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CHyMErA Cas12a Nuclease Optimization and Large-Scale Exon Deletion hgRNA Libraries
(Pooled Libraries #209734, #209735, #209736)

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  • Purpose

    Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA) lentiviral libraries express hybrid Cas9-Cas12a guide RNAs under a single U6 promoter.

    The CHyMErA Cas12a Nuclease Optimization hybrid guide (hgRNA) libraries allow the user to assess and compare the efficacy of different LbCas12a (#209734) or AsCas12a nucleases (#209735) in the context of the Cas9-Cas12a CHyMErA system (Gonatopoulos-Pournatzis et al., 2020).

    The CHyMErA Large-Scale Exon Deletion hgRNA library (#209736) screens for frame-preserving exons that affect cell fitness in human cells, using the CHyMErA screening platform (Gonatopoulos-Pournatzis et al., 2020) with SpCas9 and AsCas12a nucleases.

  • Vector Backbone

    #209734 and #209735: pLCHKOv2 - does not express Cas9 or Cas12a

    #209736: pLCHKOv3 - does not express Cas9 or Cas12a

Ordering

Item Catalog # Description Quantity Price (USD)
Pooled Library 209734 CHyMErA LbCas12a nuclease optimization hgRNA library† 1 $430 Add to Cart
Pooled Library 209735 CHyMErA AsCas12a nuclease optimization hgRNA library† 1 $430 Add to Cart
Pooled Library 209736 CHyMErA large-scale exon deletion hgRNA library† 1 $850 Add to Cart
Available to Academic and Nonprofits Only

Library Details

Pooled Library Genes Targeted No. gRNAs/element hgRNAs
209734 844 1–10 18,000
209735 844 1–10 18,000
209736 12,126 exons in 2,095 genes 6–18 300,000
  • Species
    Human
  • Controls
    1,503 dual intergenic and 100 non-coding targeting hgRNAs in each library
  • Lentiviral Generation
    3rd

Library Shipping

This library is 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
    50 ng/µL

Resource Information

Depositor Comments

The Cas12a Nuclease Optimization hgRNA libraries can be used to assess dropout and enrichment of guides targeting reference core essential and nonessential genes via single and combinatorial genome editing. Each nuclease optimization hgRNA library consists of 18,000 hgRNAs targeting 482 core essential and 362 nonessential genes through gene knockout (9,129 hgRNAs) or deletion of frame-disruptive exons (3,236 hgRNAs). In addition, the libraries contain controls including intronic-intergenic (4,032 hgRNAs), dual intergenic (1,503 hgRNAs) and non-coding (100 hgRNAs) targeting hgRNAs.

The Large-Scale Exon Deletion hgRNA library aims to delete all targetable frame-preserving exons in DepMap common essential genes (5,333 exons; 85,188 hgRNAs) along with 2,489 frame-preserving exons in additional genes (41,044 hgRNAs). This library also targets 4,304 frame-disruptive exons (i.e., exons whose deletion results in gene inactivation) in common essential and additional genes (42,560 hgRNAs) to evaluate the gene phenotype. The library contains all hgRNAs included in the CHyMErA Cas12a Nuclease Optimization hgRNA libraries to determine the efficacy of Cas9 and Cas12a for gene knockout and exon deletion.

A diagram depicts a cell expressing both Cas12a and Cas9 nucleases. Lines indicate that we are looking at a magnified view of the nucleus. The schematic structure of the hgRNA is shown, consisting of a U6 promoter, Cas9 guide (Cas9 spacer and tracrRNA), and Cas12a guide (direct repeat and Cas12a spacer). An arrow points from the hgRNA to a diagram in which Cas9 hgRNA or Cas12a hgRNA complexes are bound to target DNA. Scissors show cut sites within exons or flanking an exon.

  • Figure 1. CHyMErA system overview. Cells express SpCas9 and either LbCas12a or AsCas12a and an hgRNA fusion of Cas9 and Cas12a gRNAs. Cas12a RNA processing activity generates individual Cas9 and Cas12a gRNAs which bind to their respective nucleases and effect combinatorial targeting.

Deconvolution algorithms for analyzing NGS data (Link opens in a new window) are available at GitHub.

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:

    CHyMErA Cas12a Nuclease Optimization hgRNA Library or Large-Scale Exon Deletion hgRNA Library was a gift from Thomas Gonatopoulos-Pournatzis (Addgene #)

  • For your References section:

    Genome-scale exon perturbation screens uncover exons critical for cell fitness. Xiao M-S, Damodaran AP, Kumari B, Dickson E, Xing K, On TA, Parab N, King HE, Perez AR, Guiblet WM, Duncan G, Che A, Chari R, Andresson T, Vidigal JA, Weatheritt RJ, Aregger M, Gonatopoulos-Pournatzis T. Mol Cell. 2024 June 24. doi: 10.1016/j.molcel.2024.05.024.
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