Bosch et. al. 2019 report in biorxiv a homology-independent genomic site-specific gene knock-in method that can be applied both in vitro and in vivo.
Traditional methods of site-specific knock-in use homologous repair, which requires cloning of long homology arms that flank the insertion cassette. Such knock-in relies on a double strand break in the genomic DNA made by a Cas9 protein guided by gRNA.
The method discussed in the article also uses a Cas9 protein to create double stranded break (DSB), however a DSB is introduced to both the genomic DNA and the donor plasmid. This method first described by Schmid-Burgk et. al. (2016) as CRISPaint (CRISPR-assisted insertion tagging) and shown to be useful in mammalian cells has three components, a plasmid expressing a single sgRNA that targets the genomic region of choice, a second plasmid expressing a sgRNA targeting a specific reading frame of the donor sequence, and a third universal donor plasmid containing the sequence to be inserted. The authors use this method to insert various ‘tags’ into genes (GFP, Gal4 etc) but it could be used to insert any sequence. In this study Cas9 was provide from the expression of an integrated transgene but it could be provided in other ways as well.
One plasmid expresses a sgRNA that targets Cas9 to cleave the genomic DNA target site while another plasmid expresses a sgRNA that targets Cas9 to cleave a donor plasmid with the sequences to be inserted. A unique design in the donor plasmid includes a frame-selector sgRNA target site so the sgRNA can guide linearization of the plasmid in different reading frames which makes insertion into the appropriate translation frame of the target gene in the genomic DNA easy. Once the donor plasmid is linearized, the cassette is inserted in the genomic DNA using non-homologous end joining (NHEJ) instead of homologous repair. This method eliminates labor intensive construction of traditional donor plasmid and provides an effective tool to fluorescently tag endogenous proteins, generate reporters of gene expression, and disrupt gene function.
Bosch et. al. tested the effectiveness of the CRISPaint method in cultured S2R+ cells, and in Drosophila. They designed plasmids to target four genes and tag them with mNeonGreen and a puromycin resistant gene. Selecting with puromycin, they were able to quickly and efficiently derive cultures where most cells carried the insertion.
Interestingly, a portion of the puromycin selected cells had no mNeonGreen expression or the localization of fluorescence was unexpected. Upon further characterization, the authors found that only clones with correct mNeonGreen localization express fusion proteins that match the predicted molecular weights in western blotting. They concluded that only these clones contained an in-frame insertion in the correct target gene. This is a step that would be necessary for future experiments to eliminate clones with off target insertion.
To test the efficiency of CRISPaint method in vivo, the group designed sgRNA and donor plasmids for injection into Drosophila embryos. Since it was not possible to carry out negative lethal selections with puromycin in insects, an eye color marker was used for positive insertion selection.
The aim of the donor plasmid was to knock-in Gal4 in-frame with the protein so the expression can be detected with a UAS transgene reporter. Positive selection marker was detected in 5-21% progeny. Positive lines were crossed with a UAS-GFP line to identify in-frame Gal4 insertion. Several lines were created where the GFP expression matched the known expression patterns for the target genes.
The authors made some surprising findings which included that the knock-in in vivo was not seamless. Indels of variable sizes happened at all events of insertion and these indels did not hinder Gal4 expression.
They also found that while a correct orientation was important, in frame insertion was not necessary for expression. Interestingly, it was discovered that there were constructs that were effective in the cultured S2R+ cells but not in the Drosophila system.
Overall, the authors introduced a method that streamlines creation of transgenic cell and drosophila lines, and this method should also be applicable to other insects. They demonstrated that the CRISPaint method is as efficient as traditional methods but less labor intensive. The data presented in the article show multiple applications of the method where it can be used to study gene localization, expression and function.