| Antibodies |
| Anti-NCAM1 Rabbit mAb, Alexa Fluor 647 conjugate |
Cell Signaling Technology |
Cat#50831S; RRID: AB_3717968 |
| Anti-MAP2 Mouse mAb, Alexa Fluor 594 conjugate |
BioLegend |
Cat#801802; RRID: AB_2721382 |
| Anti-phospho-AKT (Thr308) Rabbit mAb |
Cell Signaling Technology |
Cat#13038T; RRID: AB_2629447 |
| Goat Anti-Rabbit IgG H&L cross-adsorbed secondary antibody, Alexa Fluor 647 conjugate |
Thermo Scientific |
Cat#A-21244; RRID: AB_2535812 |
| Anti-pan-AKT Mouse mAb |
Cell Signaling Technology |
Cat#2920; RRID: AB_1147620 |
| Anti-PTEN Rabbit mAb |
Cell Signaling Technology |
Cat#9559; RRID: AB_390810 |
| Anti-lamin A/C Mouse mAb |
BioLegend |
Cat#600001; RRID: AB_2810655 |
| StarBright Blue 520 Goat Anti-Mouse IgG |
Bio-Rad |
Cat#12005866; RRID: AB_2934034 |
| StarBright Blue 700 Goat Anti-Rabbit IgG |
Bio-Rad |
Cat#12004162; RRID: AB_3712168 |
| hFAB Rhodamine Anti-Actin Primary Antibody |
Bio-Rad |
Cat#12004164; RRID: AB_2861334 |
| Bacterial and virus strains |
| NEB 10-beta electrocompetent cells |
New England Biolabs |
Cat#C3020K |
| Chemicals, peptides, and recombinant proteins |
| DMEM |
Gibco |
Cat#11965-092 |
| DMEM/F-12 |
Gibco |
Cat#11320-033 |
| Fetal Bovine Serum (FBS) |
Hyclone |
Cat#SH30071.03 |
| Penicillin/Streptomycin |
Gibco |
Cat#15140-122 |
| OPTI-MEM |
Gibco |
Cat#31985-070 |
| DPBS |
Gibco |
Cat#14190-144 |
| Versene |
Gibco |
Cat#15400-054 |
| Trypsin-EDTA 0.05%, Phenol Red |
Gibco |
Cat#25300-054 |
| TransIT-293 |
Mirus |
Cat#MIR 2700 |
| Dimethyl Sulfoxide (DMSO) |
Sigma-Aldrich |
Cat#D8418-50mL |
| CryoStor CS10 Cell Freezing Medium |
STEMCELL Technologies |
Cat#100-1061 |
| Matrigel Matrix |
Corning |
Cat#354234 |
| mTeSR Plus |
STEMCELL Technologies |
Cat#100-0276 |
| ROCK Inhibitor Y-27632 |
SelleckChem |
Cat#S1049 |
| Puromycin Dihydrochloride |
Gibco |
Cat#A11138-03 |
| 0.45 um Syringe Filter |
VWR |
Cat#76479-012 |
| SpCas9 2NLS Nuclease (300 pmol) |
Synthego |
N/A |
| Poly-L-ornithine |
Sigma-Aldrich |
Cat#P3655 |
| Accutase |
STEMCELL Technologies |
Cat#07922 |
| Doxycycline hyclate |
Sigma-Aldrich |
Cat#D9891 |
| Recombinant Human NT-3 |
Peprotech |
Cat#450-03 |
| Recombinant Human BDNF |
Peprotech |
Cat#450-02 |
| GlutaMAX Supplement 100X |
Gibco |
Cat#35050-061 |
| MEM Non-Essential Amino Acid Solution 100X |
Gibco |
Cat#11140-050 |
| B-27 Supplement 50X |
Gibco |
Cat#17504-044 |
| N-2 Supplement 100X |
Gibco |
Cat#17502-048 |
| KnockOut DMEM/F-12 |
Gibco |
Cat#12660-012 |
| Neurobasal-A Medium |
Gibco |
Cat#12349-015 |
| Laminin Mouse Protein |
Gibco |
Cat#23017-015 |
| RPMI 1640 Medium |
Invitrogen |
Cat#11875135 |
| DMEM, no glucose |
Gibco |
Cat#11966-025 |
| Sodium-L-Lactate |
Sigma-Aldrich |
Cat#71718 |
| Vitronectin XF |
STEMCELL Technologies |
Cat#100-0763 |
| CHIR-99021 |
TOCRIS |
Cat#4423 |
| Wnt-C59 |
Selleck Chemicals |
Cat#S7037 |
| Bovine Serum Albumin (BSA) |
Sigma-Aldrich |
Cat#A9418 |
| Ascorbic Acid |
Sigma-Aldrich |
Cat#A8960 |
| B27 + Insulin |
Invitrogen |
Cat#17504044 |
| TrypLE Select 10X |
Gibco |
Cat#A12177-01 |
| Paraformaldehyde 20% Solution |
Electron Microscopy Sciences |
Cat#15713-S |
| Triton X-100 |
Sigma-Aldrich |
Cat#Y8787-50ML |
| Tween-20 |
Sigma-Aldrich |
Cat#P1279-500ML |
| Glutaraldehyde 25% Solution |
Electron Microscopy Sciences |
Cat#16210 |
| Deoxynucleotide (dNTP) Solution Mix 10 mM |
New England Biolabs |
Cat#N0447L |
| Ultrapure BSA |
Thermo Scientific |
Cat#AM2616 |
| Ribolock RNase Inhibitor |
Thermo Scientific |
Cat#EO0381 |
| RevertAid H Minus Reverse Transcriptase |
Thermo Scientific |
Cat#EP0451 |
| Ampligase Buffer |
Biosearch Technologies |
Cat#SS000015-D3 |
| Taq-IT Polymerase |
Qiagen |
Cat#P7620L |
| Ampligase |
Biosearch Technologies |
Cat#E0001-100D2 |
| RNase H |
New England Biolabs |
Cat#M0297L |
| Phi29 DNA Polymerase |
Thermo Scientific |
Cat#EP0094 |
| Glycerol |
Fisher Scientific |
Cat#G33-1 |
| Formamide |
Fisher Scientific |
Cat#BP227-500 |
| Nuclease-Free Water |
Thomas Scientific |
Cat#C001X09 |
| 20X SSC |
Invitrogen |
Cat#15557036 |
| 10X HBSS |
Gibco |
Cat#14065-056 |
| Bovine Serum Albumin (fraction V) |
Sigma-Aldrich |
Cat#A2153-100G |
| Sodium azide |
Sigma-Aldrich |
Cat#S-8032 |
| Ascorbic Acid |
Sigma-Aldrich |
Cat#A92902-100G |
| 4’,6-Diamidino-2-phenylindole (DAPI) |
Fisher Scientific |
Cat#D1306 |
| Sodium borohydride |
Sigma-Aldrich |
Cat#452882 |
| Phalloidin-CF568 conjugate |
Biotium |
Cat#00044-T |
| Wheat Germ Agglutinin (WGA)-CF555 conjugate |
Biotium |
Cat#29076-1 |
| Concanavalin A-Alexa Fluor 750 conjugate |
Thermo Scientific |
Cat#C56127
|
| Carbenicillin Disodium, USP Grade |
GoldBio |
Cat#C-103-250 |
| Kanamycin |
Sigma-Aldrich |
Cat#K1537-25G |
| Q5 High-Fidelity 2X Master Mix |
New England Biolabs |
Cat#M0492L |
| Klenow Fragment, 3’-5’ exo-minus |
New England Biolabs |
Cat#M0212L |
| NEBuilder HiFi DNA Assembly Master Mix |
New England Biolabs |
Cat#E2621L |
| T4 DNA Ligase |
New England Biolabs |
Cat#M0202M |
| BsaI-HFv2 |
New England Biolabs |
Cat#R3733L |
| SapI |
New England Biolabs |
Cat#R0569L |
| Esp3I |
New England Biolabs |
Cat#R0734L |
| BsmBI-v2 |
New England Biolabs |
Cat#R0739S |
| NheI-HF |
New England Biolabs |
Cat#R3131L |
| MluI-HF |
New England Biolabs |
Cat#R3198S |
| EcoRI-HF |
New England Biolabs |
Cat#R3101S |
| NotI-HF |
New England Biolabs |
Cat#R3189S |
| SacII |
New England Biolabs |
Cat#R0157S |
| RIPA Buffer |
Abcam |
Cat#ab156034 |
| Halt Protease Inhibitor Cocktail |
ThermoFisher Scientific |
Cat#1860932 |
| Laemmli Sample Buffer |
Bio-Rad |
Cat#1610737 |
| β-Mercaptoethanol |
Bio-Rad |
Cat#1610710 |
| 8–16% Criterion TGX Stain-Free Protein Gel |
Bio-Rad |
Cat#5678103 |
| Tris/Glycine/SDS Running Buffer |
Bio-Rad |
Cat#1610732 |
| Trans-Blot Turbo Midi 0.2 um PVDF Transfer Packs |
Bio-Rad |
Cat#1704157 |
| Critical commercial assays |
| Glass 6-well Plate |
Cellvis |
Cat#P06-1.5H-N |
| Corning Untreated 245 mm Square BioAssay Dishes |
Fisher Scientific |
Cat#07-200-600 |
| Lonza 4D Nucleofector SE Kit S |
Lonza |
Cat#V4XC-1032 |
| Lonza 4D Nucleofector P3 Kit S |
Lonza |
Cat#V4XP-3032 |
| Lonza 4D Nucleofector P3 Kit X |
Lonza |
Cat#V4XP-3024 |
| Illumina MiSeq v2 Nano 500 Kit |
Illumina |
Cat#MS-103-1003 |
| Pierce BCA Protein Assay Kit |
ThermoFisher Scientific |
Cat#23225 |
| SMRTbell Prep Kit 3.0 |
Pacific Biosciences |
Cat#102-182-700 |
| Deposited Data |
| Impact scores, distinguishability scores, landmark features |
MaveDB; Rubin et al. |
URN#s: 00001243-a, 00001244-a, 00001244-b
|
| Variant-level features, EMDs, p-values, scores |
Zenodo |
Cat#15787685; https://zenodo.org/records/15787685
|
| Phenotype image data, single-cell features and genotypes, CellProfiler pipelines |
Biolmage Archive |
Accession#S-BIAD3095; https://www.ebi.ac.uk/biostudies/bioimages/studies/S-BIAD3095
|
| Experimental models: Cell lines |
| Human embryonic kidney 293T (HEK 293T) |
ATCC |
Cat#CRL-3216; RRID: CVCL_0063 |
| U2OS cells |
ATCC |
Cat#HTB-96; RRID: CVCL_0042 |
| U2OS C11 cells |
Hasle et al. |
N/A |
| WTC11 iPS cells |
Coriell |
Cat#GM25256; RRID: CVCL_Y803 |
| NGN2-inducible WTC11 iPS cells (NWTC11.G3-WT) |
Gladstone Institute; gift from Li Gan lab |
N/A |
| Oligonucleotides |
| See Table S6
|
|
|
| Recombinant DNA |
| pHSG299 |
Takara Bio |
Cat#3299 |
| pHR-UCOE-EF1a-Zim3-dCas9-BFP |
Addgene; Replogle et al. |
Cat#188775; RRID: Addgene_188775 |
| pJBL_051 |
Gift from Jay Shendure lab; Lalanne et al. |
N/A |
| lentiGuide-Puro |
Addgene; Sanjana et al. |
Cat#52963; RRID: Addgene_52963 |
| pCAG-NLS-HA-Bxb1 |
Addgene; Hermann et al. |
Cat#51271; RRID: Addgene_51271 |
| Hyperactive piggyBac transposase expression vector (hyPBase) |
Gift from Jay Shendure lab; Yusa et al. |
N/A |
| pMD2.G |
Addgene |
Cat#12259; RRID: Addgene_12259 |
| psPAX2 |
Addgene |
Cat#12260; RRID: Addgene_12260 |
| PBv2b |
This publication |
VIS-seq expression vector. N/A |
| PBv2b-NSapI |
This publication |
VIS-seq expression vector. Cat#252243; RRID: Addgene_252243 |
| PBv2b-CSapI |
This publication |
VIS-seq expression vector. Cat#252244; RRID: Addgene_252244 |
| PBv2c-NSapI |
This publication |
VIS-seq expression vector. Cat#252245; RRID: Addgene_252245 |
| Software and algorithms |
| Cellpose v2.2.1 |
Stringer et al. |
https://www.cellpose.org/; RRID: SCR_021716 |
| StarDist v0.8.5 |
Schmidt et al. |
https://github.com/stardist/stardist
|
| STARCall |
Bradley et al. |
https://github.com/FowlerLab/starcall-workflow
|
| CellProfiler v4.2.6 |
Carpenter et al. |
https://cellprofiler.org/; RRID: SCR_007358 |
| Pycytominer v1.2.1 |
Serrano et al. |
https://github.com/cytomining/pycytominer
|
| SciPy v1.14.1 |
Virtanen et al. |
https://scipy.org/; RRID: SCR_008058 |
| NumPy v1.24.4 |
Harris et al. |
https://numpy.org/; RRID: SCR_008633 |
| Scikit-learn v1.5.2 |
Pedregosa et al. |
https://scikit-learn.org/; RRID: SCR_002577 |
| Scikit-image v0.24.0 |
van der Walt et al. |
https://scikit-image.org/; RRID: SCR_021142 |
| Matplotlib v3.9.2 |
Hunter et al. |
https://matplotlib.org/; RRID: SCR_008624 |
| NetworkX v3.4.2 |
Hagberg et al. |
https://networkx.org/; RRID: SCR_016864 |
| Snakemake v7.32.4 |
Molder et al. |
https://snakemake.readthedocs.io/; RRID: SCR_003475 |
| Pandas v2.2.3 |
McKinney et al. |
https://pandas.pydata.org/; RRID: SCR_018214 |
| Python v3.11 |
Python Software Foundation |
https://www.python.org/; RRID: SCR_008394 |
| XGBoost v3.0.1 |
Chen and Guestrin |
https://xgboost.ai/; RRID: SCR_021361 |
| PyMOL v3.1 |
Schrodinger LLC |
https://www.pymol.org; RRID: SCR_000305 |
| Pacybara |
Weile et al. |
https://github.com/rothlab/pacybara
|
