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CRISPR Screen library screening

An important technological platform for high-throughput gene functional studies and the discovery of novel drug targets.

Efficient and precise

High-throughput sequencing/bioinformatics analysis

Multi-level verification

In Vivo Screening / In Vitro Screening / In Silico Library Screening

Widely applicable

Disease mechanisms / drug targets / signaling pathways
Analysis

Data reliability

High-throughput sequencing/bioinformatics analysis

Technical Overview

CRISPR‑screen library screening is currently a key technological platform for high‑throughput studies of gene function and the discovery of novel drug targets. By constructing CRISPR libraries containing large numbers of sgRNAs, researchers can systematically perturb gene functions across the entire genome or within specific functional gene sets. Combined with high‑throughput sequencing and bioinformatics analysis, this approach enables the identification of critical regulatory factors that influence disease phenotypes, cellular states, and drug responses.

Aniphe BioLab ConstructionIn silico virtual prediction, in vitro screening, and in vivo validationThe Trinity CRISPR Screen technology platform enables multi‑level, multidimensional dissection of gene functions, spanning computational‑assisted design, high‑throughput screening at the cellular level, and functional validation in animal disease models.

The platform supports the identification of disease‑related key genes, elucidation of drug mechanisms of action, screening of therapeutic targets, and functional validation, thereby accelerating the translation of basic research findings into drug development.

Principles of the CRISPR-Cas9 Core Editing Technology

The CRISPR‑Cas9 system recognizes the target genomic sequence via an artificially designed single guide RNA (sgRNA) and directs the Cas9 protein to precisely bind to the target DNA region. Following Cas9‑induced double‑strand DNA breaks, cellular DNA repair mechanisms mediate gene editing, resulting in functional modifications of the target gene. Depending on experimental requirements, the following outcomes can be achieved:

Gene knockout
(CRISPR Knock-out)

The CRISPR-Cas9 system is used to disrupt the coding region of a target gene, thereby abolishing its original function.

Applicable to:

  • Gene Function Research
  • Disease-Driven Gene Screening
  • Discovery of Drug Sensitivity-Related Genes

Gene knock-in
(CRISPR Knock-in)

By homologous recombination or site-specific editing, exogenous genes, disease‑associated mutations, fluorescent tags, or functional tags can be inserted at specific loci in the genome.

Applicable to:

  • Disease Model Construction
  • Protein Localization Tracking
  • Gene function validation

Gene Suppression and Activation
CRISPRi/CRISPRa)

Using the deactivated Cas9 (dCas9) system, target gene expression can be regulated without cleaving DNA.

Applicable to:

  • Research on Gene Expression Regulation
  • Gene Dosage Effect Analysis
  • Analysis of Complex Regulatory Networks

Multiplex Editing

By simultaneously introducing multiple sgRNAs, simultaneous editing of multiple genes can be achieved.

Applicable to:

  • Gene family research;
  • Signal pathway analysis;
  • Study of polygenic interactions.

In Vivo Screen

Disease-Model–Driven In Vivo Functional Screening

In Vivo Screen integrates CRISPR library‑based screening with animal disease models to investigate gene function in physiological and pathological contexts that more closely mimic the course of disease. Compared with in vitro screening, in vivo screening accounts for immune cell–cell interactions, tissue microenvironments, angiogenesis, pharmacological treatment pressures, and intercellular competition, thereby identifying key regulatory factors that influence disease progression and therapeutic response.

Technological Advantages

Higher conversion value

Assessing the impact of gene function in a complex in vivo environment

More comprehensive mechanism analysis

It is possible to identify regulatory relationships that are difficult to model in vitro.

Higher conversion value

It helps to screen candidate targets with therapeutic potential for diseases.

Main applications

Related to tumor initiation and progression
Gene screening

Tumor immune evasion
Mechanism Study

Related to immunotherapy response
Target discovery

Drug tolerance and combination therapy
Therapeutic target screening

IV. In Vitro Screen

High-throughput gene function screening at the cellular level

In vitro screening is primarily conducted using cell culture systems. By introducing an sgRNA library into a population of cells stably expressing Cas9, researchers can simultaneously perturb a large number of genes and, through phenotypic screening combined with NGS-based sequencing analysis, identify key regulatory genes that influence cellular functions. This approach offers advantages such as well-controlled experimental conditions, short screening timelines, and high throughput, making it an important technical strategy for studying gene function and for the initial identification of drug targets.

Technological Advantages

High-throughput screening

Supports whole-genome or custom sub-library screening.

Controllable conditions

Facilitates the study of specific stimuli, pharmacological challenges, or changes in cellular states.

Relatively efficient

Candidate regulatory genes can be rapidly identified, providing guidance for subsequent validation studies.

Main applications

Screening of Genes Associated with Tumor Cell Proliferation and Survival

Drug Sensitivity and Resistance
Mechanism Study

Functional Analysis of Cellular Signaling Pathways

Screening for genes associated with specific phenotypes

V. In Silico Library Screening

Computational-Assisted Library Design and Target Prediction

In silico library screening leverages bioinformatics, multi-omics data analysis, and computational predictions to optimize candidate genes, sgRNA design, and screening strategies prior to experimental implementation. By integrating public databases, multi-omics datasets, and predictive models, it enables early assessment of candidate target relevance, refinement of library design, and enhanced efficiency in subsequent experimental screening.

Technological Advantages

Optimize the screening strategy

Pre-screen high-value candidate genes

Improve editing efficiency

Optimize sgRNA design through algorithms to reduce the risk of off-target effects.

Reduce experimental costs

Supports customized Yaku designs to reduce ineffective screening.

Main applications

CRISPR Library Design Optimization

Candidate Target Prioritization

Multi-omics–Integrated Target Discovery

Multi-omics–Integrated Target Discovery

VI. Advantages of the Ailinfei CRISPRScreen Platform

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VII. Standard CRISPR Screen Protocol

CRISPR Screen enables systematic functional genomic screening through genome-wide perturbations and high-throughput sequencing analysis.

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VIII. Technical Application Scenarios

Main applications

Screening of Genes Associated with Tumor Initiation, Progression, and Metastasis

Novel anti-tumor drug
Target discovery

Synthetic Lethality Screening

Research on the Mechanisms Underlying Tumor Immunotherapy

Analysis of Drug Resistance Mechanisms

Research on Genes Associated with Neurodegenerative Diseases

Viral infection of the host
Factor screening

Signaling pathways and regulation
Network Resolution

IX. Literature and Case Studies

In vivo epigenetic CRISPR screen identifies Asf1a as an immunotherapeutic target in Kras-mutant lung adenocarcinoma

Journal published in: Cancer Discovery (IF = 29.7)

Research Design

The research team constructed a custom CRISPR library targeting 524 epigenetic regulatory factors and performed in vivo screening in a KRAS-mutant lung adenocarcinoma animal model.

Key Findings

Asf1a is an important negative regulator that promotes tumor immune evasion.

Asf1a deficiency enhances the polarization of inflammatory M1-type macrophages.

Combination anti-PD-1 therapy can significantly enhance antitumor efficacy.

The complete response rate in the combination therapy group increased from 10% to 60%.

Significance of the Study

The in vivo CRISPR screen can identify therapeutic targets that are difficult to capture through conventional in vitro screening, even within the complex tumor immune microenvironment, thereby providing critical evidence for the development of precision immunotherapy strategies.

X. Frequently Asked Questions (FAQ)

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Q1: How does the Tau P301S mouse differ from other tau models, such as Tg4510?

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A: The P301S (PS19) driver is Thy1, with tau expression more concentrated in the forebrain. This model exhibits a milder onset but rapid progression, and its motor phenotype progresses slightly more slowly than that of Tg4510. Tg4510 is driven by CaMKIIα-tTA, allowing for reversible expression; however, it suffers from transgene‑induced disruption of the Fgf14 gene. Consequently, P301S offers greater advantages for studying tau aggregation and propagation in the absence of such interference.
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Q2: Why is it recommended to use a wild-type from the same litter as the control?

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A: Breeding hemizygous transgenic mice yields both positive and wild-type littermates that share the same genetic background and maternal environment, thereby minimizing batch effects, cage‑site effects, and microecological differences, and enhancing the statistical power of experiments.
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Q3: Can this strain be used in tau propagation/inoculation experiments?

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A: Yes. Injection of human brain homogenates from Alzheimer’s disease, chronic traumatic encephalopathy, or progressive supranuclear palsy, or of recombinant tau fibrils, into the hippocampus or striatum of P301S mice induces accelerated propagation of tau pathology both at the injection site and in distal connected regions, making this a classic model for studying prion-like transmission and strain-specific properties of tau.
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Q4: How can phenotypic variability among mice be controlled?

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A: For each experimental cohort, use individuals of the same sex and born around the same time whenever possible, and prior to the start of the experiment, screen out and exclude animals with clearly impaired motor function based on baseline behavioral assessments. The sample size should generally be ≥12–15 animals per group (accounting for a post‑hoc coefficient of variation in pathology of approximately 20–30%). The experimental report must specify the level of transgene expression (if required, brain‑derived human tau levels may be measured by ELISA or Western blot) as a covariate for post‑hoc analyses.
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Q1: At what age do SD senescent mice begin to be considered elderly?

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A: In experimental studies, SD rats are typically classified as aged at 18 months or older, in the mid-to-late stages of aging between 21 and 24 months, and as very old beyond 24 months. The temporal sequence of aging varies across different tissues and organs; the specific age range should be aligned with the study’s primary endpoint.
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Q2: Which types of research are SD senescent mice best suited for? How should one choose between senescent mice and genetically modified progeria models?

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A: The most suitable models for studying normal physiological aging and age‑related chronic progressive diseases—such as age‑associated cognitive decline, sarcopenia, and metabolic syndrome—are those that closely mimic these conditions. If the goal is to target specific molecular pathways—e.g., telomere dysfunction or DNA repair defects—then corresponding genetically engineered progeroid models are more appropriate. However, when evaluating the ultimate efficacy of anti‑aging therapeutics, the spontaneously aging SD model remains the indispensable gold standard.