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Human CRISPRi-v2 Dual Guide Libraries
(Pooled Libraries #197348, #197349)

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  • Purpose

    These dual sgRNA libraries contain a fixed element (either a non-targeting guide or EMC2 guide) and the well-established Human Genome-wide CRISPRi-v2 library in tandem. One library has a non-targeting control guide and the second library has a guide targeting EMC2, a protein subunit of the ER membrane complex (EMC) that when depleted, leads to degradation of the EMC. New dual guide libraries can be generated by cloning desired fixed guides into the pJR152 backbone and combining with the Human Genome-wide CRISPRi-v2 library.

  • Vector Backbone

    pJR152 (Plasmid #196280) - does not express Cas9

Ordering

Item Catalog # Description Quantity Price (USD)
Pooled Library 197348 CRISPRi-v2-ctrl dual guide library† 1 $640 Add to Cart
Pooled Library 197349 CRISPRi-v2-EMC2 dual guide library† 1 $640 Add to Cart
Available to Academic and Nonprofits Only

  • † A Cas9 plasmid is NOT included with this item and will have to be ordered separately. Can be used in conjunction with pHR-UCOE-SFFV-Zim3-dCas9-P2A-BFP (Addgene #188767), pHR-UCOE-SFFV-dCas9-XTEN80-KRAB(Kox1)-P2A-EGFP (Addgene #188765), or otherwise used with cell lines already expressing inhibitory dCas9.

Library Details

  • Species
    Human
  • Genes targeted
    18,905
  • gRNAs per gene
    5
  • Control gRNAs
    1,895
  • Total gRNAs
    103,269
  • Lentiviral Generation
    3rd

Library Shipping

These libraries are delivered as suspended DNA in a microcentrifuge tube on blue ice. The tube's contents will not necessarily be frozen. For best results, minimize freeze/thaws.

  • Volume
    ∼20 µL
  • Concentration
    25 ng/µL

Resource Information

Depositor Comments

The schematic has two parts, A and B. Part A shows a circular plasmid with mU6 promoter, CR1V2, hU6 promoter, CR3fixed, EF1a promoter, puro, t2A, and BFP features labelled. Part B shows 3 linear pieces of DNA. The top one is labelled CRISPRi V2 library and from left to right contains mU6 promoter, CR1V2, BamHI site, NotI site, EF1a promoter, puro, T2A, and BFP features. Dotted lines from the cut sites lead to the middle piece of DNA labelled insert. From left to right the insert contains the BamHI site, hU6 promoter, CR3fixed, and NotI site. Next to the middle DNA is labelled BstXI/BlpI annealing and cloning of constant guide. The bottom piece of DNA, labelled dual library, contains all of the features from left to right listed in the plasmid. Under the mU6 promoter is the forward indexed primer depicted as an arrow pointing from left to right. Under the hU6 promoter is the reverse primer (binds hU6) depicted as an arrow pointing from right to left.

Figure 1: Dual-guide library design and construction. (A) Schematic of the dual sgRNA vector. Expression of the randomized CRISPRi-v2 sgRNA is driven by a mU6 promoter and the fixed guide is driven by a hU6 promoter, each flanked by unique guide constant regions (CR). Downstream, the EF1a promoter drives the expression of the puromycin resistance selectable marker and BFP. (B) Cloning a dual genome-wide library is comprised of two steps. First, a guide of interest is inserted using standard oligo annealing and ligation into a BstXI/BlpI cut backbone. Second, both CRISPRi-v2 library and the fixed guide are digested with complementary restriction sites (BamHI/NotI) and ligated at scale, resulting in an mU6-'V2 guide'-hU6-'fixed guide' library design. To sequence the resulting library, a standard 5' indexed primer is coupled with a reverse primer that anneals to the hU6 region upstream of the inserted fixed guide. This strategy ensures only guides containing the fixed region are amplified for sequencing. Image reused from Guna et al. bioRxiv 2023, under a Creative Commons CC-BY-NC-ND License (Link opens in a new window).

Algorithm used for analyzing the sequencing results is available at https://github.com/mhorlbeck/ScreenProcessing (Link opens in a new window).

Please visit https://doi.org/10.1101/2023.01.22.525086 (Link opens in a new window) for bioRxiv preprint.

How to cite this pooled library ( Back to top )

These pooled libraries were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    Human CRISPRi-v2 Control Dual Guide Library was a gift from Rebecca Voorhees (Addgene #197348)
    Human CRISPRi-v2 EMC2 Dual Guide Library was a gift from Rebecca Voorhees (Addgene #197349)

  • For your References section:

    A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens. Guna A, Page KR, Replogle JM, Esantsi TK, Wang ML, Weissman JS, Voorhees RM. BMC Genomics. 2023 Oct 30;24(1):651. doi: 10.1186/s12864-023-09754-y. PubMed 37904134 (Link opens in a new window)
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