Mammalian cell culture and nucleofection
HCT116 cells (ATCC, CCL-247) were cultured in McCoy’s 5 A medium supplemented with 10% FBS. HEK293T (ATCC, CRL-3216) and U2OS (ATCC, HTB-96) cells (a generous gift from Dr. Andrew Belmont at UIUC) were cultured in Advanced DMEM supplemented with 10% FBS and 1 × GlutaMax (ThermoFisher, 35050061). Primary human Mesenchymal Stem Cells (hMSC) was obtained from ATCC (PSC- 500-012) and cultured in Mesenchymal Stem Cell Basal Medium (PSC-500-030) supplemented with Bone Marrow-Mesenchymal Stem Cell Growth Kit Components (PSc-500-041). All cells were cultured at 37 °C in a 5% CO2 incubator. To form the RNP, nuclease and crRNA (sgRNA) were diluted with PBS to a total volume of 10 µL followed by the incubation at room temperature for 10–20 min. For U2OS and HCT116 cell lines, 1 × 10^6 cells were transfected using SE Cell Line Nucleofector Kit with RNP containing 320 pmol crRNA and 192 pmol AsCas12a Nuclease (IDT, 1081069), AsCas12a Ultra Nuclease (IDT, 10001273), or LbCas12a Nuclease (IDT, 10007923) using DN-100 program and EN-113 program respectively on a Lonza 4D Nucleofector according to manufacturer’s instructions. 1 × 10^6 HEK293T cells were transfected using SF Cell Line Nucleofector Kit with RNP complex containing 320 pmol crRNA and 192 pmol
AsCas12a Nuclease or AsCas12a Ultra Nuclease using DS-150 program. All nucleofections were supplemented with 300 pmol Cas12a Electroporation Enhancer (IDT, 1076301). Cas12a orthologs validation was conducted on HEK293T cells with the same condition of AsCas12a mentioned above, involving FnCas12a, TsCas12a, Mb2Cas12a, Mb3Cas12a, BsCas12a, HkCas12a, PxCas12a, and ErCas12a (generous gifts from Dr. Piyush K. Jain at the University of Florida). For hMSCs, 0.5 × 10^6 cells were transfected using P1 Primary Cell Nucleofector Kit with RNP containing 320 pmol crRNA and 192 pmol AsCas12a Nuclease (IDT, 1081069) using FF-104 on a Lonza
4D-Nucleofector according to manufacturer’s instructions. For Cas9 based genome editing experiments, U2OS cells were transfected using SE Cell Line Nucleofector Kit with RNP containing 320 pmol sgRNA and 192 pmol SpCas9 Nuclease (IDT, 1081059) using DN-100 program on a Lonza 4D-Nucleofector according to manufacturer’s
instructions. All nucleofections were supplemented with 300 pmol Cas9 Electroporation Enhancer (IDT, 1075916).

HDR mediated reporter gene knock-in
IRES-EGFP containing dsDNA donor was amplified from the plasmid (generated in our group, Supplementary Data File 1) using PrimeSTAR Max polymerase (Takara, R045A) with homology arm-containing primers. The 50 bp homology arms are incorporated with primers listed in Supplementary Data File 1. PCR products were initially gel-purified using GeneJET gel extraction kit (Thermofisher, K0691) followed by another round of PCR to scale up the amount of the donor. Lyophilization was used to condense donors to get a concentration around 2 µg/µL. 10 µg of each donor was used in EGFP knock-in nucleofection. 1 × 10^6 HEK293T cells were transfected using SF Cell Line Nucleofector Kitwith Cas12a RNP using DS-150 programon a Lonza 4D-Nucleofector according to the manufacturer’s instruction. All nucleofections were supplemented with 300 pmol Cas12a Electroporation Enhancer, and medium was supplemented with HDR enhancer (IDT, 10007910). 

Multiplex genome editing
Multiple crRNAs (total 320 pmol) were used in RNP formation. 1 × 10^6 U2OS cells were transfected using SE Cell Line Nucleofector Kit with RNP containing 320 pmol crRNAs (or sgRNAs) and 192 pmol AsCas12a (or SpCas9) Nuclease using DN-100 program on a Lonza 4D Nucleofector according to manufacturer’s instructions. 1×10^6
HEK293T cells were transfected using SF Cell Line nucleofector Kit with RNP using DS-150 program. All nucleofections were supplemented with 300 pmol Cas12a (or Cas9) Electroporation Enhancer.