Journal: bioRxiv
Article Title: PROTAC molecule-mediated SpCas9 protein degradation for precise genome editing
doi: 10.1101/2025.01.07.631496
Figure Lengend Snippet: A .Optimized CASPROTAC 6 degraded dCas9 proteins. 293T-Tet-On 3G cells with stable integration of dCas9-APEX2 were pretreated with doxycycline (500 ng/mL) for 9 h to the simultaneous expression of Cas9-APEX2 protein complex, 15 hrs after removing doxycycline, CASPROTAC 6 was added for 24 hrs in a dose manner. The protein level of dCas9-APEX2 was monitored by Western blotting. B . Quantification of the intensity of the staining from A. Anti-Flag antibody was used to detect the level of dCas9-APEX-Flag proteins. Anti-β-actin was used as the loading control. C . The 293T-nCas9-PE2 bulk cells were pretreated with doxycycline (500 ng/mL) for 24 h, then removing doxycycline, CASPROTAC 6 was added for 24 hrs. The protein level of nCas9-PE2 was monitored by Western blotting. D . Quantification of the intensity of the staining from C. Anti-Cas9 antibody was used to detect the level of dCas9-APEX-Flag proteins. Anti-β-actin was used as the loading control. E . 293T-nCas9-BE3RA bulk cells were pretreated with doxycycline (500 ng/mL) for 24 h. Then, after removing doxycycline, CASPROTAC 6 was added for another 24 hrs. The protein level of nCas9-BE3RA was monitored by Western blotting. F . Quantification of the intensity of the staining from E. Anti-Cas9 antibody was used to detect the level of dCas9-APEX-Flag proteins. Anti-β-actin was used as the loading control. Bars show mean value□±□s.e.m. and significance was calculated using Student’s t-test (n = 2 or 3). *p < 0.05, **p < 0.005, and ***p < 0.0001 (versus the control). G . CASPROTAC 6 reduced CRISPR editing efficiency. FANCF-gRNA and SpCas9 protein were mixed at a 1:1 molar ratio (20 pmol SpCas9+ 20 pmol FANCF-gRNA for 1 million cells) and incubated at room temperature for 20 min to allow the formation of the CRISPR ribonucleoprotein complexes. 293T cells were electroporated with FANCF-RNP. Six hours after electroporation, cells were incubated with CASPROTAC 6 for 24 hrs. CRISPR editing efficiency was measured by amplicon sequencing. H . The reduction of CRISPR/SpCas9-mediated editing based on the amplicon sequencing results. I . Batch output mode comparing CRISPR editing of control and CASPROTAC 6-treated cells. Each column represents the base composition of a single nucleotide at the FANCF site 2 locus. The percentage of each base or indel at each nucleotide is shown as a proportion of each row (n=1). *p < 0.05, **p < 0.005, and ***p < 0.0001 (versus the control).
Article Snippet: For 293T-nCas9-PE2 bulk cells, the wild-type 293T cell line was grown in 6 well plates (50% confluency) and co-transfected with 1.0 μg Xlone-PE2 (Addgene 136463) plasmid and 1.0 μg PiggyBac Transposase vector 6 µg PEI (Polyscience, 23966) in 50 µl of serum-free medium and selected with 3□μg ml -1 Blasticidin (Biomol) for 2-3 days.
Techniques: Expressing, Western Blot, Staining, Control, CRISPR, Incubation, Electroporation, Amplification, Sequencing
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