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

• 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

• Depositing Labs: Thomas Gonatopoulos-Pournatzis
• Publication: Xiao et al. Mol Cell. 2024 June 24. doi: 10.1016/j.molcel.2024.05.024.

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

• Protocols:
  • Library Transformation and NGS Protocols (DOCX, 22 KB)
• Depositor Data:
  • Distribution of NGS Reads:
    • LbCas12a Nuclease Optimization Library (PDF, 149 KB)
    • AsCas12a Nuclease Optimization Library (PDF, 126 KB)
    • Exon Deletion Library (PDF, 124 KB)
  • hgRNA sequences:
    • LbCas12a and AsCas12a Nuclease Optimization hgRNA sequences (XLSX, 3.1 MB)
    • Large-Scale Exon Deletion hgRNA sequences (XLSB, 25.2 MB)

Terms and Licenses

Academic/Nonprofit Terms:

• UBMTA

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.

• 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 are available at GitHub.