A Simple and Scalable Strategy for Analysis of Endogenous Protein Dynamics

The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically, it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose, a large set of proteins representing diverse structures and functions, some of which are known or potential drug targets, were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86% of the targets, as determined by luminescence-based plate assays, blotting, and imaging. In order to determine whether endogenously tagged proteins yield more representative models, cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases, only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches.


Supplementary
Integration of HiBiT was directed to the C-terminus using 2 different guides (Guide 1 and Guide2) and internally at Thr300 using one guide (Guide 3). Data are represented as mean luminescence values normalized to cell number (n = 3) as measured in lytic format with variability expressed as SD. SBR for each condition is represented in parentheses. Dashed line indicates luminescence produced by unedited U-2 OS cells. Values to the left of each blot represent the migration of the corresponding size standard. Blots were cropped for each target and analyzed using Fiji with linear contrast adjustments to view each protein band. Uncropped blots are available in Fig. S6a-j.

C-terminal C-terminal
Supplementary Figure S6. Full-size unedited HiBiT blots shown in in Fig. 3 and Fig. S5a-j. Left panels show colorimetric images of membranes with visible molecular weight ladder. Right panels show luminescence images of the same membranes. Luminescence images were collected using BioRad Image Lab 5.2.1 software with exposures of 5 min (e, g), 10 min (b, f), 15 min (a, c, d), and 60 min (h, i, j).
Red boxes indicate where blots were cropped to generate figures.
Supplementary Figure S7. Bioluminescence imaging of HiBiT tagged targets in HeLa cells. Top panels show pseudo-colored bioluminescence images of HiBiT fusions in edited HeLa pools. Bottom panels are an overlay of the bioluminescence image with the corresponding brightfield image. Objectives, EM, and exposure times for each image are found in Table S8. Scale bar = 20 µm.
Supplementary Figure S8. Signal stability testing of Jurkat HiBiT pools for 69 targets over a period of 60 days (15 passages). HiBiT Luminescence was measured in lytic format at each passage. Data are represented as percent of luminescence signal relative to day 11 (100%). Green represents targets that maintained at least 33% of the original signal at passage 10, and gray represents those with at least 10% of the starting signal. Red represents targets that maintained less than 10% of signal by passage 10.  Figure S9. Indel distribution in edited K-562 pools. Next-generation sequencing was performed on amplicons spanning the cut site for the (a) AKT1, (b) FOS, (c) IRAK4, and (d) MAPK8 targets. Bars indicate the number of reads for each indel size. Blue bar denotes an indel size of 0 bp, while red represents indel size of 39 bp (HiBiT). Indel sizes with read counts above 100 (dashed line) were used to calculate percent of wild type, HiBiT, or indel in the pools (see Supplementary Table S9). Figure S10. Comparison of luminescence between edited pools and clones. Data represent single luminescence read relative to cell number obtained from either pools (white bars) or clones (blue bars) of the indicated target in (a) HeLa, (b) K-562, and (c) Jurkat cells. Dashed line denotes luminescence of unedited cells. Figure S11. HiBiT signal stability in clones. (a) HeLa, (b) K-562, and (c) Jurkat clones were maintained in culture for the indicated time period. At each passage, luminescence was measured in lytic format. Data represent individual luminescence reads normalized to cell number for the specified target. Figure S12. 2D Droplet-digital PCR plots of 11 clones (a-k). Fluorescence amplitude plots show droplets containing the target gene (blue) and droplets containing the HiBiT insertion and target insertion (orange). Negative ddPCR droplets are in gray. Total alleles, HiBiT alleles, and HiBiT frequency are summarized in Supplementary Table S11.