Programmable RNA Targeting Using CasRx in Flies

Abstract

CRISPR-Cas genome editing technologies have revolutionized the fields of functional genetics and genome engineering, but with the recent discovery and optimization of RNA-targeting Cas ribonucleases, we may soon see a similar revolution in the study of RNA function and transcriptome engineering. However, to date, successful proof of principle for Cas ribonuclease RNA targeting in eukaryotic systems has been limited. Only recently has successful modification of RNA expression by a Cas ribonuclease been demonstrated in animal embryos. This previous work, however, did not evaluate endogenous expression of Cas ribonucleases and only focused on function in early developmental stages. A more comprehensive evaluation of this technology is needed to assess its potential impact. Here we report on our efforts to develop a programmable platform for RNA targeting using a Cas ribonuclease, CasRx, in the model organism Drosophila melanogaster. By genetically encoding CasRx in flies, we demonstrate moderate transcript targeting of known phenotypic genes in addition to unexpected toxicity and lethality. We also report on the off-target effects following on-target transcript cleavage by CasRx. Taken together, our results present the current state and limitations of a genetically encoded programmable RNA-targeting Cas system in Drosophila melanogaster, paving the way for future optimization of the system.

Fig. 1.

CasRx-mediated target transcript reduction in restricted tissue types using the binary Gal4/UAS system. (A) Representative genetic crossing schematic. (B) Inheritance rates of triple transheterozygous flies inheriting three transgenes (UASt-CasRx or UASt-dCasRx, gRNA^array, and Gal4-driver), corresponding to flies highlighted in the red box in panel A. Significant differences in inheritance between CasRx and dCasRx groups were observed in all three gene targets (gRNA^w, P = 0.00595; gRNA^N, P = 0.00402; gRNA^y, P = 0.02205). (C) Phenotypes of the triple transheterozygous flies. The white arrow identifies chitin pigment reduction in the thorax resulting from y targeting. Black and white fly with “X” represents a lethal phenotype with no live adults able to be scored or imaged. CasRx, Cas ribonuclease; gRNA^array, guide RNA array; gRNA^N, guide RNA targeting the Notch gene; gRNA^w, guide RNA targeting the white gene; gRNA^y, guide RNA targeting the yellow gene.

Fig. 2.

Genetic assessment of programmable CasRx-mediated transcript reduction in flies. (A) Representative genetic crossing schematic to generate transheterozygotes. (B) Inheritance and penetrance rates of transheterozygous flies inheriting both Ubiq-CasRx (or Ubiq-dCasRx) and gRNA^array corresponding to the red box in panel A. Phenotype penetrance rate is depicted by blue shading in the box plot. Significant differences in inheritance between CasRx and dCasRx groups were observed in all three groups (P values: gRNA^w = 0.00135; gRNA^N = 0.00006; gRNA^y = 0.00016). (C) Brightfield images of transheterozygous flies with representative phenotypes for each cross. Corresponding genotype for each image is dictated by the combination of constructs on top of the columns and the side of the rows. Arrows point to tissue necrosis in the eye. Black and white fly with “X” represents lethality phenotype where no transheterozygote adults emerged. dCasRx, catalytically inactive negative control CasRx.

Fig. 3.

Robust CasRx-mediated reduction of GFP. (A) Representative bidirectional genetic crossing schematic. (B) Box plot of transheterozygote inheritance rates resulting from bidirectional crosses between Ubiq-CasRx (or Ubiq-dCasRx) and gRNA^GFP-OpIE2-GFP flies. (C) Images of F₁ larvae from paternal crosses demonstrating significant reduction in GFP expression for transheterozygous larvae expressing both Ubiq-CasRx and gRNA^GFP-OpIE2-GFP compared to control transheterozygotes expressing Ubiq-dCasRx and gRNA^GFP-OpIE2-GFP or without expressing a CasRx protein. (Left-right) Ubiq-CasRx/gRNA^GFP transheterozygous larvae, heterozygous gRNA^GFP larvae from Ubiq-CasRx cross, Ubiq-dCasRx/gRNA^GFP transheterozygous larvae, heterozygous gRNA^GFP larvae from Ubiq-dCasRx cross. CyO, ; dsRed, red fluorescent protein; GFP, green fluorescent protein; M, maternal inheritance of CasRx; P, paternal inheritance of CasRx; RFP, red fluorescent protein.

Fig. 4.

Quantification of potential CasRx-mediated on/off target activity. (A) Maximum a posteriori estimates for the logarithmic fold change of transcripts. DESeq2 pipeline was used for estimating shrunken Maximum a posteriori logarithmic fold changes. Wald test with Benjamini-Hochberg correction was used for statistical inference. Grey dots represent transcripts not significantly differentially expressed between Ubiq-CasRx and Ubiq-dCasRx group (P > 0.05). Red dots represent transcripts significantly differentially expressed between CasRx and dCasRx group (P < 0.05). Pink dot identifies the respective CasRx target gene for each analysis (P-value indicated in th/by inset). (B) Transcript expression levels (TPM) of transcripts targeted with CasRx or dCasRx. Student's t-test was used to calculate significance (w: P = 0.07; N: P = 0.04; y: P = 0.006; GFP: P = 0.008). (C) Percentage of transcripts significantly differentially expressed resulting from CasRx cleavage. A pairwise two-sample test for independent proportions with Benjamini-Hochberg correction was used to calculate significance. LFC, logarithmic fold change; MAP, Maximum a posteriori.