal Effects Caused by CRISPR/Cas9 Transgenic Components in Drosophila melanogaster Chun-Chieh Lin and Christopher J. Potter G3 g3.116.034884; Early Online September 16, 2016, doi:10.1534/g3.116.034884 

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