Pcas9 gfp

Cells were transfected by 4D Nucleofector Amaxa technology (Lonza) using the cell line nucleofector (solution P1, FF-104) with 1 μg of plasmid pCAS9-GFP (44719; Addgene) and 1 μg of each plasmid (MLM3636, 43860; Addgene) encoding for the different gRNAs (2 μg for gRNA^EWS). For Cas9/gRNA RNP complexes, MSC^Pat and hMPCs were transfected directly with the different combination of gRNAs and Cas9-GFP protein (ratio 2:1). gRNA sequences are listed in the Supplementary material.

Genome editing was performed utilizing a CRISPR/Cas9 protocol adapted from previous studies (Cohn et al., 2019 (link); Hinson et al., 2015 (link)). 8×10⁶ iPSCs were electroporated with 20 μg pCas9-GFP (Addgene 44719), 20 μg of the appropriate hU6-driven sgRNA (designed using https://zlab.bio/guide-design-resources), and 20 μg of the HR targeting vector to generate ACTN2-BirA* knock-in iPSCs (Ding et al., 2013 (link)). Electroporated cells were transferred to a Matrigel-coated 100 mm dish containing mTeSR1 and 10 μM Y-27632. The following day, selection was started with 50 μg/mL Hygromycin B (Invitrogen 10687010) to isolate single iPSC clones, which were then manually picked, expanded, and screened via Sanger sequencing. Isogenic knockout of TNNT2 was performed similarly, but in the absence of an HR vector and with GFP-based FACS enrichment in place of antibiotic selection.

The BE3-encoding gene and synthetic polyadenylation sequence from pCMV-BE3, the CAG reporter from pCas9_GFP (Addgene plasmid #44719), and the U6 promoter-driven gRNA cassette from pGuide (Addgene plasmid #64711) with the protospacer sequence 5’-CAGGTTCCATGGGATGCTCT-3’ (for Pcsk9 studies), the protospacer sequence 5’-CATTCAACGTCACAACCACC-3’ (for the Hpd studies), or the protospacer sequence 5’-GGTGCTAGCCTTGCGTTCCG-3’ (control studies: irrelevant protospacer not matching any sequence in the mouse genome) were cloned into pDUAL-Basic expression vector. For R26^mTmG/+ experiments, which used SpCas9 and not the BE3, the mTmG protospacer (5’-ATTATACGAAGTTATATTAA-3’) was cloned into plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (a gift from Feng Zhang; Addgene plasmid # 42230). Vector Biolabs (Malvern, PA) used these constructs to generate recombinant adenovirus type 5 particles. Premade adenovirus type 5 particles containing the GFP transgene or Cre recombinase under a CMV promoter were obtained from Vector Biolabs. Ad viral vectors are referred to as Ad.BE3.Pcsk9, Ad.BE3.Hpd, Ad.BE3.Null, Ad.SpCas9.mTmG, Ad.GFP, and Ad.Cre, and the titers are indicated in Supplementary Table 2.