CRISPR Clover-LMNA HDR Assay Protocol

The CRISPR Clover-LMNA HDR assay was adapted from refs. 17 (link) and 18 (link). U2OS cells were seeded and transfected with siCtrl or PALB2 small interfering RNA (siRNA) (Supplementary Information) for a final concentration of 50 nM, using Lipofectamine RNAiMAX (Invitrogen). Twenty-four hours post-transfection, 1 × 10⁶ cells were retransfected with 1 μg of pCR2.1-CloverLMNAdonor, 1 μg pX330-LMNAgRNA1, 1 μg pcDNA3 empty vector or the indicated siRNA-resistant flag-tagged PALB2 construct (Supplementary Information), 0.1 g of piRFP670-N1 (used as transfection control), and 200 pmol of siRNA using the 4D-Nucleofector X (Lonza).^{19 (link)} After 48 hours, cells were replated on glass coverslips, fixed with 4% paraformaldehyde, and expression of mClover-LMNA (indicative of successful HDR) was assayed by fluorescence microscopy. Data represent the mean percentages (± SEM) of mClover-LMNA–positive cells over the iRFP670-positive population from five independent experiments (total n > 500 iRFP-positive cells/condition).