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Figures
- Fig. 1. TALEN- and CRISPR/Cas9-mediated precise genomic deletion and nucleotides replacement.
(A,E) TALEN-mediated miR-281 deletion and molecular identification. (A) The pair of scissors indicates where the TALENs cut at the miR-281 locus. Dashed red line indicates the deleted genomic region (0.32 kb). (E) The genomic DNAs of two homozygous lines, 17-12 and 200-11, were used as PCR templates. Arrows in opposite directions indicate where the primers are located. The appearance of a 0.31 kb PCR product indicates successful deletion. (B,F) TALEN-mediated SmaI replacement at the chameau locus and molecular identification of positive events. (B) The pair of scissors indicates where the TALENs cut at the chameau locus. (F) Genomic DNAs of two heterozygous F1 lines, 24-1 and 24-6, were used to show PCR and positive SmaI digestion results. Arrows in opposite directions indicate where the primers used for PCR are located (B). PCR products were digested by SmaI; the two cleaved fragments, 0.29 kb and 0.2 kb, represent successful nucleotide replacements. (C,G) CRISPR/Cas9-mediated loxP replacement at the CG4221 locus and molecular characterization. (C) The scissors indicate where the CRISPR/Cas9 cleaves at the CG4221 locus. The empty pentagon box represents the loxP site. (G) Genomic DNAs of two heterozygous F1 lines, 23-1 and 23-7, were used for PCR examination. The primers are indicated by the two opposite arrows in panel C. The appearance of a 0.17 kb band indicates successful loxP replacement. (D,H) CRISPR/Cas9-mediated HindIII replacement at CG5961 and molecular characterization. (D) The scissors indicate where the CRISPR/Cas9 cleaves at the CG5961 locus. (H) Genomic DNAs of two homozygous F1 lines, 1-3 and 1-5, were used for PCR and following HindIII enzyme digestions. The primers for PCR are indicated by the two opposite arrows in panel D. The appearance of two cleaved fragments, 0.26 kb and 0.20 kb, indicates successful HindIII replacement. M: DNA marker; NC: negative control, the corresponding PCR products were amplified from Lig4169 genomic DNA with particular primers in each case. PC: positive control, the corresponding PCR products were amplified from the donor plasmid DNAs with particular primers in each case; k: kilo-base pair; HA-L and HA-R: left homologous arm and right homologous arm; HR: homologous recombination; donor plasmid: circular donor plasmid containing HA-L and HA-R and additional elements if any, the pBSK vector backbone is omitted here; all the dashed black lines indicate the homologous regions in the fly genome and on the donor plasmid; the empty boxes in panel A indicate the genomic regions of miR-281; all the filled boxes with numbers indicate the sequential coding sequences of the corresponding genes in each case, not necessarily representing the full coding sequences.
- Fig. 2. Comparison of TALEN- and CRISPR/Cas9-mediated HDR at the yellow locus.
(A) CRISPR/Cas9- or TALEN-mediated yellow deletion. The CRISPR/Cas9 binding sequence is 5′-GGGTTTTGGACACTGGAACCG-3′ (underlined in panel A); PAM sequence is marked in red. One pair of TALEN binding sites is marked by boxes. The pairs of scissors indicate the Cas9 or TALEN cutting site at the yellow locus. The dashed red lines represent the deleted yellow genomic sequence (0.18 kb). (B) Molecular identification of the yellow deletions. The genomic DNAs of two heterozygous F1 lines, 46-3 (CRISPR/Cas9-mediated mutagenesis) and 8-1 (TALEN-mediated mutagenesis) were used as examples. The pair of primers used for PCR is shown in panel A (opposing arrows). The appearance of a 0.46 kb PCR band indicates successful deletion. (C) Frequencies of CRISPR/Cas9- and TALEN-mediated HDR at the yellow locus. The deletion-yielding events in both F0 and F1 are scored based on the appearance of the 0.64 kb short PCR product shown in panel B. The legends for the rest of the elements/labels are the same as in Fig. 1.
- Fig. 3. In vivo tagging at the chameau and CG4221 loci.
(A,C) chameau C-terminal eGFP tagging. (A) The cartoon scissors represent the CRISPR/Cas9 and indicate where it cuts at the chameau locus. The empty pentagon box indicates loxP site. The filled green box marked with eGFP indicates where the eGFP fragment is fused. The empty box marked by STOP indicates the stop codon of the chameau gene. (C) The genomic DNAs of two heterozygous F1 lines, 15-13 and 15-24 were used for PCR templates. The pair of primers used for PCR is shown in panel A as the two opposing arrows. The appearance of 0.72 kb PCR products indicates successful eGFP tagging (precise insertions). (B,D) CG4221 C-terminal Myc tagging. (B) The cartoon scissors indicate the Cas9 nuclease and its cutting site at the CG4221 locus. The empty triangle indicates the FRT site. The empty box marked with Myc indicates the Myc sequence. The empty box marked by STOP represents the stop codon of the gene CG4221. (D) The genomic DNAs of two heterozygous F1 lines, 55-1 and 55-2 were used as templates for PCR detection. The pair of primers used for PCR is shown as the two opposing arrows in panel B. The positive 0.2 kb PCR band indicates successful Myc tagging (precise insertions). (E–G) Immunostaining detection of the expression of Chameau-eGFP. (E) 3rd instar larval wing discs of line 15-13 were stained with the anti-eGFP antibody. (F) Knockdown of chameau by vg>chameauIR led to loss of GFP signals in the vg-Gal4 expression regions. The genotype in panel F is w; chameau-eGFP/vg-Gal4; chameauIR/+. The wing disc boundary is marked by a dotted white line in panel F. (G) Expression pattern of vg-Gal4 in 3rd instar larval wing discs. vg>GFP: w; vg-Gal4/UAS-eGFP. GFP: green fluorescent protein; eGFP: enhanced green fluorescent protein. (H) Detection of the expression of CG4221-Myc fusion protein by Western blot. Embryos of w1118 and CG4221-Myc line 55-1 were collected for Western blot at 0–3 hours. Anti-Myc antibodies were used to detect CG4221-Myc and tubulin was used as a loading control. See Fig. 1 legend for the common elements or labels that are not explained here.
- Fig. 4. The easy-to-screen white platform for the yellow mutagenesis.
(A) Schematic representation of the generation of HDR-mediated yellow mutagenesis using the easy-to-screen white platform. The cartoon scissors indicate the TALENs and their cutting site at the yellow locus. The pair of TALEN binding sites is marked by the boxes. The empty triangles are two FRT sites located in the 5′ regulatory region and the intron of the white gene, and are oriented in the same direction. The two red-filled boxes indicate two genomic parts of the white gene separated by the second FRT site. Arrows with the names of LF, LR, RF, RR indicate the primers used for PCRs to get the L (left) and R (right) fragments as indicated in panel B. (B) Molecular identification of the white knock-in at the yellow locus, resulting in a simultaneous mutation in yellow. The genomic DNA of heterozygous F1 line, 61-1, was used for showing the positive PCR results. LF and LR were used as primers to get the L fragment. RF and RR were used as primers to get the R fragment. (C–E) Removal of the white+ marker carried in the yellow mutants. (C) An eye of Lig4169 flies. (D) An eye of the yellow mutant line, 61-1, which carried the white knock-in. (E) A mosaic white eye phenotype induced by heat-shock on the offspring of line 61-1 that was crossed with hs-Flp. See Fig. 1 legend for the common elements or labels not explained here.
- Fig. 5. Removal of “ends-in” recombination resultant from CG4221 mutagenesis.
(A) “Ends-in” and “ends-out” homologous recombination (HR) were generated via the HDR pathway in the process of CG4221 mutagenesis, leading to either two copies of the CG4221 mutations or one copy of the CG4221 mutation in the fly genome, respectively (see also supplementary material Fig. S11). The Cre recombinase was introduced into the “ends-in” line to remove the duplicated copy of CG4221 by flipping out the DNAs between the two loxP sites, converting the “ends-in” into the “ends-out” events. The short arrows with names indicate the primers used in panels B,D. (B) PCR assays to distinguish the “ends-in” and “ends-out” HR events occurred during CG4221 mutagenesis. The genomic DNAs of heterozygous F1 lines 23-1, 32-5 and 56-7 were used for PCR assays. T7f and e1r detect a corresponding 0.36 kb “ends-in” band, while nothing for the “ends-out”. (C) “Ends-in” and “ends-out” HR ratio of CG4221 mutagenesis, based on the PCR analyses as described in panel B. (D) Molecular confirmation of the removal of the “ends-in” products. Line 32-5 was selected as an example to show successful removal of additional sequences between the two loxP sites by Cre recombinase, as indicated by the loss of the band amplified with T7f and er. Primer pairs used for PCRs are indicated on the right of the gel. Rp49 genomic primers were used for genomic DNA quality control. See the legends to Fig. 1 for the common elements or labels that are not explained here.
Tables
- Table 1. Summary of HDR frequencies mediated by TALEN or CRISPR/Cas9 at different loci
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