Pspgrna

xCas9(3.7)-ABE(7.10) (#108382), xABEmax (#119813), NG-ABEmax (#124163), SpG-ABE (#140002), NG-ABE8e (#138491), and pSPgRNA (#47108) plasmids were purchased from Addgene (Watertown, MA). To generate the SpG-ABE8e plasmid, NG-ABE8e was digested by NotI and EcoRV and subcloned into the SpG-ABE plasmid backbone by In-Fusion cloning (Takara Bio, Mountain View, CA). SgRNA-A6 and sgRNA-A8 targeting the A→G R560C mutation site on exon 13 of Pde6b gene in the mouse genome were designed by an online webtool (https://benchling.com). All sgRNAs constructed were generated by T4 ligation of annealed oligos into the BbsI digested pSPgRNA plasmid. Both the N-NG-ABE8e and C-NG-ABE8e.sgRNA-A8 vectors used the CBh promoter and were generated by In-Fusion cloning of PCR amplification inserted into restriction enzyme-digested backbones. The coding sequences of split-intein ABE are shown in Tables S4–S6. All constructed plasmids were verified by sequencing.

The CBL^C404Y (c.1211 G>A) variant was inserted at the endogenous locus in the C12 WT hiPSC using Cytidine base editing (CBE) with CBE enzyme BE3-FNLS (46). Briefly, the sgRNA for CBE was designed to target the non-coding strand and introduce the position 6 “C-to-T” conversion, to create the G-to-A conversion on the coding strand. The sgRNA target sequence was cloned into the pSPgRNA (Addgene plasmid # 47108) (47) to make the gene targeting construct. To introduce the CBL C404Y variants, the WT hiPSC (C12) were dissociated using Accutase (Innovative Cell Technologies) and electroporated (1 x10⁶ cells per reaction) with 4 μg sgRNA-construct plasmid and 4 μg CBE enzyme coding vector BE3-FNLS (Addgene plasmid # 112671) (46) using Lonza 4D-Nucleofector and the Nucleofector solution (Lonza V4XP-3034) following our previously reported protocol (48). The cells were then seeded, and 4 days later, the hiPSC were dissociated into single cells by Accutase and re-plated at a low density (4 per well in 96-well plates) to get the single-cell clones. 10 days later, individual colonies were picked, expanded and analyzed by PCR and DNA sequencing to identify the clones carried the desired CBL^C404Y heterozygous variant and the isogenic WT control clones. The sgRNA target, PCR and sequencing primers are listed below.

HEK293T (ATCC Number: CRL-3216) and MCF7 (ATCC Number: HTB-22) cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum; cell lines were maintained at 37°C and 5% CO₂ atmosphere. Cells were transfected with TransIT-X2 Transfection Reagent (Roche) following the manufacturer's instructions. After 30 h in culture, plasmid transfected cells were used for experimentation.
pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid. The oligonucleotides containing the target sequences were hybridized, phosphorylated and cloned into the plasmid using BbsI sites. The target sequences are provided in Supplemental Table S1.