Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors
- Medical Scientist Training Program, University of California, San Francisco, United States
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, United States
- Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States
- Whitehead Institute for Biomedical Research, United States
- Department of Microbiology, Harvard Medical School, United States
- Institute for Neurodegenerative Disease, University of California, San Francisco, United States
- Center for Alzheimer's Disease and Related Dementias, National Institutes of Health, United States
- National Institute on Aging, National Institutes of Health, United States
- National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
- Department of Urology and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, United States
- Arc Institute, United States
- Department of Biochemistry and Biophysics, University of California, San Francisco, United States
- Department of Biology, Massachusetts Institute of Technology, United States
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, United States
Figures
Figure 1 with 1 supplement
Design and validation of ultra-compact dual-single guide RNA (sgRNA) CRISPR interference (CRISPRi) libraries.
(A) Schematic of growth screen used to compare single- and dual-sgRNA libraries. (B) Schematic of dual-sgRNA library sequencing strategies. (C) Comparison of growth phenotypes for DepMap essential …
Figure 1—figure supplement 1
Additional comparisons of pilot single- and dual-single guide RNA (sgRNA) library screens.
(A) Comparison of growth phenotypes for all elements between our pilot single-sgRNA library and Horlbeck et al. data, merged by gene name (n=20,228 elements). Growth phenotypes are reported as γ …
Figure 2 with 1 supplement
CRISPR interference (CRISPRi) effectors containing SID or MeCP2 domains have non-specific effects on cell viability and gene expression.
(A) Schematics of CRISPRi transcription repressor domains and general lentiviral expression construct used for all CRISPRi effectors. UCOE = ubiquitous chromatin opening element; SFFV = spleen …
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Figure 2—source data 1
p-Values and growth defects depicted in Figure 2C.
- https://cdn.elifesciences.org/articles/81856/elife-81856-fig2-data1-v2.xlsx
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Figure 2—source data 2
Data depicted in Figure 2E.
- https://cdn.elifesciences.org/articles/81856/elife-81856-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
Design of constructs for CRISPR interference (CRISPRi) effector expression.
Figure 3 with 1 supplement
Zim3-dCas9 and dCas9-Kox1-MeCP2 mediate strongest knockdown.
(A) Experimental design to measure knockdown mediated by different CRISPR interference (CRISPRi) effectors by delivering single guide RNAs (sgRNAs) targeting either essential genes or cell surface …
Figure 3—figure supplement 1
Additional measurements of on-target activity of CRISPR interference (CRISPRi) effectors.
(A) Depletion of K562 cells expressing essential gene-targeting single guide RNAs (sgRNAs) and different CRISPRi effectors, measured as the ratio of mCherry-positive (sgRNA-expressing) to …
Figure 4 with 2 supplements
Validation of a suite of optimized Zim3-dCas9 cell lines.
(A) Distribution of anti-B2M signal intensity (APC) in individual RPE1 (left) and Jurkat (right) cells expressing indicated CRISPR interference (CRISPRi) effectors and single guide RNAs (sgRNAs). …
Figure 4—figure supplement 1
Single-cell distributions of knockdown in different Zim3-dCas9 cell lines.
(A) Distribution of anti-CD151, anti-CD55, and anti-CD29 signal intensities (APC) in HepG2 cells expressing Zim3-dCas9. Data from three independent transductions are shown. A weak targeting single …
Figure 4—figure supplement 2
Growth of different Zim3-dCas9-expressing cell lines.
Growth rates of HepG2, HuTu-80, and HT29 cells expressing Zim3-dCas9, measured as the log2 of the ratio of BFP-negative (effector-expressing) to BFP-positive (not effector-expressing) cells …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Human) | K562 CRISPRi (BFP) | This paper | N/A | Stable cell line with SFFV Zim3-dCas9 P2A BFP |
| Cell line (Human) | K562 CRISPRi (GFP) | This paper | N/A | Stable cell line with SFFV Zim3-dCas9 P2A GFP |
| Cell line (Human) | K562 CRISPRi (no fluorescent marker) | This paper | N/A | Stable cell line with SFFV Zim3-dCas9 P2A Hygro |
| Cell line (Human) | RPE1 CRISPRi (Zim3) | Replogle et al., 2022 | N/A | Stable cell line with SFFV Zim3-dCas9 P2A BFP |
| Cell line (Human) | Jurkat CRISPRi (Zim3) | This paper | N/A | Stable cell line with EF1alpha Zim3-dCas9 P2A mCherry |
| Cell line (Human) | HepG2 CRISPRi | This paper | N/A | Stable cell line with EF1alpha Zim3-dCas9 P2A mCherry |
| Cell line (Human) | HT29 CRISPRi | This paper | N/A | Stable cell line with EF1alpha Zim3-dCas9 P2A mCherry |
| Cell line (Human) | HuTu-80 CRISPRi | This paper | N/A | Stable cell line with EF1alpha Zim3-dCas9 P2A mCherry |
| Antibody | Anti-human CD55 (clone JS11, mouse monoclonal), APC | BioLegend | Cat#: 311311; RRID:AB_2075857 | Flow cytometry; 1:100 in PBS with 5% (v/v) FBS |
| Antibody | Anti-human CD81 (clone 5A6, mouse monoclonal), APC | BioLegend | Cat#: 349509; RRID:AB_2564020 | Flow cytometry; 1:100 in PBS with 5% (v/v) FBS |
| Antibody | Anti-human CD151 (clone 50–6, mouse monoclonal), APC | BioLegend | Cat#: 350405; RRID:AB_10661726 | Flow cytometry; 1:100 in PBS with 5% (v/v) FBS |
| Antibody | Anti-human CD29 (clone TS2/16, mouse monoclonal), APC | BioLegend | Cat#: 303007; RRID:AB_314323 | Flow cytometry; 1:100 in PBS with 5% (v/v) FBS |
| Antibody | Anti-human B2M (clone 2M2, mouse monoclonal), APC | BioLegend | Cat#: 316312; RRID:AB_10641281 | Flow cytometry; 1:100 in PBS with 5% (v/v) FBS |
| Antibody | Mouse IgG1, κ Isotype Ctrl (clone MOPC-21, mouse monoclonal), APC | BioLegend | Cat#: 981806 | Flow cytometry; 1:100 in PBS with 5% (v/v) FBS |
| Recombinant DNA reagent | Plasmid pJB120_pHR-UCOE-SFFV-EGFP | This paper | Addgene: 188900 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pJB074_pHR-UCOE-SFFV-dCas9-HA-2xNLS-P2A-EGFP | This paper | Addgene: 188898 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pJB069_pHR-UCOE-SFFV-Zim3-NLS-dCas9-HA-2xNLS-P2A-EGFP | This paper | Addgene: 188899 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pCL63_pHR-UCOE-SFFV-SID4x-dCas9-HA-2xNLS-XTEN80-KRAB(Kox1)-P2A-EGFP | This paper | Addgene: 188901 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pCL51_pHR-UCOE-SFFV-dCas9-NLS-KRAB(Kox1)-MeCP2-P2A-EGFP | This paper | Addgene: 188902 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pCL35_pHR-UCOE-SFFV-dCas9-HA-2xNLS-XTEN80-KRAB(Kox1)-P2A-EGFP | This paper | Addgene: 188765 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pAG389_pHR-UCOE-SFFV-Zim3-NLS-dCas9-HA-2xNLS-P2A-Hygro | This paper | Addgene: 188768 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pNM1130_pHR-UCOE-EF1a-Zim3-NLS-dCas9-HA-2xNLS-loxP-P2A-EGFP-loxP | This paper | Addgene: 188773 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pNM1129_pHR-UCOE-SFFV-Zim3-NLS-dCas9-HA-2xNLS-loxP-P2A-EGFP-loxP | This paper | Addgene: 188774 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pNM1128_pHR-UCOE-EF1a-Zim3-NLS-dCas9-HA-2xNLS-mTagBFP2 | This paper | Addgene:188775 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pNM1127_pHR-UCOE-SFFV-Zim3-NLS-dCas9-HA-2xNLS-mTagBFP2 | This paper | Addgene: 188776 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pNM1125_pHR-UCOE-EF1a-Zim3-NLS-dCas9-HA-2xNLS-P2A-mTagBFP2 | This paper | Addgene:188777 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pNM1124_pHR-UCOE-EF1a-Zim3-NLS-dCas9-HA-2xNLS-P2A-EGFP | This paper | Addgene: 188778 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pNM1123_pHR-UCOE-SFFV-Zim3-NLS-dCas9-HA-2xNLS-P2A-mCherry | This paper | Addgene: 188779 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pJB109_pHR-UCOE-EF1a-Zim3-NLS-dCas9-HA-2xNLS-P2A-mCherry | This paper | Addgene: 188766 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pJB108_pHR-UCOE-SFFV-Zim3-NLS-dCas9-HA-2xNLS-P2A-mTagBFP2 | This paper | Addgene: 188767 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pRAP0006_pHR-UCOE-EF1a-dCas9-HA-2xNLS-XTEN80-KRAB(Kox1)-IRES-mCherry | This paper | Addgene: 188769 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pRAP0003_pHR-UCOE-SFFV-dCas9-HA-2xNLS-XTEN80-KRAB(Kox1)-IRES-mCherry | This paper | Addgene: 188770 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pCL75_pHR-UCOE-EFS-dCas9-HA-2xNLS-XTEN80-KRAB(Kox1)-P2A-EGFP | This paper | Addgene: 188771 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pCL74_pHR-UCOE-CMV-dCas9-HA-2xNLS-XTEN80-KRAB(Kox1)-P2A-EGFP | This paper | Addgene: 188772 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | Plasmid pCRISPRia-v2 (parent vector) | DOI: 10.7554/eLife.19760 | Addgene: 84832 | |
| Recombinant DNA reagent | Plasmid pU6-sgRNA EF1alpha-puro-T2A-mCherry | This paper | Addgene: 188780 | Further information in Supplementary file 7 |
| Recombinant DNA reagent | pJR98 | This paper | Addgene: 187239 | CR3 constant region – hU6 sgRNA promoter flanked by BsmBI sites. Further information in Supplementary file 7 |
| Recombinant DNA reagent | pJR100 | This paper | Addgene: 187240 | Lentiviral sgRNA vector for Perturb-seq with mU6 sgRNA promoter, CR1 constant region with CS1 capture sequence in stem loop, and UCOE EF1alpha driving PURO-BFP marker expression. Further information in Supplementary file 7 |
| Recombinant DNA reagent | pJR101 | DOI: 10.1016/j.cell.2022.05.013/this paper | Addgene: 187241 | Lentiviral sgRNA vector for Perturb-seq with mU6 sgRNA promoter, CR1 constant region with CS1 capture sequence in stem loop, and UCOE EF1alpha driving PURO-GFP marker expression. Further information in Supplementary file 7 |
| Recombinant DNA reagent | pJR103 | This paper | Addgene: 187242 | Lentiviral sgRNA vector with mU6 sgRNA promoter, CR1 constant region, and UCOE EF1alpha driving PURO-BFP marker expression. Further information in Supplementary file 7 |
| Recombinant DNA reagent | pJR104 | This paper | Addgene: 187243 | Lentiviral sgRNA vector with mU6 sgRNA promoter, CR1 constant region, and UCOE EF1alpha driving PURO-GFP marker expression. Further information in Supplementary file 7 |
| Recombinant DNA reagent | pJR106 | This paper | Addgene: 187244 | Lentiviral sgRNA vector for CROP-seq with mU6 sgRNA promoter, CR1 constant region, and UCOE EF1alpha driving PURO-BFP marker expression. Further information in Supplementary file 7 |
| Recombinant DNA reagent | pJR107 | This paper | Addgene: 187245 | Lentiviral sgRNA vector for CROP-seq with mU6 sgRNA promoter, CR1 constant region, and UCOE EF1alpha driving PURO-GFP marker expression. Further information in Supplementary file 7 |
| Commercial assay or kit | Direct-zol RNA Miniprep | Zymo Research | Cat#: R2051 | |
| Commercial assay or kit | Qubit RNA Broad Range (BR) Kit | Thermo Fisher Scientific | Cat#: Q10211 | |
| Commercial assay or kit | NucleoSpin Blood kit (XL or L) | Macherey-Nagel | Cat#: 740950 (XL) or 740954 (L) | Purification of genomic DNA from cell pellets |
| Commercial assay or kit | NEBNext Ultra II Q5 PCR MasterMix | NEB | Cat#: M0544 | PCR amplification of dual-sgRNA elements from genomic DNA |
| Other | TransIT-LT1 Transfection Reagent | Mirus Bio | Cat#: MIR 2300 | Transfection reagent for lentivirus production |
| Software, algorithm | Python scripts to count dual-sgRNA elements in sequencing data and remove recombined elements | This paper | N/A | https://github.com/josephreplogle/CRISPRi-dual-sgRNA-screens, Replogle, 2022 |
| Sequence-based reagent | Sequences of individual sgRNAs to target benchmarking genes | This paper | N/A | Sequences listed in Supplementary file 8 |
| Sequence-based reagent | Library dJR004, Pilot genome-wide single-sgRNA human CRISPRi library | This paper | N/A | sgRNA targeting sequences in Supplementary file 1 |
| Sequence-based reagent | Library dJR020, Pilot genome-wide dual-sgRNA human CRISRi library | This paper | N/A | sgRNA targeting sequences in Supplementary file 1 |
| Sequence-based reagent | Library dJR072, Final genome-wide, sgRNA 1+2, dual-sgRNA human CRISPRi library with UMIs | This paper | Addgene: 187246 | sgRNA targeting sequences in Supplementary file 4 |
| Sequence-based reagent | Library dJR073, Final genome-wide, sgRNA 3+4, dual-sgRNA human CRISPRi library with UMIs | This paper | Addgene: 187247 | sgRNA targeting sequences in Supplementary file 4 |
| Sequence-based reagent | Library dJR074, Final genome-wide, sgRNA 5+6, dual-sgRNA human CRISPRi library with UMIs | This paper | Addgene: 187248 | sgRNA targeting sequences in Supplementary file 4 |
| Sequence-based reagent | Library dJR075, Final genome-wide, sgRNA 1+2, dual-sgRNA human CRISPRa library with UMIs | This paper | Addgene: 187249 | sgRNA targeting sequences in Supplementary file 9 |
| Sequence-based reagent | Library dJR076, Final genome-wide, sgRNA 3+4, dual-sgRNA human CRISPRa library with UMIs | This paper | Addgene: 187250 | sgRNA targeting sequences in Supplementary file 9 |
| Sequence-based reagent | Library dJR077, Final genome-wide, sgRNA 5+6, dual-sgRNA human CRISPRa library with UMIs | This paper | Addgene: 187251 | sgRNA targeting sequences in Supplementary file 9 |
Additional files
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Supplementary file 1
Table with dual- and single-single guide RNA (sgRNA) sequences used for preliminary comparison.
- https://cdn.elifesciences.org/articles/81856/elife-81856-supp1-v2.xlsx
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Supplementary file 2
Table with read counts and growth phenotypes from pilot screen.
- https://cdn.elifesciences.org/articles/81856/elife-81856-supp2-v2.csv
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Supplementary file 3
Table with aggregated CRISPR interference (CRISPRi) single guide RNA (sgRNA) performance across screens.
- https://cdn.elifesciences.org/articles/81856/elife-81856-supp3-v2.xlsx
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Supplementary file 4
Table with finalized dual-single guide RNA (sgRNA) human CRISPR interference (CRISPRi) libraries.
- https://cdn.elifesciences.org/articles/81856/elife-81856-supp4-v2.xlsx
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Supplementary file 5
List of integration barcodes.
- https://cdn.elifesciences.org/articles/81856/elife-81856-supp5-v2.xlsx
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Supplementary file 6
Table with Dolcetto versus dual-single guide RNA (sgRNA) Perturb-seq comparison.
- https://cdn.elifesciences.org/articles/81856/elife-81856-supp6-v2.xlsx
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Supplementary file 7
Table of plasmids.
- https://cdn.elifesciences.org/articles/81856/elife-81856-supp7-v2.xlsx
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Supplementary file 8
Table with sequences of single guide RNAs (sgRNAs) used for individual validation.
- https://cdn.elifesciences.org/articles/81856/elife-81856-supp8-v2.xlsx
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Supplementary file 9
Table with dual-single guide RNA (sgRNA) human CRISPR activation (CRISPRa) libraries.
- https://cdn.elifesciences.org/articles/81856/elife-81856-supp9-v2.xlsx
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Supplementary file 10
Table with in silico dual-single guide RNA (sgRNA) mouse CRISPR interference (CRISPRi) libraries.
- https://cdn.elifesciences.org/articles/81856/elife-81856-supp10-v2.xlsx
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Supplementary file 11
Table with in silico dual-single guide RNA (sgRNA) mouse CRISPR activation (CRISPRa) libraries.
- https://cdn.elifesciences.org/articles/81856/elife-81856-supp11-v2.xlsx
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Supplementary file 12
Table with dual-single guide RNA (sgRNA) human CRISPR interference (CRISPRi) library targeting DepMap Common Essential genes.
- https://cdn.elifesciences.org/articles/81856/elife-81856-supp12-v2.csv
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MDAR checklist
- https://cdn.elifesciences.org/articles/81856/elife-81856-mdarchecklist1-v2.docx
