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CRASP-Seq Pooled Libraries
(Pooled Libraries #232068, #232069, #232070)

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  • Purpose

    The CRASP-Seq Gene KO library (#232069) enables CRASP-Seq experiments by providing a flexible platform for studying splicing regulation. This library leverages the CHyMErA platform, which is based on dual Cas9 and Cas12a nuclease expression alongside Cas9-Cas12a hybrid gRNAs (hgRNAs) expressed under a single U6 promoter. Researchers can clone their minigene reporter of interest into this genome-wide knockout library to create a custom CRASP-Seq vector. This allows the systematic evaluation of how each protein-coding gene impacts the splicing of the selected reporter, offering a powerful tool for understanding splicing mechanisms and regulatory factors for the splicing event of interest. In this library, Cas9-Cas12a hybrid gRNAs (hgRNAs) are expressed under a single promoter. The library also enables combinatorial knockouts of paralogous and other gene pairs using Cas9 and Cas12a, while additionally targeting selected non-coding RNAs by deleting their promoters.

    The CRASP-Seq BE Tiling library (#232070) includes tiling Cas9 guide RNAs (gRNAs) designed to target 39 splicing-related genes. It is designed for conducting CRASP-Seq experiments combined with high-throughput base editing mutagenesis using Cas9 base editors, enabling precise and scalable investigations into protein regions that affect splicing regulation.

    The CRASP-Seq CBC Backbone library (#232068) is designed to accommodate the cloning of diverse gRNA libraries. Unlike the non-barcoded pLCHKO-CRASP (Addgene plasmid #231928) backbone, the CRASP-Seq CBC backbone, this library includes a 12-nucleotide cell barcode (CBC), which enhances its functionality by enabling library diversification and the generation of replicates during library preparation. Unlike the CRASP-Seq Gene KO (#232069) and BE Tiling (#232070) libraries, this vector does not express a predefined CRISPR gRNA library. Instead, it serves as a barcoded backbone for cloning custom libraries tailored to any CRASP-Seq modulation approach. This includes various CRISPR screening strategies such as knockout, CRISPRi, CRISPRa, and base editing, allowing users to construct and implement the appropriate library for their specific experimental needs.

  • Vector Backbone

    CRASP-Seq CBC Backbone library (Pooled Library #232068) (GB, 22 KB) - does not express Cas9 or Cas12a

Ordering

Item Catalog # Description Quantity Price (USD)
Pooled Library 232069 CRASP-Seq Gene KO library† 1 Pending
Pooled Library 232070 CRASP-Seq BE Tiling library† 1 Pending
Pooled Library 232068 CRASP-Seq CBC Backbone library‡ 1 Pending
Available to Academic and Nonprofits Only

  • † A Cas9 plasmid is NOT included with this item and will have to be ordered separately. #232069 can be used in conjunction with the CHyMErA system and requires BOTH Lenti‐Cas9‐2A‐Blast (Addgene #73310) and pLenti-opCas12a-6xNLS (Addgene #209022). #232070 can be used in conjunction with pLenti-nSpCas9-TadA8e (Addgene #209044) or pLenti-nSpCas9-evoCDA1 (Addgene #209042).

  • ‡ Please note that this is not gRNA-containing library and does not require an additional Cas9 plasmid. It is a barcoded backbone intended for downstream cloning.

Library Details

  • CBC backbone barcode
    12 nucleotide barcode; > 107 theoretical diversity
Pooled Library Genes Targeted Targeting gRNAs Control gRNAs
232069 22,987* 95,463 430
232070 39* 6,984 1,610
  • *See Depositor Comments for additional details
  • Species
    Human
  • Lentiviral generation
    3rd

Library Shipping

Each library is delivered 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

CRASP-Seq Gene KO library details (#232069)

Note: hgRNAs are Cas9-Cas12a hybrid gRNAs expressed under a single promoter

  • Genes targeted:
    • 19,628 protein coding genes
    • 2,415 paralogous gene pairs
    • 625 hand-picked gene pairs
    • 319 non-coding RNAs
  • gRNAs per element:
    • 4 hgRNAs per protein-coding gene
    • 4 hgRNAs for each gene pair
    • Varying number of hgRNAs targeting non-coding RNAs
  • Control gRNAs:
    • 331 hgRNAs targeting intergenic regions
    • 99 non-targeting hgRNAs

CRASP-Seq BE Tiling library details (#232070)

Note: gRNAs are for Cas9

  • Genes targeted:
    • 39 protein coding genes
  • gRNAs per element:
    • Varying number of gRNAs targeting each gene (tiling library)
  • Control gRNAs:
    • 1,520 gRNAs targeting intergenic regions
    • 90 non-targeting gRNAs

A non-barcoded version of the CRASP-Seq CBC Backbone library is available at Addgene, as pLCHKO-CRASP (Addgene plasmid #231928). Please note that the CRASP-Seq libraries all use the barcoded version as a backbone.

CRASP-Seq (CRISPR-based identification of Alternative Splicing Regulators with Phenotypic Sequencing) is a platform that integrates genome-wide genetic perturbations with deep sequencing of splicing reporters. This approach enables a comprehensive and quantitative assessment of how every protein-coding human gene influences alternative splicing events of interest. By linking CRISPR-induced gene perturbations to quantifiable splicing phenotypes using high-throughput RNA sequencing, CRASP-Seq allows precise measurement of each perturbation’s impact on splicing outcomes. The method is highly versatile, enabling the investigation of diverse alternative splicing events—including cassette exons, mutually exclusive exons, and alternative 5′ and 3′ splice sites—under various CRISPR-based perturbations, such as gene knockouts, base editing, CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa). CRASP-Seq provides an efficient and scalable framework for uncovering splicing regulators, making it a valuable tool for functional genomics and RNA biology research.

Vector schematic shows 5 prime and 3 prime LTRs and BGH. The hybrid guide RNA is composed of a Cas9 guide (Cas9 spacer and tracrRNA) and Cas12a guide (Cas12a direct repeat and Cas12a spacer) under a U6 promoter, followed by the CBC barcode. In the opposite orientation is a DOX-inducible TRE promoter for the splicing reporter (exon and alt exon and exon). In that same orientation is puromycin resistance, 2A, and rtTA (for dox inducible system) under a PGK promoter. Vector is then spliced at splicing reporter and sequenced in two orientations.

Figure 1. CRASP-Seq Gene KO library schematic of the CRASP-Seq vector and sequencing strategy.

A Cas9 and Cas12a protein are shown bound to a stretch of DNA using respective guide RNAs. Where proteins are bound, the DNA is unwound, and scissors indicate that cuts will be made to DNA. Combinatorial editing is used for dual-targeting of protein-coding genes, targeting of paralog pairs, and gene segment deletion.

Figure 2. Schematic of combinatorial genome editing using Cas9 and Cas12a.

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:

    The CRASP-Seq CBC Backbone library was a gift from Thomas Gonatopoulos-Pournatzis (Addgene #232068 ; http://n2t.net/addgene:232068 ; RRID:Addgene_232068)
    The CRASP-Seq Gene KO library was a gift from Thomas Gonatopoulos-Pournatzis (Addgene #232069 ; http://n2t.net/addgene:232069 ; RRID:Addgene_232069)
    The CRASP-Seq BE Tiling library was a gift from Thomas Gonatopoulos-Pournatzis (Addgene #232070 ; http://n2t.net/addgene:232070 ; RRID:Addgene_232070)
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