V5-PPP2R1A knock-in THP-1 cells
5 x 10^5 THP-1 cells were resuspended in 20 µL nucleofection solution (16.4µL SG nucleofector solution + 3.6µL supplement 1) from the SG Cell Line 4D-NucleofectorTM X kit (Lonza, V4XC-3032). 300 pmol PPP2R1A sgRNA (Synthego, Key Resources Table) was incubated with 100pmol Alt-R S.p. HiFi Cas9 Nuclease V3 (Integrated DNA Technologies, 1081061) at room temperature for 20 minutes prior to the addition of 566ng mCherry-P2A-V5-PPP2R1A donor template DNA (Integrated DNA Technologies, Key Resources Table) on ice (6mL total volume). THP-1 cells in SG buffer were mixed with the sgRNA/Cas9/template complex and transferred to a 16- well nucleocuvette strip. Nucleofection was performed using the FF-100 program of the Amaxa 4D Nucleofector X Unit (Lonza, AAF- 1002X) and cells were incubated at 37°C for 10 minutes post-nucleofection before addition of culture media. Following expansion, mCherry cells were FACS-selected using a BD FACSAriaTM Fusion sorter.

Generation of THP-1 CDK9AS/AS cells

sgRNAs targeting CDK9 (300 pmol; Key Resources Table) were incubated for 20 minutes at room temperature with 100pmol Alt-R SpCas9 nuclease (Integrated DNA Technologies, 1074182) prior to the addition of 100pmol of the CDK9 Phe-103-Ala dsDNA HDR template (Key Resources Table; final volume - 5 mL). The ribonucleoprotein complex was added to 5x10^6 THP-1 cells resuspended in 20 µL Nucleofector solution (16.4 µL SG nucleofector solution + 3.6 µL supplement 1) from the SG Cell Line 4D-NucleofectorTM Kit (Lonza, V4XC-3032). Cells were transferred to a 16-well NucleocuvetteTM strip and electroporated using the Amaxa 4D-Nucleofector TM X Unit (program FF-100, AAF-1002X). Electroporated cells were expanded in THP-1 culture media prior to isolation of single cell clones in 96-well culture plates using the Becton Dickinson Fusion FACS sorter.

Competitive Proliferation and Cell Death Assays

For HeLa, MM1.S, BJ-T and HS5 competition assays, cells stably expressing either the FUCas9Cherry or FUCas9CFP vector were electroporated with Alt-R S.p. HiFi Cas9 Nuclease V3 and INTS6-A targeting sgRNA or sgSCR (Synthego, Key Resources Table) respectively. 100pmol Alt-R S.p. HiFi Cas9 Nuclease V3 and 300pmol synthetic guide RNA were pre-incubated with sterile, nuclease-free water (snRNP reaction; 5 µL volume) at room-temperature for 20 minutes. 0.5 x10^6 cells were resuspended in 16.4 µL electroporation buffer plus 3.6 µL supplement (see below), mixed with the snRNP reaction, transferred to a 16-well NucleocuvetteTM strip and electroporated using the Amaxa 4D-NucleofectorTM X Unit (AAF-1002X). HeLa cells were prepared using SE buffer (Lonza V4XC-1012) and the CN-114 program, MM1.S cells were prepared using SG buffer (Lonza, V4XC-3032) and the FF-100 program, BJ-T and HS5 primary cells were prepared using P3 Primary Cell buffer (Lonza V4XP-3032) and the DS-138 program.