US 20210108193A1IN IN ( 19 ) United States ( 12 ) Patent Application Publication ( 10 ) Pub . No .: US 2021/0108193 A1 Mali et al . ( 43 ) Pub . Date : Apr. 15 , 2021 ( 54 ) METHODS FOR SCREENING GENETIC ( 52 ) U.S. CI . PERTURBATIONS CPC C12N 15/1065 ( 2013.01 ) ; C12N 5/069 ( 2013.01 ) ; C12N 15/86 ( 2013.01 ); CI2N ( 71 ) Applicant: The Regents of the University of 2740/15052 ( 2013.01) ; C12N 2506/45 ( 2013.01 ) ; C12N 2740/15043 ( 2013.01 ); A61K California , Oakland , CA ( US ) 35/44 ( 2013.01 ) ( 72 ) Inventors: Prashant Mali , La Jolla , CA (US ) ; ( 57 ) ABSTRACT Udit Parekh , La Jolla , CA (US ); Yan Understanding the complex effects of genetic perturbations Wu , La Jolla , CA (US ); Kun Zhang , on cellular state and fitness in human pluripotent stem cells La Jolla , CA ( US ) ( hPSCs) has been challenging using traditional pooled screening techniques which typically rely on unidimensional phenotypic readouts . Here , Applicants use barcoded open reading frame (ORF ) overexpression libraries with a ( 21 ) Appl. No .: 17 / 028,836 coupled single - cell RNA sequencing ( scRNA - seq ) and fit ness screening approach , a technique we call SEUSS ( Scal ( 22 ) Filed : Sep. 22 , 2020 comprehensiveable fUnctional assaying Screening platform by Sequencing . Using this) , systemto establish , Appli a cants perturbed hPSCs with a library of developmentally critical transcription factors ( TFs ), and assayed the impact of Related U.S. Application Data TF overexpression on fitness and transcriptomic cell state across multiple media conditions. Applicants further lever ( 60 ) Provisional application No. 62 / 904,614 , filed on Sep. aged the versatility of the ORF library approach to system 23 , 2019 . atically assay mutant gene libraries and also whole gene families. From the transcriptomic responses, Applicants built genetic co - perturbation networks to identify key altered gene modules . Strikingly , we found that KLF4 and SNAI2 Publication Classification have opposing effects on the pluripotency gene module, ( 51 ) Int. Ci . highlighting the power of this method to characterize the C12N 15/10 ( 2006.01 ) effects of genetic perturbations. From the fitness responses , C12N 5/071 ( 2006.01 ) Applicants identified ETV2 as a driver of reprogramming C12N 15/86 ( 2006.01 ) towards an endothelial - like state . A61K 35/44 ( 2006.01 ) Specification includes a Sequence Listing . Patent Application Publication Apr. 15 , 2021 Sheet 1 of 12 US 2021/0108193 A1 Reprogrammingandregulatory network analysis Matrix .100-04P11P1,21,3010-21P2,1P2,2P2,3 -P2,0001-00P3,1P32P3,3 -P3.N 000-40PN,TPN2PN,3-PNN CoefficientDesign Genes Unsupervised Clustering TF's Concluding YNNAAMMMNNNA 003-0+1) Estimateof 3LTR Expression A 020-0 040-5 LUNO( Change Fold Log FIG.1A Barcode fromamplified BarcodeHygromycinTFP2A TFBarcode fragmentationpre- CLNA Barcodedsingletranscrirecell libraries TE Barcode O00 basedDroplet singlecellcapture, transcriptionreverse andbarcoding LibraryAlternateMedia selectionLibraryand 5LTR PooledTF Gene Family Library Mutant ProteinTransduction Patent Application Publication Apr. 15 , 2021 Sheet 2 of 12 US 2021/0108193 A1 KLF4 CDX2 FOXP1 MYOG MYCL ASCL5 ASCL4 mCherrySNAI2 NEUROG3 TRX5 MYG05 HCXB4 ESARG SRY FOXA3 ONECUT1 2 LHX3 NEUROG5 MESP1 GATA2 -2 MEF2G ASCL5 ASCL1nnnnn HOXA1 PAX7 SPI1 FOXA2 GATA1 ERG NEUROG5 GATA6 FOXAS MYCH ETV2 Pluripotent Unilineage Multilineage FIG . 1B Patent Application Publication Apr. 15 , 2021 Sheet 3 of 12 US 2021/0108193 A1 Pluripotent SP11 Medium SNA12 RUNX1 PAX7 OTX2 08 ONECUT1 NEUROG3 NEUROG1 NEURO01 MYOG MYOD1 06 MYC 610 07 MESP1 20 mCherry 2 LHX3 FOXA2 CRX 04 CDX2 ASCL5 ASCL41 FIG . 1C ASCL1ASCL3 Unilineage C12978885883 C11 Medium ONECUT1 NEUROG3 NEURO01 MYC mCherry 60 KLF4 20 GATA4 GATA2 FOXA2 COX2 880883 Multilineage FIG . 1D 01 03 TBX5 mCherry 60 KLF4 HNFIB C6 CDX2 ATF7 C2 FIG . 1E Patent Application Publication Apr. 15 , 2021 Sheet 4 of 12 US 2021/0108193 A1 TBX5 Medium SPI1 Unilineage SNAI2 RUNX1 Multilineage PAX7 Pluripotent OTX2 T ONECUT1 NEUROG3 NEUROG1 NEURO01 MYOG MYOD1 MYC MESP1 . Genotype mCherry LHX31 KLE4 HNF1B GATA4 GATA2 FOXA2 CRX CDX2 ASCL51 ASCL4 ASCL3 ASCL1 2000 3000 4000 # of Differentially Expressed Genes FIG . 1F Patent Application Publication Apr. 15 , 2021 Sheet 5 of 12 US 2021/0108193 A1 0.00 Pluripotentstate coefAvg 0.04 Ribosome FIG2B. ma accessibility COX2 Multilineage biogenesisSignaling Chromatin ONECUT1 NEUROG3 Notchpathway NEURO01 andmotility pathways KLFA Cytoskeleton GATA4 Unilineage Neuron GATA2 differentiation FOXA2 CDX2 SNAIZ Embryonic lontransport RUNX1 FIG.2C development metabolism H PAX7 Growthfactorresponse mitochondrial ZX10 NEUROG3 NEUROG1 NEURO01 Pluripotent Gene moduleGene- SNNnetworkdetection Gene3a Genet Gene2Genel Gene6 Gene5 by Gene? FIG.2A lontransport statePluripotent Signalingpathways Notchpathway Growthfactorresponse CytoskeletonandmotilityRibosomebiogenesis 22.1P2,2P2,3-P2,N - Chromatinaccessibility differentiationNeuron Embryonicdevelopment P3.1P3,2P3.3-P3,N CoefficientMatrix My PIN1,2P1,3-1,1P PN,TPN2PN,3-PNA Translationandmitochondrialmetabolism Genes Patent Application Publication Apr. 15 , 2021 Sheet 6 of 12 US 2021/0108193 A1 coefAvg 0.050 0.025 0.000 -0.025 -0.050 coefAvg 0.01 0.00 -0.01 FIG.2E FIG.2G lontransport statePluripotent PluripotentstateNotchpathway lontransport Growthfactorresponse Notch pathway Signalingpathways Ribosomebiogenesis Cytoskeletonandmotility RibosomebiogenesisCytoskeleton andmobility EmbryonicdevelopmentdifferentiationNeuron Growth factorresponseSignaling pathways Chromatinaccessibility Neurondifferentiation Embryonicdevelopment Translationandmitochondrialmetabolism Chromatinaccessibility Translationandmitochondrialmetabolism KLF11,KLF9KLF10 KLF1,KLF5KLFOKLF7KLF2,KLFA KLF12,KLF3KLF8 KLF13,KLF14KLF15 Group GroupII GroupIl VGroup KLF15,KLF17 CTerminal FIG.2D AcetylaseBindingAbilitySin3A*BindingSiteBC2H2ZincFingerDomainsCrBP Binding Site FIG2F. b MBILNLB N-Terminal Patent Application Publication Apr. 15 , 2021 Sheet 7 of 12 US 2021/0108193 A1 FIG3C. FIG.3F 3HFIG. OKLF4 NSNAI2 Mesenchymal markers CDH5CDHS/DAPI - SPP1 que LAMC1 Epithelial EPCAM CDH2 mo Control 0.6 0.4 0.2 0.2 Control )coefficient ( size Effect Cadherins FIG.3G FIG.3E SNA12 2020909090 EGMETV2 FIG3B. KOR EGMControl KL-4 PC1:EMT-likesignature 2 ControlPluripotent 62Day EGMETV2 EGMControl DPPA4 NANOG 501 251 ControlPluripotent DNMT3B POU5F1 3AFIG. Control SNAI%effects 29492222 EGMETV2 PECAM1 10000EGM Control SOX2 2 LMX1A 6000 3000 ControlPluripotent SCRNAVSSONA: GU1 NANOG SCRNAfitnesslogFC 3DFIG. CDH5 EGMETV2 DNMT3B POU5F1 POU5F1HNFSB ETV2 2 EGMControl KLF4effects MYCN GATA6 400 200 ControlPluripotent -2 Tood SOX2 SALL2 logFC fitness gDNA 5000 Patent Application Publication Apr. 15 , 2021 Sheet 8 of 12 US 2021/0108193 A1 Hpal 5 ' LTR mCherry P2A Hygromycin 3 LTR Gibson Assembly ? + TF Barcode Bambi Bambi 5 LTR Efia mCherry |P2A |Hygromycin |TF Barcode 3'LTR Gibson Assembly + Bambi BamHI Efla P2A Hygromycin TF Barcode 3'LTR FIG . 4 Patent Application Publication Apr. 15 , 2021 Sheet 9 of 12 US 2021/0108193 A1 SNA2 ONECUTI MYOG OTX2 CRX Unilineage Medium CDX2 FOXP1 LUX1A SCRNA VS GDNA : R = 0.7 MYCL ASCL3 2 TBX5 inCherrySIX1 0000 SIX2 RUNX1 gDNAfitnesslogFC MYC01 -2 ASCL4 POU5F1 ESRRG G?1 SOX2 LHX3 -2 2 MEF2C NEUROG3 SRY SCRNA fitness logFC FOXA3 SP1 2 HOXB6 FIG . 5B ASCL1 MYC 0 ASCL5 SOX3 Multilineage Medium GATA2 GATA1 SCRNA VS ODNA : R = 0.43 MESP , NEUROD1 NEUROG1 GATA4 ? HOXA1 HAND2 .0 TFAP20 logFCgDNAFitness NEUROD1 HOXA11 -2.5 HNFAA ERG FOXA1 SPIB ERG -5.0 -ETV URODA SOX2 FOXAT FOXA2 -2.5 0.0 2.5 MYCN SOX10 SCRNA fitness logFC SP1C FOXA1 FIG . 5C SOX2 GATA6 POU5F1 Pluripotent Unilineage Multilineage FIG . 5A Patent Application Publication Apr. 15 , 2021 Sheet 10 of 12 US 2021/0108193 A1 2.5 -2.5 FIG.6C 0 SPI1 SNAI2 TBX5 KLF4 CDX2 ATF7 RUNX1 mCherry ? ONECUT11 NEUROG1 2.5 -2.5 NEUROD1 FIG.6D mCherry.MESP1 LHX3 KLF4 FIG.6B FOXA2 CDX2 1 ASCL5 CDX2 Ctx2m Kif4m Cdx2m ONECUT1 NEUROG3 NEUROD1 mCherry GATA4 GATA2 FOXA2 Myod1m Ascl1m 2.5 0.0 -2.5 -5.0 FIG.6A SP11 SNA2 RUNX1 PAX7 OTX2 ONECUT NEUROG3 NEUROG1 NEUROD1 MYOG MYOD1 MYC MESP1 mCherryLHX3 FOXA2 CRX CDX2 ASCL5 ASCL4 ASCL3 ASCL1 Patent Application Publication Apr. 15 , 2021 Sheet 11 of 12 US 2021/0108193 A1 R 0.877 R = 0.843 R 0.859 0.31 0.41 0.2 0.2 0.2 0.14 0.01 0.04 0.0 3-0.2 -0.2 -0.2 -0.5 0.0 0.5 -0.5 0.0 0.5 Pluripotent medium Pluripotent medium Pluripotent medium batch 2 batch 3 FIG . 7A FIG . 7B FIG . 70 R 0.867 R 0.87 R 0.681 0.41 0.4 0.4 0.24 0.2 batch2 0.2 mediumPluripotent mediumPluripotent 0.0 mediumMultilineage 0.0 combined -0.2 combined -0.2 -0.21 -0.5 0.0 0.5 -0.5 0.0 0.5 -0.5 0.0 0.5 Pluripotent medium Pluripotent medium Multilineage medium FIG . 7D FIG . 7E FIG . 7F Pluripotent Medium Endothelial Medium Multilineage Medium SNAI2 SNAI2 COX2 2 2.5 KLF4 KLF4 ONECUT1 KLF4 MYC 0 Fitnesslogfold-change 1 Fitnesslogfold-change GATA4 CDX2 Fitnesslogfold-change CDX2 2 SPIC POU5F1 -2.51 SOX10 ENFIB BOOK GATAO SOX2 . HNFIA SRIC POU5F1 500 1000 1500 500 1000 0 500 1000 1500 # of differentially of differentially of differentially expressed genes expressed genes expressed genes FIG , 8A FIG . 8B FIG . 8C Patent Application Publication Apr. 15 , 2021 Sheet 12 of 12 US 2021/0108193 A1 SNAI2 FIG9C. SALL2 2A } 0.2 0.6 Control FIG.10B w hPSCscreen SNAI2 MYC:R=0.49 ? weer KLF4 -0.2 SNAI2 WS 0.0 -0.2 KLF 2.01 1.5 1.0 0.51 Control mutants MYC 1.6 1.41 1.2 Control anderen w SNA12 LAMC1 WWW KLF4 ,SNAI2 2.01 1.6 1,2 Control FIG.10A DNMT3B MarkersEpithelial KLF4:R=0.63 -0.50.0 hPSCscreen 0.6 0.41 Control om KLF4SNAI2 0.5 0.0 -0.5 SNAI2 1.2 0.8 0.4Control family KLF NANOG KLF4 1.15, 1.10 1.05 0.950.90 Control SNAI2 FIG.9A FIG.9B VIM KLF4 VSNA12 ]Contro KLF4 2|SNA 1.21 0.8 0.6 0.41Control KLF4 SNAI2 *010100OOOO ***** KLF4 2 FIG.9D 31 1 Control MarkersMesenchymal 1.2 0.6 0.4Control endlocopoco SNAI2 SNA12 KLF4 TPM2 SNA2 NetworkPluripotency SOX2 MS KLFA Cadherins KLF4 1.2 Control ??????? 0.80.6 0.4Control 2.0 1.6 1.2 Control US 2021/0108193 A1 Apr.