Fluorescence Amplification Method for Forward Genetic Discovery of Factors in Human mRNA Degradation

Abstract

Nonsense-mediated decay (NMD) degrades mRNAs containing a premature termination codon (PTC). PTCs are a frequent cause of human genetic diseases, and the NMD pathway is known to modulate disease severity. Since partial NMD attenuation can potentially enhance nonsense suppression therapies, better definition of human-specific NMD is required. However, the majority of NMD factors were first discovered in model organisms and then subsequently identified by homology in human. Sensitivity and throughput limitations of existing approaches have hindered systematic forward genetic screening for NMD factors in human cells. We developed a method of in vivo amplification of NMD reporter fluorescence (Fireworks) that enables CRISPR-based forward genetic screening for NMD pathway defects in human cells. The Fireworks genetic screen identifies multiple known NMD factors and numerous human candidate genes, providing a platform for discovery of additional key factors in human mRNA degradation.

Figure 1. Fireworks in vivo NMD reporter system

(A) Schematic of the Fireworks approach is shown for the “green” Fireworks cell line. Each Fireworks reporter expresses a single polyprotein consisting of (1) 10 or 5 FPs (RFP or GFP), (2) TEV protease, and (3) PEST-β-globin. The β-globin gene lacks the first intron and either carries or lacks a PTC (PTC39) upstream of its last (second) intron. The resulting polyprotein contains 7 TEV protease cleavage sites: (i) between each copy of fluorescent protein, (ii) between the last fluorescent protein and the protease, (iii) within the protease, and (iv) between the protease and the PEST-β-globin. As a result, in vivo translated FPs are separated from each other, the protease, and the PEST-β-globin through in vivo proteolytic cleavage. The PEST degradation sequence (Rogers et al., 1986) was added to shorten the β-globin protein half-life. (B) HeLa cells with genome-integrated Fireworks reporters produce strong fluorescence signals, which are above the calibration range of a FACS instrument. The fluorescence is stable, uniform, and virtually immune to silencing. Lower-right insert: Pellets of Fireworks cells at ambient lighting. (C) Fluorescence of stable orthogonal Fireworks cell lines observed with an epifluorescence microscope. Each (HeLa) cell line carries two FRT-integrated Fireworks reporters that differ by a single (PTC39) nucleotide: the cell line on the left carries PTC39 in the second exon of the β-globin sequence in the RFP-containing reporter; the cell line on the right carries the identical PTC39 in the GFP-containing reporter. mRNAs expressed by the PTC39-containing reporters are NMD substrates and yield lower fluorescence; mRNAs of the control Fireworks reporters are unaffected, producing the green (left lower panel) and red (right upper panel) signal. The brown color of the cells in the lower right panel results from the leakage of red color into the green channel. (D) Pools of HeLa cells with genome-integrated Fireworks reporters appropriately respond to the introduction of PTC39 by a 7- to 9-fold fluorescence drop (no attempt was made to fine-tune color compensation). See also Figure S1.

Figure 2. Fireworks reporters permit rounds of efficient forward genetic screening for factors required for human NMD

(A) NMD inhibition via expression of UPF1-targeting shRNA results in an 8- fold increase in red fluorescence of the Fireworks “green” cell line (shown in Figure 1A and the left panel of Figure 1C). (B) NMD inhibition via expression of Cas9 and UPF1-targeting sgRNA causes a 4- fold increase in red fluorescence of the Fireworks “green” cell line. (C) Schematic of sgRNA library enrichment using FACS sorting of Fireworks cells. Importantly, sgRNA-specific effects are amplified, whereas cell-specific effects are reset at the beginning of each enrichment round. (D) Enrichment of sgRNAs that affect human NMD is evident after FACS sorting of the GeCKO-Lt-CRISPR sgRNA library transduced into “green” Fireworks cells. An increase in the NMD-defective cell population is seen in the blue gates after sequential rounds of enrichment (round 1: 0.21%, round 2: 0.56%, and round 3: 2.96% of total cells).

Figure 3. FACS analysis of the fluorescence shift produced by individual sgRNAs identified by Fireworks screening of the GeCKO-LtCRISPR sgRNA library for factors affecting human NMD

(A) sgRNAs obtained from the genome-wide screen (Table 1) were individually transduced into the “green” Fireworks cell line (Figure 1A) and the resulting cells were FACS-analyzed for an increase in red fluorescence. Populations of cells with increased red fluorescence (nearly not observable [0.9–1.0%] in the corresponding negative controls) appear in the red gate. Fractions (percent) of cells in the original (black gates) and shifted (red gates) populations are indicated. (B) To exclude possible off-target effects of sgRNAs, two of the genes not previously implicated in NMD, AVEN and RPS8, were targeted by shRNAs. FACS-analyses of the shRNA-transduced “green” Fireworks cell line are shown; populations of cells with increased red fluorescence appear in the red gate (FACS-analysis of the orthogonal “red” Fireworks cell line transduced with shRNA targeting RPS8 is shown in Figure S2B). See also Figure S2.