REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
TAF1	Cell Signaling Technology	Cat#12781S; RRID:AB_2798025
TAF5	ThermoFisher Scientific	Cat#MA3–076; RRID:AB_2633321
TAF6	ThermoFisher Scientific	Cat#A301–276A-M; RRID:AB_2779789
TAF10	Sigma	Cat#MABE1079; RRID:AB_10952566
TAF12	Proteintech	Cat#12353–1-AP; RRID:AB_2271582
TBP	Proteintech	Cat#22006–1-AP; RRID:AB_10951514
CCT2	Proteintech	Cat#24896–1-AP; RRID:AB_2879783
SpCas9	Diagenode	Cat#C15200229; RRID:AB_2889848#C15310258; RRID:AB_2715516
FLAG M2	Sigma	Cat#F3165; RRID:AB_259529
HA tag	Sigma	Cat#H3663; RRID:AB_262051
Myc tag	Proteintech; Sigma-Aldrich	Cat#60003–2-Ig; RRID:AB_2734122 Cat#M4439; RRID:AB_439694
GAPDH	Proteintech	Cat#10494–1-AP; RRID:AB_2263076
β-Tubulin	Proteintech	Cat#10094–1-AP; RRID:AB_2210695
CD46-BV421	BD Biosciences	Cat#743776; RRID:AB_2741744
RNA polymerase II	ThermoFisher Scientific	Cat#A300653A; RRID:AB_519334
Rabbit IgG	Proteintech	Cat#30000–0-AP; AB_2819035
Anti-rabbit IgG, HRP-linked Antibody	Cell Signaling Technology	Cat#7074; RRID:AB_2099233
Anti-mouse IgG, HRP-linked Antibody	Cell Signaling Technology	Cat#7076; RRID:AB_330924
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488	ThermoFisher Scientific	Cat#A32731; RRID:AB_2633280
Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 647	ThermoFisher Scientific	Cat#A32728; RRID:AB_2633277
Bacterial and virus strains
NEB Stable competent E. coli cells	New England Biolabs	Cat#C3040H
Endura electrocompetent cells	LGC Biosearch Technologies	Cat#60242–2
Chemicals, peptides, and recombinant proteins
TRIzol	Sigma-Aldrich	Cat#T3934
Paraformaldehyde	ThermoFisher Scientific	Cat#28908
Formaldehyde	Pierce	Cat#PI28908
Glycine	Sigma-Aldrich	Cat#G8898
Puromycin	ThermoFisher Scientific	Cat#A1113803
G418 Sulfate	Gibco	Cat#10131027
Blasticidin S	Gibco	Cat#A1113903
Accutase	Sigma-Aldrich	Cat#A6964
Hygromycin	ThermoFisher Scientific	Cat#10687010
Doxycycline	Sigma	Cat#D9891
D-sorbitol	Sigma-Aldrich	Cat#S1876
Tunicamycin	Sigma-Aldrich	Cat#SML1287
Hydrogen peroxide	Sigma-Aldrich	Cat#H1009
Mitoxantrone	MedChemExpress	Cat#HY-13502
Critical commercial assays
RNeasy Plus Mini Kit	Qiagen	Cat#74136
RNA loading dye	ThermoFisher Scientific	Cat#LC6876
10% TBE-Urea gel	ThermoFisher Scientific	Cat#EC68752BOX
Hybond-N+ membrane	Amersham	Cat#NV0796
ULTRAhyb-Oligo hybridization buffer	ThermoFisher Scientific	Cat#AM8663
North2South Chemiluminescent Hybridization and Detection reagents	ThermoFisher Scientific	Cat#17097
P3 Primary Cell 4D-Nucleofector™ X Kit S	Lonza	Cat#V4XP-3032
Wizard Genomic DNA Purification Kit	Promega	Cat#A1120
NEBNext Ultra II Q5 Master Mix	New England Biolabs	Cat#M0544X
QIAquick PCR Purification Kit	Qiagen	Cat#28104
GeneJET PCR purification column	ThermoFisher Scientific	Cat#K0701
GeneJET Gel Extraction Kit	ThermoFisher Scientific	Cat#K0692
KAPA HiFi HotStart DNA polymerase	Roche	Cat# KK2601
PrimeSTAR® Max DNA Polymerase	Takara Bio	Cat#R045B
SPRIselect beads	Beckman	Cat#B23318
D1000 ScreenTape	Agilent	Cat#5067–5582; Cat#5067–5583
High Sensitivity D1000 ScreenTape	Agilent	Cat#5067–5584
High Sensitivity D1000 Reagents	Agilent	Cat#5067–5585
Qubit dsDNA HS assay	ThermoFisher Scientific	Cat#Q32851
PhiX	Illumina	Cat#FC-110–3001
MiSeq Reagent Micro Kit v2 (300 cycles)	Illumina	Cat#MS-103–1002
NextSeq 2000 P2 Reagents (200 Cycles) v3	Illumina	Cat#20046812
NovaSeq 6000 S1 platform (200 cycles kit)	Illumina	Cat#20028318
NovaSeq 6000 S1 platform (100 cycles kit)	Illumina	Cat#20028319
Illumina Stranded mRNA Prep kit	Illumina	Cat#15031047
In-Fusion Snap Assembly Master Mix	Takara Bio	Cat#638948
NEBuilder HiFi DNA Assembly Master Mix	New England Biolabs	Cat#E2621L
X-tremeGENE 9 DNA Transfection Reagent	Sigma-Aldrich	Cat#6365809001
Zombie NIR viability dye	BioLegend	Cat#423106
one-step SensiFAST real-time PCR kit	Bioline	Cat#BIO-72001
Maxima H Minus First Strand cDNA Synthesis Kit	ThermoFisher Scientific	Cat#K1652
SensiFAST™ SYBR No-ROX Kit	Bioline	Cat#BIO-98050
Accel-NGS 2S Plus DNA Library Kit	Swift Biosciences	Cat#21096
Maxi-prep plasmid purification kit	Invitrogen	Cat#K210016
BP clonase II	ThermoFisher Scientific	Cat#11789020
LR clonase II	ThermoFisher Scientific	Cat#11791020
BveI	ThermoFisher Scientific	Cat#FD1744
Esp3I	ThermoFisher Scientific	Cat#FD0454
XhoI	New England Biolabs	Cat#R0146L
CsiI	ThermoFisher Scientific	Cat#FD2114
MluI	ThermoFisher Scientific	Cat#FD0564
FastAP	ThermoFisher Scientific	Cat#EF0651
T4 DNA ligase	New England Biolabs	Cat#M0202
Lipofectamine RNAiMax	ThermoFisher Scientific	Cat#13778150
Streptavidin Sepharose Beads	ThermoFisher Scientific	Cat#20353
Dynabeads protein G	ThermoFisher Scientific	Cat#10004D
Proteinase-K	ThermoFisher Scientific	Cat#EO0492
RNaseA	Invitrogen	Cat#12091021
Bicinchoninic acid (BCA) assay	Pierce	Cat#23225
Bradford reagent	BioRad	Cat#5000006
NuPAGE LDS Sample Buffer (4x)	ThermoFisher Scientific	Cat# NP0007
cOmplete protease inhibitors	Roche	Cat#11836145001
4–12% Bis‐Tris gels	Life Technologies	Cat#NP0323BOX
Immobilon-P PVDF membrane	Sigma-Aldrich	Cat#IPVH00010
SuperSignal West Pico PLUS chemiluminescence reagent	ThermoFisher Scientific	Cat#34580
DMEM with high glucose and pyruvate	Gibco	Cat#11995073
heat-inactivated fetal bovine serum (HI-FBS)	Gibco	Cat#16140071
penicillin-streptomycin	Gibco	Cat#15140122
trypsin-EDTA	Gibco	Cat#25200056
Opti-MEM	Gibco	Cat#31985062
Deposited data
CRISPR screen sequencing	This study	GEO: GSE244337
GUIDE-seq data	This study	GEO: GSE262849
HAP1 and RPE1 RNA-Seq data	This study	GEO: GSE244340
TAF5 RNA-Seq	This study	GEO: GSE244357
ChIP-seq data	This study	GEO: GSE244373
All sequencing data produced in this study	This study	GEO: GSE244374
TAF5 AP-MS data	This study	MassIVE: MSV000092798
TAF5 miniTurboID data	This study	MassIVE: MSV000092798
Microscopy data	This study	DOI: 10.17632/3sdsc83vsn.2
HAP1 cells ribosome profiling data	Malecki et al.84	GEO: GSE93133
Lymphoblastoid cell lines (GM19204 and GM19238) ribosome profiling data	Raj et al.85	GEO: GSE75290
Primary human reticulocytes ribosome profiling data	Mills et al.86	GEO: GSE85864
hES cells (H1) ribosome profiling data	Werner et al.87	GEO: GSE62247 (SRR1610244 to SRR1610259)
BJ fibroblast cell lines (EH, EL and ELR) ribosome profiling data	Ji et al.88	GEO: GSE65885 (SRR1802146 to SRR1802148; SRR1802152 to SRR1802154)
HCT116 cells ribosome profiling data	Fijałkowska et al.89	GEO: GSE87328
HEK293 cells ribosome profiling data	Oh et al.90	GEO: GSE70804
U2OS human osteosarcoma cell line ribosome profiling data	Jang et al.91	GEO: GSE56924
RPE1 cells ribosome profiling data	Tanenbaum et al.92	GEO: GSE67902
Proteomics data	Sinitcyn et al.8	PMID: 36959352
Experimental models: Cell lines
HAP1	Horizon Discovery	Cat#C631
HAP1 Cas9	Horizon Discovery	Cat#Cas9-011
HAP1 Cas9/Cas12a	This study	N/A
RPE1 hTERT	ATCC	Cat# CRL-4000
RPE1 hTERT TP53 −/− Cas9	Hart et al.29	N/A
RPE1 hTERT TP53 −/− Cas9/Cas12a	This study	N/A
HEK293T	ATCC	Cat#ACS-4500
HEK293 Flp-In T-REx cells	Invitrogen	Cat#R78007
Oligonucleotides
See Table S4	This study	N/A
Recombinant DNA
See Table S4 & Methods	This study	N/A
Software and algorithms
AlphaFold2 2.3.1	Jumper et al.121, Varadiet al.122	https://alphafold.ebi.ac.uk/download
BEDTools 2.30.0	Quinlan and Hall109	https://bedtools.readthedocs.io/en/latest/
Biomart	Kinsella et al.140	http://useast.ensembl.org/biomart/martview/62cd2a1c43671bf181f1a4d18e66b210
BLAT	UCSC Genome Browser	https://hgdownload.soe.ucsc.edu/downloads.html#utilities_downloads
bowtie 1.3.1	Langmead et al.128	https://bowtie-bio.sourceforge.net/manual.shtml
bowtie2 2.5.1	Langmead and Salzberg108	https://bowtie-bio.sourceforge.net/bowtie2/manual.shtml
CRISPResso2	Clement et al.113	https://github.com/pinellolab/CRISPResso2
cutadapt 1.18	Martin107	https://cutadapt.readthedocs.io/en/stable/
deepTools 3.5.2	Ramírez et al.120	https://deeptools.readthedocs.io/en/develop/
g:Profiler	Kolberg et al.118	https://biit.cs.ut.ee/gprofiler/gost
GuideScan	Perez et al.126	https://guidescan.com
MAGeCK mle module	Li et al.50	https://sourceforge.net/p/mageck/wiki/Home/
MaxEntScan	Yeo and Burge148	http://hollywood.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html
MISO 0.5.4	Katz et al.124	https://miso.readthedocs.io/en/fastmiso/
Python 2.7.15	Python Software Foundation	https://www.python.org
Python 3.8.5	Python Software Foundation	https://www.python.org
R version 4.3.1	R Foundation	https://www.r-project.org
R package PRROC	Grau et al.130	https://cran.r-project.org/web/packages/PRROC/index.html
RibORF	Ji150	https://github.com/zhejilab/RibORF
Rule Set 2	Doench et al.125	https://portals.broadinstitute.org/gpp/public/software/sgrna-scoring-help#rs2
Samtools 1.16.1	Li et al.119	http://www.htslib.org/download/
scikit-learn 1.3.0	Pedregosa et al.131	https://scikit-learn.org/stable/install.html
STAR 2.7.11a	Dobin et al.149	https://github.com/alexdobin/STAR/releases
UCSC liftOver	UCSC Genome Browser	https://genome.ucsc.edu
UCSC Multiz Refseq protein alignment	Blanchette et al.141	https://hgdownload.soe.ucsc.edu/goldenPath/hg38/multiz100way/
UCSC table browser	UCSC Genome Browser	https://genome.ucsc.edu
UCSF ChimeraX 1.6.1	Pettersen et al.123	https://www.cgl.ucsf.edu/chimerax/
VastDB	Tapial et al.6	https://vastdb.crg.eu/wiki/Main_Page
Whippet	Sterne-Weiler et al.117	https://github.com/timbitz/Whippet.jl
FlowJo software	BD Biosciences, version 10.8.1	https://www.flowjo.com/solutions/flowjo
Cytoscape v3.9.1	Shannon et al.114	https://cytoscape.org/
STRING plugin v2.0.1	Doncheva et al.115	https://apps.cytoscape.org/apps/stringapp
Partek Flow software (version 10.0.23.0531)	N/A	https://www.partek.com/partek-flow/
GraphPad Prism Version 9.0	GraphPad Software Inc.	https://www.graphpad.com/
Adobe Illustrator v28.4.1	Adobe	https://www.adobe.com/products/illustrator.html
Affinity Designer 2 v2.3.0	Affinity Designer	https://affinity.serif.com/en-us/designer/
BioRender	BioRender	https://www.biorender.com/
Other
Semi-dry transfer	Bio-Rad	Cat#1703940
UV Crosslinker	VWR	Cat#89131–484
Lonza 4D-Nucleofector Transfection System	Lonza	Cat#AAF-1003B, AAF-1003X
M220 Focused Ultrasonicator	Covaris	Cat#500295
4150 TapeStation System	Agilent	Cat#G2992AA
BTX Gemini Electroporator	BTX	Cat#452042
Bioruptor Plus sonicator	Diagenode	Cat#B01020002
Mini Gel Tank	Life Technologies	Cat#A25977,
Mini Blot Module	Life Technologies	Cat#B1000
VeritPro Thermal Cycler	Applied Biosystems	Cat#A48141
CFX96 Touch Real-Time PCR	BioRad	Cat#1855195
MiSeq Sequencing System	Illumina	N/A
NextSeq 2000 Sequencing System	Illumina	N/A
NovaSeq 6000 Sequencing System	Illumina	N/A
EVOS™ M5000 Imaging System	Invitrogen	Cat#AMF5000
iBright CL1500 Imaging System	Invitrogen	Cat#A44114
Vi-Cell BLU Cell Viability Analyzer	Beckman Coulter	Cat#C19196
Incucyte SX1	Sartorius	Cat#4837
LSRFortessa cell analyzer	BD Biosciences	N/A