REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Cas9 (1:1000)	Abcam	Cat. # ab191468
Bacterial and virus strains
5-Alpha competent E. coli (high efficiency)	NEB	Cat. # C2987H
One Shot™ Stbl3™ Chemically Competent E. coli	Invitrogen	Cat. # C737303
Endura™ ElectroCompetent Cells	Lucigen	Cat. # 60242-2
Chemicals, peptides, and recombinant proteins
Dulbecco’s modified Eagle’s medium (DMEM)	Corning	Cat. # 10-017-CV
Phenol-red-free DMEM	Cytiva	Cat. # SH30284.01
PBS, 1× (w/o Ca2+, Mg2+)	Corning	Cat. # 21-040-CV
Fetal bovine serum (FBS)	Gemini Bio	Cat. # 100-500
Bovine serum albumin (BSA)	Sigma-Aldrich	Cat. # A1595
Absolute ethanol (200 proof)	Fisher Bioreagents	Cat. # BP-2818-500
RNase Away	Molecular Bioproducts	Cat. # 21-236-21
ELIMINase	Decon Labs	Cat. # 1101
Puromycin dihydrochloride	Thermo Fisher Scientific	Cat. # A1113803
Blasticidin S HCl	Thermo Fisher Scientific	Cat. # A1113903
O'GeneRuler (100 bp DNA ladder, ready-to-use)	Thermo Fisher Scientific	Cat. # SM1143
6× loading dye	Thermo Fisher Scientific	Cat. # R0611
UltraPure 10× TBE Buffer	Thermo Fisher Scientific	Cat. # 15581-044
Gibco™ Trypsin-EDTA (0.05%), phenol red	Thermo Fisher Scientific	Cat. # 25300062
Gibco™ TrypLE™ Express Enzyme (1×)	Thermo Fisher Scientific	Cat. # 12605010
SYTOX™ Green Dead Cell Stain	Thermo Fisher Scientific	Cat. # 34860
UltraPure Agarose	Thermo Fisher Scientific	Cat. # 16500100
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	Cat. # 15140122
Qiagen Protease (7.5 AU)	Qiagen	Cat. # 19155
Nuclease-Free Water	Qiagen	Cat. # 129117
Bovine serum albumin (fatty acid free, low endotoxin)	Sigma-Aldrich	Cat. # A8806
Hexadimethrine bromide	Sigma-Aldrich	Cat. # 107689-10G
(1S,3R)-RSL3	Cayman Chemical	Cat. # 19288
Ethylenediaminetetraacetic acid (EDTA)	Fisher Scientific	Cat # 15575-020
TransIT-LT1 transfection reagent	Mirus	Cat. # MIR 2300
BstXI	New England Biolabs	Cat. # R0113S
BlpI	New England Biolabs	Cat. # R0585S
T4 DNA Ligase	New England Biolabs	Cat. # M0202S
Critical commercial assays
Herculase II Fusion Enzyme with dNTP Combo (400 rxn)	Agilent	Cat. # 600679
Qiagen QiaAmp DNA Blood Maxi Kit	Qiagen	Cat. # 51194
QIAquick Gel Extraction Kit	Qiagen	Cat. # 28706
HiSpeed Plasmid Kits	Qiagen	Cat. # 12663
Qubit dsDNA HS Assay Kits	Thermo Fisher Scientific	Cat. # 32851
Deposited data
U2-OS CRISPR Screen Result	Li et al.2	CRISPRlipid, http://crisprlipid.org
MDA-MB-453 CRISPR Screen Result	Li et al.2	CRISPRlipid, http://crisprlipid.org
Experimental models: Cell lines
HuH7	Kind gift from Dr. Holly Ramage (University of Pennsylvania)	N/A
U-2 OS Tet-On	Clontech	Cat. # 630919
HEK293T	UC Berkeley Cell Culture Facility	N/A
Oligonucleotides
sgCherry	Self-cleaving mCherry	GGCCACGAGTTCGAGATCGA
sgSAFE_5784	Safe-targeting guide	AAATTTCATGGGAAAATAG
oMCB1562	PCR-1 Forward	aggcttggatttctataacttcgtatagcatacattatac
oMCB1563	PCR-1 Reverse	ACAtgcatggcggtaatacggttatc
oMCB1439	PCR-2 Forward	caagcagaagacggcatacgagatgcacaaaagg aaactcaccct
oMCB1440-AD002	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTGAA CTCCAGTCACCGATGTCGACTCGG TGCCACTTTTTC
oMCB1440-AD003	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTGAA CTCCAGTCACTTAGGCCGACTCG GTGCCACTTTTTC
oMCB1440-AD004	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTGAA CTCCAGTCACTGACCACGACTCG GTGCCACTTTTTC
oMCB1440-AD005	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTGA ACTCCAGTCACACAGTGCGAC TCGGTGCCACTTTTTC
oMCB1440-AD006	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTG AACTCCAGTCACGCCAATCGA CTCGGTGCCACTTTTTC
oMCB1440-AD007	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTGA ACTCCAGTCACCAGATCCGACT CGGTGCCACTTTTTC
oMCB1440-AD009	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTGA ACTCCAGTCACGATCAGCGACT CGGTGCCACTTTTTC
oMCB1440-AD010	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTGA ACTCCAGTCACTAGCTTCGACT CGGTGCCACTTTTTC
oMCB1440-AD012	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTGA ACTCCAGTCACCTTGTACGACT CGGTGCCACTTTTTC
oMCB1440-AD013	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTG AACTCCAGTCACAGTCAACGA CTCGGTGCCACTTTTTC
oMCB1440-AD014	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTG AACTCCAGTCACAGTTCCCGAC TCGGTGCCACTTTTTC
oMCB1440-AD015	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTG AACTCCAGTCACATGTCACGA CTCGGTGCCACTTTTTC
oMCB1440-AD016	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTG AACTCCAGTCACCCGTCCCGA CTCGGTGCCACTTTTTC
oMCB1440-AD018	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTG AACTCCAGTCACGTCCGCCGA CTCGGTGCCACTTTTTC
oMCB1440-AD019	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTG AACTCCAGTCACGTGAAACGA CTCGGTGCCACTTTTTC
oMCB1440-AD021	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTG AACTCCAGTCACGTTTCGCGA CTCGGTGCCACTTTTTC
oMCB1440-AD022	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTG AACTCCAGTCACCGTACGCGA CTCGGTGCCACTTTTTC
oMCB1440-AD025	PCR-2 Reverse with Barcode	aatgatacggcgaccaccgagatctacac GATCGGAAGAGCACACGTCTG AACTCCAGTCACACTGATCGAC TCGGTGCCACTTTTTC
oMCB1672_new10gCRKO	Illumina Sequencing Primer	GCCACTTTTTCAAGTTGATAACG GACTAGCCTTATTTAAACTTGCT ATGCTGTTTCCAGCTTAGCTCTTAAAC
Recombinant DNA
lentiCas9-Blast	Addgene	Cat. # 52962
Bassik Human CRISPR Knockout Library	Addgene	Cat. # 101926, # 101927, # 101928, # 101929, # 101930, # 101931, # 101932, # 101933, # 101934
pMDLg/pRRE	Addgene	Cat. # 12251
pRSV-Rev	Addgene	Cat. # 12253
pMD2.G	Addgene	Cat. # 12259
pMCB320	Addgene	Cat. # 89359
Software and algorithms
Image Lab v6.0.1	Bio-Rad Laboratories	https://www.bio-rad.com/en-us/product/image-lab-software?ID=KRE6P5E8Z
IncuCyte v2020A	Sartorius	https://www.sartorius.com/en/products/live-cell-imaging-analysis/live-cell-analysis-software
CasTLE statistical framework v1.0	Morgens et al.8	https://github.com/elifesciences-publications/dmorgens-castle
BowTie 2 v1.1.3.1	Langmead et al.9; Langmead and Salzberg10	https://bowtie-bio.sourceforge.net/index.shtml
FlowJo v10	BD Biosciences	https://www.flowjo.com/solutions/flowjo
Other
245 mm square TC-treated culture dish	Corning	Cat. # 431110
96-well flat clear bottom black polystyrene TC-treated microplates	Corning	Cat. # 3904
0.45 μm filter	Thermo Fisher Scientific	Cat. # 158-0045