Single-Cell Sequencing Service | High-Throughput Sequencing

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●  Knockout Vector Construction ●  Point Mutation Vector Construction ●  Knock-In Vector Construction ●  Overexpression Vector Construction

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CRISPR Library Screening

CRISPR Library Screening Service

CRISPR-scRNA-seq integration
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CRISPR Detection Service

crRNA Design & Synthesis

CRISPR Detection Service

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Single-cell sequencing works by conducting high-throughput sequencing on the transcriptome of individual cells to analyze the differences in gene expression between cells. This technology overcomes the limitations of traditional bulk sequencing by providing cell-level resolution, revealing heterogeneity among cells within the same population. Single-cell sequencing holds tremendous potential in multiple fields, such as studying cell differentiation trajectories during development, tumor heterogeneity, immune cell diversity, and gene expression regulatory networks.

Combining single-cell sequencing with CRISPR library screening is an important application. With single-cell resolution, it is possible to directly establish the relationship between gene perturbation, gene expression profiles, and phenotypes by linking sgRNA to transcriptome information.

EDITGENE offers a Chromium-based single-cell CRISPR screening solution from 10x Genomics, providing services such as plasmid library construction, viral library packaging, pooled library cell construction, functional screening, single-cell sequencing, and bioinformatics analysis. Additionally, we provide conventional single-cell sequencing services.

10x Genomics Chromium Single-cell CRISPR Screening Principle (Direct capture Perturb Seq)

Direct capture PerturbSeqDirect Capture PerturbSeq is based on the 5′ and 3′ end library preparation methods developed by 10x Genomics, offering two sgRNA detection strategies. 5′ End Detection Strategy: A reverse transcription primer is designed for the constant sequence following the sgRNA, synthesizing sgRNA-cDNA during reverse transcription. The sgRNA-cDNA is then combined with other mRNA-cDNA molecules to bind to the TSO sequence on gel beads, linking with the cell barcode (CBC) and unique molecular identifiers (UMI) for library preparation and sequencing. 3′ End Detection Strategy: Gel beads are pre-loaded with CBC, UMI, and reverse transcription primers targeting a specific sequence in the constant region. During reverse transcription, sgRNA-cDNA is synthesized, tagged with the TSO sequence, and processed with other mRNA-cDNA molecules for library preparation and sequencing.

Replogle, Joseph M et al. “Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing.” Nature biotechnology vol. 38,8 (2020): 954-961

Advantages of 10x Genomics Chromium Single-cell CRISPR Screening

1. Single-cell resolution detection: Establishes a direct link between sgRNA, gene expression profiles, and phenotypes at the single-cell level.

2. Higher validation efficiency: Compared to traditional CRISPR screening techniques or single-gene edited cell models, it offers greater efficiency in verifying gene functions.

3. Mature Direct Capture PerturbSeq platform: The library preparation and sequencing strategy of Direct Capture PerturbSeq is well-established and yields more accurate experimental results. Additionally, by using this strategy, a single lentiviral vector can carry multiple U6-sgRNA expression cassettes, significantly enhancing the efficiency of gene perturbation.