-
Purpose
Dual sgRNA libraries containing a fixed element (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 subunit of the ER membrane complex that when depleted, leads to degradation of the EMC. New libraries can be generated by cloning desired fixed guides into pJR152 and combining with the Human Genome-wide CRISPRi-v2 library.
-
Vector Backbone
pJR152 - does not express Cas9
-
Depositing Labs
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 | |
† 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
-
SpeciesHuman
-
Genes targeted18,905
-
gRNAs per gene5
-
Control gRNAs1,895
-
Total gRNAs103,269
-
Lentiviral Generation3rd
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
-
Concentration25 ng/µL
Resource Information
-
Protocols
-
Depositor Data
-
Terms and Licenses
Depositor Comments
Algorithm used for analyzing the reads is available here: https://github.com/mhorlbeck/ScreenProcessing (Link opens in a new window).
- 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 from bioRxiv 2023.01.22.525086, reprinted under the terms of the Creative Commons Attribution License (Link opens in a new window).)
Please visit https://www.biorxiv.org/content/10.1101/2023.01.22.525086v1 (Link opens in a new window) for bioRxiv preprint.
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 Dual Guide libraries were a gift from Rebecca Voorhees (Addgene #) -
For your References section:
A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens. Guna A, Page KR, Replogle JR, Esantsi TK, Wang ML, Weissman JS, Voorhees RM. bioRxiv. 2023 Jan 22:2023.01.22.525086. doi: 10.1101/2023.01.22.525086. Preprint. PubMed 36711738