Sf cell line solution

MV411 cells were modified by CRISPR-HDR to express a C-terminal FKBP12^F36V (dTAG) fusion of MYB. A donor DNA construct, including the knock-in cassette and ca. 400 homology arms, was commercially synthesized (Genewiz, Burlington, MA) and cloned into the pAAV-MCS2 plasmid vector obtained from Addgene (Watertown, MA). rAAV packaging was performed at the Boston Children’s Hospital Viral Core. MV411 cells were electroporated with Cas9/sgRNA complexes targeting the HDR insertion using a Lonza SF Cell Line 4D Nucleofector (Lonza V4XC-2032). RNP complexes were formed by mixing 8.5 μg of TrueCut Cas9 Protein v2 (Invitrogen A36499) and 120 pmol sgRNA. 0.3×10⁶ cells were washed with PBS and resuspended in 20 μL of SF Cell Line solution (Lonza). Ten μL of crude rAAV lysate was added to the cells immediately after electroporation^{63 (link)}. After a 5-7 day incubation period the cells were sorted for mScarlet fluorescence. Single clones were then obtained by single-cell dilution microwell plating and screened for bi-allelic donor insertion by PCR. Clones were validated by Western blotting and Sanger sequencing. TF degradation was induced by adding 500 nM of dTAG^v-1 as previously described^{38 (link)} and followed by FACS measurement of mScarlet fluorescence and Western blotting.