CRISPR screening in hematology research: from bulk to single-cell level

Meyers, Sarah; Demeyer, Sofie; Cools, Jan

doi:10.1186/s13045-023-01495-5

Journal of Hematology & Oncology

Table 1 Overview of the technical details of the different published sc-CRISPR studies

From: CRISPR screening in hematology research: from bulk to single-cell level

	Cell type	gRNA capture	Perturbation type	Cas	Number of gRNAs	gRNA multiplexing	Number of cells	Delivery	Sequencing chemistry	Readout
Transcriptomics
CROP-seq [46]	Jurkat	Poly-A	CRISPRko	Cas9	119	–	5798 (+ 1320 cells with NT gRNA)	Lentiviral	DROP-seq	RNA
Perturb-seq [43, 44]	K562	Barcode	CRISPRi	dCas9-KRAB	UPR epistasis screen: 9 triplet combinations UPR Perturb-seq experiment: 91	Up to 3 gRNAs in a single vector	UPR epistasis screen: 15006 UPR Perturb-seq experiment: 65337	Lentiviral	10X Genomics 3′-seq	RNA
Perturb-seq [43, 44]	BMDCs, K562	Barcode	CRISPRko	Cas9	BMDC: 67 K562: 46 + 36	MIMOSCA can study interaction effects based on cells with multiple gRNAs	BMDC: 70000 K562: 104000 + 26000	Lentiviral	10X Genomics 3′-RNA-seq DROP-seq	RNA
CRISP-seq [45]	Bone marrow cells, LSK cells	Barcode	CRISPRko	Cas9	Bone marrow: 57 In vivo: 5 genes + 2 control gRNAs	Multiplexing 2 gRNAs using 2 vectors with each a different fluorophore	Bone marrow: 6144 In vivo: 2768	Lentiviral	MARS-seq	RNA
Mosaic-seq [47]	K562	Barcode	CRISPRi	dCas9-KRAB	241	High MOI	12444	Lentiviral	DROP-seq	RNA
Direct-capture Perturb-seq [74]	K562, iPSCs	gRNA-specific primer, capture sequence	CRISPRko CRISPRi CRISPRa	Cas9 dCas9-KRAB-dCas9-SunTag/scFV-VP64	K562 UPR screen: 32 iPSCs: 40 K562 interaction screen: 92 gRNA pairs K562 multiplexed screen: 87 CRISPRi, 49 CRISPRa gRNAs	gRNA pairs	K562 UPR screen: ~ 50000 iPSCs:5300 K562 interaction screen: ~ 30000 K562 multiplexed screen: ~ 30000	Lentiviral	10X Genomics 3′-seq with feature barcoding 10X Genomics 5′ seq	RNA
TAP-seq [84]	K562	Poly-A	CRISPRi	dCas9-KRAB	7055	–	231667	Lentiviral	10X Genomics 3′-seq DROP-seq	RNA
In vivo Perturb-seq [113]	Mouse embryo forebrain ventricles	Barcode	CRISPRko	Cas9	38 gRNA pairs	2 gRNAs	46770	Lentiviral injection in the forebrain	10X Genomics 3′-seq	RNA
Genome-wide Perturb-seq [136]	K562, Rpe1	Capture sequence	CRISPRi	dCas9-KRAB	11294 (K562 all expressed genes) 2291 (K562 essential genes) 2688 (Rpe1 essential genes)	2 gRNAs per vector	> 2.5 million cells	Lentiviral	10X Genomics 3′-seq with feature barcoding	RNA
Direct-seq [73]	HEK293T, Jurkat, K562	8A8G tag	CRISPRko, CRISPRa	Cas9 dCas9–VP64 MS2–p65–HSF1	12472 gRNA pairs	gRNA pairs	13435	Lentiviral	Fluidigm C1 10X Genomics 3′ and 5′-seq	RNA
Sc-Tiling [106]	MLL-AF9 murine leukemic cells	Capture sequence	CRISPRko	Cas9	602	–	4943	Lentiviral	10X Genomics 3′-seq with feature barcoding	RNA
Sc-eVIP* [111]	A549 lung cancer cells	Barcode	Coding variant overexpression	/	100 TP53 variants 101 KRAS variants	–	162314 (TP53) 150044 (KRAS)	Lentiviral	10X Genomics 3′-seq	RNA
POKI-seq [143]	Primary human T cells	Barcode	Custom HDR template	Cas9	36 knock-in templates	–	> 40000	Electroporation of Cas9 RNP with HDR templates	10X Genomics 3′-seq	RNA
Deaminase screening [108]	A375	Poly-A	base editing	BE3	420	–	13218	Lentiviral	Drop-seq	RNA
Genga et al. [48]	human embryonic stem cells	Poly-A	CRISPRi	dCas9-KRAB	160	–	16110	Lentiviral	10X Genomics 3′-seq	RNA
Norman et al. [49]	K562	Barcode	CRISPRa	dCas9-SunTag	28680 gRNA pairs	Yes, dual-gRNA vector	~ 104000	Lentiviral	10X Genomics 3′-seq	RNA
Tian et al. [50]	Human iPSC derived neurons	Poly-A	CRISPRi and CRISPRa	dCas9-KRAB DHFR-dCas9-VPH	CRISPRi: 374 CRISPRa: 206	–	CRISPRi: ~ 58000 CRISPRa: ~ 38000	Lentiviral	10X Genomics 3′-seq	RNA
Alda-Catalinas et al. [51]	Mouse embryonic stem cells	Poly-A	CRISPRa	dCas9-VP64 + MS2-p65-HSF1	475	–	203894	Lentiviral	10X Genomics 3′-seq	RNA
Belk et al. [52]	OT-1T cells	gRNA-specific primer	CRISPRko	Cas9	48	–	70646	Retroviral	10X Genomics V(D)J 5′ scRNA with feature barcoding	RNA
Giladi et al. [53]	LSK cells	Barcode	CRISPRko	Cas9	21	–	23641	Lentiviral	MARS-seq	RNA
DoNick-seq [54]	HEK293T, human intestinal crypt-like cells	Barcode	CRISPRko	Cas9 nickase	16 × 4	2 gRNA pairs	25602	Lentiviral	10X Genomics 3′-seq	RNA
PerturbSci-Kinetics [140]	HEK293	gRNA-specific primer	CRISPRi	dCas9-KRAB-MeCP2	699	–	161966	Lentiviral	Combinatorial indexing	Nascent RNA
Compressed perturb-seq [103]	THP-1	Poly-A	CRISPRko CRISPRi	Cas9 dCas9-KRAB	339	Via overloading or high MOI	Cell-pooled: 32700 guide-pooled: 24192	Lentiviral	10X Genomics 3′-seq	RNA
Proteomics
Perturb-CITE-seq [132]	Melanoma cells cocultured with TILs	Poly-A	CRISPRko	Cas9	744	–	> 218000	Lentiviral	10X Genomics 3′-seq	RNA + protein
ECCITE-seq [131]	PBMC, MyLa, Sez4, NIH/3T3, K562	gRNA-specific primer	CRISPRko	Cas9	Species mixing: 20 K562: 13	–	Species mixing: 5935 K562: 4120	Lentiviral	10X Genomics 5′ V(D)J	RNA + protein + TCR/BCR
Perturb-map [123]	Kras^G12D p53^−/− lung cancer cells, 4T1	ProCode	CRISPRko	Cas9	101	–	8442439	Lentiviral	10X Genomics Visium	RNA + protein + imaging
Rothenberg et al. [144]	LSK cells	Capture sequence	CRISPRko	Cas9	23 gRNA pairs	gRNA pairs	~ 5000	Retroviral	10X Genomics 3′-seq with feature barcoding	RNA + protein
CaRPool-seq [104]	HEK293FT, THP1	bcgRNA	RNA silencing	RfxCas13d	29 crRNA arrays 385 crRNA arrays	CRISPR array processed by Cas13	9355 31308	Lentiviral	10X Genomics 3′-seq with feature barcoding	RNA + protein
(bee)STING-seq [107]	K562	gRNA-specific primer	CRISPRi CRISPR base editor	KRAB-dCas9-MeCP2 SpRY-Cas9-FNLS-BE3	STING-seq: 1905 beeSTING-seq: 338	–	STING-seq v1: 15285 STING-seq v2: 82339 beeSTING-seq: 39049	Lentiviral	10X Genomics 5′ seq	RNA + protein
Epigenomics
Perturb-ATAC [101]	GM12878, primary human keratinocytes	Barcode	CRISPRkoCRISPRi	Cas9 dCas9-KRAB	B cells: 40 keratinocytes: 7	Yes	B cells: 2936 keratinocytes: 1356	Lentiviral	Fluidigm C1	Chromatin accessibility
CRISPR-sciATAC [124]	HEK293FT, NIH/3T3, K562	Poly-A	CRISPRko	Cas9	Species mixing: 20 Chromatin modifiers: 66 Chromatin remodeling complexes: 255	–	~ 30000	Lentiviral	Combinatorial indexing	Chromatin accessibility
SPEAR-ATAC [125]	K562, GM12878, Mcf7	gRNA-specific primer	CRISPRi	dCas9-KRAB	414	–	104592	Lentiviral	10X Genomics ATAC-seq	Chromatin accessibility

*sc-eVIP is a single-cell screen but does not involve the use of CRISPR gRNAs

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