Extended Data Fig. 10: Application of RfxCas13d/gRNA to interfere RNA expression during mouse preimplantation development.
From: Screening for functional circular RNAs using the CRISPR–Cas13 system
a, KD of Brg1 and Kras by RfxCas13d/gRNA in zygotes. Expression of mRNAs was detected by qRT-PCR and normalized to Actb. b, Representative images of reduced blastocyst formation 96 h after microinjection of RfxCas13d mRNA and the gRNA targeting Brg1 mRNA into mouse zygotes. An example of failed blastocyst formation is shown by red arrows and enlarged view. c, Representative images of embryogenesis after microinjection of RfxCas13d mRNA and the gRNA targeting Kras mRNA into mouse zygotes. No aberrant mouse preimplantation development was observed. d, Effect of Brg1 (b) and Kras (c) KD on blastocyst formation 96 h after microinjection of RfxCas13d mRNA and BSJ-gRNAs into mouse zygotes. e, Diagrams of mouse circMan1a2 and human circMAN1A2 are shown as magenta cylinders. f, KD of circMan1a2 in zygotes led to reduced blastocyst formation 72 h after microinjection of RfxCas13d mRNA and BSJ-gRNAs into mouse zygotes. Representative images of at 2-cell, 4-cell, morula and blastocyst stages are shown; an example under each condition is highlighted by red line and enlarged view. g, KD of circDcbld2 by RfxCas13d/BSJ-gRNA in zygotes. Expression of circDcbld2 was detected by qRT-PCR and normalized to Actb. h, Images of normal embryonic morphologies at 2-cell, 4-cell, morula and blastocyst stages under circDcbld2 KD by RfxCas13d/BSJ-gRNA in zygotes. i, Statistics of circDcbld2 KD effectd on morula and blastocyst formation after microinjection of RfxCas13d mRNA and BSJ-gRNAs into mouse zygotes. j,k, Implantation of RfxCas13d-gRNA (circMan1a2 KD)-injected zygotes to pseudo-pregnant female mice led to retarded mouse postimplantation development in E7.5. Images of E7.5 embryos shown in (j); statistics of retarded embryos shown in (k). (a,d,g,h,i,j,k) Means ± s.d. are from three independent experiments. (b,c,f,h,j) Results are representative of three independent experiments. (a,d,g,k) *: P < 0.05; **: P < 0.01; ***: P < 0.001; ns, not significant, two-tailed student’s t-test.
