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PURPOSE
‍Ultra-compact, human genome-wide CRISPRi library targeting each gene with a single element encoding a dual sgRNA cassette.

VECTOR BACKBONE
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. CR3 constant region - hU6 sgRNA promoter flanked by BsmBI sites.

Publication
Heo SJ, Enriquez LD, Federman S, Chang AY, Mace R, Shevade K, Nguyen P, Litterman AJ, Shafer S, Przybyla L, Chow ED. Compact CRISPR genetic screens enabled by improved guide RNA library cloning. Genome Biology. 2024 Jan 19;25(1):25. doi: 10.1186/s13059-023-03132-3.
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Replogle JM, Bonnar JL, Pogson AN, Liem CR, Maier NK, Ding Y, Russell BJ, Wang X, Leng K, Guna A, Norman TM, Pak RA, Ramos DM, Ward ME, Gilbert LA, Kampmann M, Weissman JS, Jost M. Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors. eLife. 2022 Dec 28;11:e81856. doi: 10.7554/eLife.81856.

Library Details
Genes/gRNAs/plasmids: 22,580 dual sgRNA elements
Controls: 1,026 dual sgRNA control elements
Lentiviral Generation: 3rd
Aliquot Details: 40 µL per tube at a concentration of 3.776 µg/µL
Uniformity Ratio: 90/10 < 2

Library Shipping
This library is provided as suspended DNA in a microcentrifuge tube on ice. The contents may not be frozen upon arrival. To ensure optimal performance, minimize freeze-thaw cycles. The requester is responsible for initiating the Material Transfer Agreement (MTA) and covering any associated shipping costs.

PURPOSE
Human sgRNA library in backbone LGR dual guide vector targeting 20,367 loci and containing 81,776 unique sgRNAs along with 572 negative controls (495 non-targeting controls and 77 negative intron controls). The library comes with puromycin and choice of one of three fluorescent protein: BFP, mCherry or eGFP.

VECTOR BACKBONE
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.

PUBLICATION
LGR Production Methodology Unpublished
ORIGINAL SOURCE MATERIAL REFERENCE:
Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature Biotechnology. 2016 Feb;34(2):184–191. doi: 10.1038/nbt.3437‍

Library Details
LGR Brunello_BFP: 150ug/tube, conc. 2.583ug/uL, vol. 60uL
Uniformity Ratio: 90/10 < 2
Fluorescent Marker: BFP
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LGR Brunello_eGFP: 150ug/tube, conc. 2.15ug/uL, vol. 70uL
Uniformity Ratio: 90/10 < 2
Fluorescent Marker: eGFP
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LGR Brunello_mCherry: 150ug/tube, conc. 2.40ug/uL, vol. 65uL
Uniformity Ratio: 90/10 < 2
Fluorescent Marker: mCherry

Library Shipping
This library is provided as suspended DNA in a microcentrifuge tube on ice. The contents may not be frozen upon arrival. To ensure optimal performance, minimize freeze-thaw cycles. The requester is responsible for initiating the Material Transfer Agreement (MTA) and covering any associated shipping costs.

PURPOSE
Parental vector for the CRISPRi/a/n libraries. 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 and three fluorescent protein marker choices, BFP, mCherry or eGFP.

PUBLICATION
ORIGINAL SOURCE MATERIAL REFERENCE: Replogle JM, Bonnar JL, Pogson AN, Liem CR, Maier NK, Ding Y, Russell BJ, Wang X, Leng K, Guna A, Norman TM, Pak RA, Ramos DM, Ward ME, Gilbert LA, Kampmann M, Weissman JS, Jost M. Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors. eLife. 2022 Dec 28;11:e81856. doi: 10.7554/eLife.81856.

PLASMID DETAILS:
1. pLGR_dual_guide-Puro-mycNLS-BFP, (plasmid DNA sequence)
Aliquot Details: 2.00 ug/tube, cone. 100 ng/uL, vol. 20uL
Fluorescent Marker: BFP

2. pLGR_dual_guide-Puro-mycNLS-eGFP, (plasmid DNA sequence)
‍Aliquot Details: 2.00 ug/tube, cone. 100 ng/uL, vol. 20uL
Fluorescent Marker: eGFP

3. pLGR_dual_guide-Puro-mycNLS-mCherry, (plasmid DNA sequence)
Aliquot Details: 2.00 ug/tube, cone. 100 ng/uL, vol. 20uL
Fluorescent Marker: mCherry

4. Insert fragment donor plasmid: pKR98 (Plasmid #187239) - CR3 constant region - hU6 sgRNA promoter flanked by BsmBI sites.
Aliquot Details: 2.00 ug/tube, cone. 100 ng/uL, vol. 20uL
Fluorescent Marker: None

Library Shipping
This library is provided as suspended DNA in a microcentrifuge tube on ice. The contents may not be frozen upon arrival. To ensure optimal performance, minimize freeze-thaw cycles. The requester is responsible for initiating the Material Transfer Agreement (MTA) and covering any associated shipping costs.