CRISPR/Cas9 Targets Chicken Embryonic Somatic Cells In Vitro and In Vivo and generates Phenotypic Abnormalities

Abstract

Chickens are an invaluable model for studying human diseases, physiology and especially development, but have lagged in genetic applications. With the advent of Programmable Engineered Nucleases, genetic manipulation has become efficient, specific and rapid. Here, we show that the CRISPR/Cas9 system can precisely edit the chicken genome. We generated HIRA, TYRP1, DICER, MBD3, EZH2, and 6 other gene knockouts in two chicken cell lines using the CRISPR/Cas9 system, with no off-target effects detected. We also showed that very large deletions (>75 kb) could be achieved. We also achieved targeted modification by homology-directed repair (HDR), producing MEN2A and MEN2B mutations of the RET gene. We also targeted DGCR8 in neural cells of the chicken embryo by in vivo electroporation. After FACS isolation of transfected cells, we observed appropriate sequence changes in DGCR8. Wholemount and frozen section antibody labelling showed reduction of DGCR8 levels in transfected cells. In addition, there was reduced expression levels of DGCR8-associated genes DROSHA, YPEL1 and NGN2. We also observed morphological differences in neural tissue and cardiac-related tissues of transfected embryos. These findings demonstrate that precisely targeted genetic manipulation of the genome using the CRISPR/Cas9 system can be extended to the highly adaptable in vivo chicken embryo model.

Figure 1. In vitro analysis of NHEJ and HDR genome modification (arrows) mediated by sgRNA-Cas9 system in chicken cell lines.

(a) Frequency (%) of NHEJ mutation mediated by KIAA1279, Cdkn1b and Mbd3-targeting sgRNA-Cas9 system in chicken DF-1 cells by PCR and T7E1 assay. 1kM- 1 kbp DNA ladder, M- 100 bp DNA ladder. (b) Frequency (%) of NHEJ mutation mediated by KIAA1279 and Cdkn1b-targeting sgRNA-CRISPR/Cas9 system in chicken lymphoma B DT40 cells by PCR and T7E1 assay. (c) Representative gel from DF-1 cells transfected with the RET-targeting sgRNA-Cas9 and the ssODN showing efficient integration of the HDR-based BamHI and EcoRV sequence. The frequency of HDR is represented in percentages. 1-No sgRNA, 2- MEN2B sgRNA #1 plus ssODN, 3- MEN2B sgRNA #1 and #2 plus ssODN and 4- MEN2A/HSCR sgRNA 1 plus ssODN. (d) Representative gel for single cell clones derived from DF-1 cells transfected with the RET-targeting sgRNA-Cas9 and the ssODN for the MEN2B and MEN2A/HSCR HDR modifications respectively. The table shows the ratio of the monoallelic and biallelic HDR-based mutations detected with single cell clones and the overall efficiency in percentage: N = 19 for MEN2B and N = 12 for MEN2A/HSCR.

Figure 2. Targeted deletion of large genomic fragments and off-target effect analysis in chicken cells in vitro.

(a) Representative gels showing the large genomic deletions within the STMN2 locus (>24 kbp) in chicken DF-1 and DT40 lymphoma B cells, and within the RET (>8 kbp) and HIRA-DGCR8 locus (>70 kbp). (b) Frequency of off-target effects mediated by RET-MEN2B and MEN2A/HSCR, CDKN1B, KIAA1279 and STMN2-targeting sgRNA-CRISPR/Cas9 system system in chicken DF-1 cells and CDKN1B and KIAA1279 -targeting sgRNA-CRISPR/Cas9 system in DT40 cells by PCR and T7E1 assay. ND-not detected, 1 kM- 1 kbp DNA ladder.

Figure 3. Protein expression 2 days after transfection with DGCR8 CRISPR/Cas9 construct.

(a) Immunofluorescence confocal images of single and merged channels of the indicated markers from whole mount staining of DGCR8 mutant embryos, indicating reduced to no DGCR8 expression in transfected (mCherry+) cells (shown by yellow arrows). (b) Histogram of pixel counts on control embryos and DGCR8 mutants embryos relative to DAPI. A total of 540 cells and 542 (> = 100 cells/embryo) were counted from 5 control and 6 electroporated embryos respectively. The low fluorescence in the mCherry waveband in controls is tissue autofluorescence. Scale bar: 5 μm. Error bars, mean ± s.e.m. *P < 0.05, ***P < 0.001.

Figure 4. Somatic targeted genetic modification by CRISPR/Cas9 system in chickens 4 days after in vivo electroporation.

(a) Frequency (%) of NHEJ mutation mediated by DGCR8-targeting sgRNA-CRISPR/Cas9 system in DF-1 cells by PCR. Red arrows indicate the NHEJ mutation created by the CRISPR/Cas9 system. M- 100 bp DNA ladder. Representative images of sham treated (con), electroporated untransfected embryos (electro.), Tol2 GFP transfected embryos (GFP transfect) with normal head development and DGCR8 CRISPR/Cas9 transfected embryos (D1 and D2) showing midbrain (open arrow) and eye (closed arrow) abnormalities. Graph shows the difference in the midbrain dimensions of DGCR8 mutant embryos compared to control embryos- N = 14. (b) Representative image of the hearts of unelectroporated embryos, electroporated with no construct embryos, Tol2 GFP and empty Cas9 transfected embryos, STMN2 transfected embryos (negative control) showing normal heart development, and DGCR8 transfected embryos showing misshapen and reduced hearts. (c) qPCR analysis of cells isolated by FACS from DGCR8-targeted embryos demonstrating the reduced mRNA levels of DGCR8 in mCherry+ brain cells (M) relative to negatively sorted cells (N). Normalisation was done with ACTB and RPL32. ND-not detected. Error bars, mean ± s.e.m. ***P < 0.001.