CRASP-Seq Pooled Libraries
(Pooled Libraries #232068, #232069, #232070, #250494)

• Purpose:

  The CRASP-seq Gene KO v2 (#250494) library enables CRASP-seq experiments by providing a flexible platform for studying splicing regulation. This updated 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 generate a custom CRASP-seq vector. This enables systematic evaluation of how each protein-coding gene influences splicing of the selected reporter, providing a powerful approach to study splicing mechanisms and regulatory factors. The library also supports combinatorial knockouts of paralogous and other gene pairs using Cas9 and Cas12a, and enables targeting of selected non-coding RNAs through promoter deletion. Pooled library #250494 is an updated version of #232069. The depositor recommends using the KO v2 library.

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

  The CRASP-seq CBC Backbone library (#232068) is designed to accommodate cloning of diverse gRNA libraries. Unlike the non-barcoded pLCHKO-CRASP (Addgene plasmid #231928), this backbone includes a 12-nucleotide cell barcode (CBC), enabling library diversification and generation of replicates during library preparation. In contrast to the Gene KO and BE Tiling libraries, the CBC backbone does not include a predefined CRISPR gRNA library. Instead, it serves as a barcoded backbone for constructing custom libraries across a range of CRISPR modalities, including knockout, CRISPRi, CRISPRa, and base editing.

• Vector Backbone:

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

• Depositing Labs: Thomas Gonatopoulos-Pournatzis
• Publication:

  #232068, #232069, #232070:
  Behera et al. Mol Cell. 2026 Jan8;86(1):41-59.e15. doi: 10.1016/j.molcel.2025.12.003.

  #250494:
  RBM39 Modulates UPR Signaling Through Regulation of IRE1α Alternative Splicing (unpublished)

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
250494	22,266*	91,078	504
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

• Protocols:
  • Library Amplification and Sequencing Protocol (DOCX, 21 KB)
• Depositor Data:
  • 250494 List of gRNA Sequences (XLSX, 19 MB)
  • 250494 Empty Vector Map (GB, 23 KB)
  • 250494 Library Distribution (PDF, 763 KB)
  • 232070 List of gRNA Sequences (XLSX, 1.1 MB)
  • 232070 Empty Vector Map (GB, 23 KB)
  • 232068 Empty Backbone Map (GB, 22 KB)

Terms and Licenses

Academic/Nonprofit Terms:

• UBMTA

Industry Terms:

• Not Available to Industry

Trademarks:

• Zeocin® is an InvivoGen trademark.

Depositor Comments

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.

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

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

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.

The CRASP-Seq Gene KO library (#232069) has been discontinuted. If you require additional details for this library, please contact Addgene at .

CRASP-Seq Gene KO v2 library details (#250494)

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

• Genes targeted:
• 19,014 protein coding genes
• 2,560 paralogous gene pairs
• 381 hand-picked gene pairs
• 311 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:
• 412 hgRNAs targeting intergenic regions
• 92 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

Please visit https://doi.org/10.1101/2025.04.25.648738 for bioRxiv preprint.