All
  • All
  • Insight
  • Research Services
  • Preclinical CRO
  • Animal Resource Bank
CN
中文 English
Latest News

Latest News

Focusing on cutting-edge technologies and research advancements,
sharing Aniphe BioLab’s achievements in scientific innovation, Professional insights
and practical experience in the fields of laboratory animals and preclinical research.

J Exp Med (II): A Bulk CRISPR Screening Platform—Functional Dissection from T Cells to the Entire Immune System

This article focuses on Bulk CRISPR Recent advances in screening, including novel editing tools, in vivo screening strategies, and in… T Expansion of applications to immune cell types outside the cell.

 

Before delving into the technical details, let’s first clarify. Bulk The meaning. Bulk CRISPR Screening refers to: selecting those that carry different gRNA Cells are mixed and cultured or subjected to in vivo experiments, and functional genes are identified through population-level phenotypic selection—such as cell proliferation, survival, and the expression of surface markers.

 

Compared with single-cell screening, Bulk The advantages of screening are:

  • High throughput: capable of covering the entire genome ( ~20,000 one gene);
  • Low cost: No need for expensive single-cell sequencing;
  • Technical maturity: The experimental procedures are relatively standardized.

 

Its core limitation is that it can only capture the population‑level average effect and cannot distinguish differential responses among cellular subpopulations. Nevertheless, this does not preclude… Bulk Screening is a powerful first step in identifying candidate genes. New type CRISPR tool


CRISPRi/a: From Knockout to Precise Regulation

Tradition CRISPR KO By inducing double-strand breaks to disrupt gene function, whereas CRISPRi (Interference) and CRISPRa (Activated) Used for catalytic deactivation dCas9 , without cutting DNA Precise regulation of gene expression under such conditions. This regulatory mechanism is of great significance in the study of immune cells:

 

01、 It is possible to study essential genes ( Knockout It can lead to cell death);

02, It can simulate the loss of certain functions (more closely mimicking the effects of pharmacological inhibition);

03、 It can be observed that positive regulatory factors ( CRISPRa Gain function filtering).

 

Key Breakthrough: Schmidt Wait for people ( 2022 ) By optimizing the high-titer lentivirus production protocol, we successfully achieved in primary human cells… T It has been achieved in cells. CRISPRi/a Screening identified regulatory factors governing cytokine production. Another study employed a more compact approach derived from Staphylococcus aureus. dCas9 Variant ( SadCas9 ), can be AAV Packaging and delivery, overcoming the source of Streptococcus pyogenes dCas9 Restrictions on excessive volume ( McCutcheon Wait, 2023 ).

 

In addition, catalytic deactivation gRNA ( dgRNA ) is used for Cas9 Expressive T In cells, high-throughput functional screening for gain-of-function was performed, leading to the identification of enhancers. T Factors of cellular effector functions ( Ye Wait, 2022 ).

 


ORF Overexpression and Site-Specific Knock-in Library

except CRISPRa In addition, large-scale open reading frames ( ORF ) Library and modular knock-in ( AI ) The document library provides a new dimension for feature selection:

 

| ORF Representative findings from overexpression screening:

Legut Wait ( 2022 ) in T Overexpression in cells by approximately 12,000 A barcoded human being ORF , unexpectedly discovered lymphotactin -β Receptor ( LTβR ). A receptor that is typically absent in lymphocytes —— Can provide powerful drive T Cellular transcription and epigenomic remodeling enhance effector functions.

 

｜Modularization AI Representative findings from the screening:

Blaeschke Wait ( 2023 ) Developed modular pooling AI Filtering platform, filtering simultaneously 100 a transcription factor and 129 a surface receptor, in clinically relevant TCR/CAR Combination screening of genetic loci was performed. It was found that… TFAP4 and BATF The co‑knock-in can significantly enhance T Cellular adaptability.

 

Key insight: Gain-of-function screening and loss-of-function screening are complementary; the former is better suited for identifying driver factors, while the latter is more effective for uncovering brake factors. Only by combining both approaches can we achieve a comprehensive understanding of immune regulatory networks.

 


Base Editing and Prime Editing: Precision Mutation Scanning

Base editing ( Base Editing ) It enables precise single‑base substitutions without inducing double‑strand breaks, which offers significant safety advantages in clinical applications:

 

01、 In people T In cells, base editing enables the simultaneous deletion of multiple genes while minimizing the risk of chromosomal translocations. Diorio Wait, 2022 )

02, Schmidt Wait ( 2024 ) to 385 Base‑editing screening of the protein‑coding regions of a gene revealed regulatory… T Specific protein domains and amino acid residues involved in cell activation and cytokine production

 

Prime editing ( Prime Editing ) provides more precise editing capabilities: Yan Wait ( 2024 ) will edit the leader protein PE7 and RNA Binding protein The of the RNA Domain fusion significantly improves human… T The efficiency of prime editing in cells.

 

These precise editing tools are laying the groundwork for in vivo functional screening, such as introducing somatic mutations in mouse models ( Katti Wait, 2024； Ely Wait, 2024 ).


RNA Editing: A New Dimension of Cas13d

and DNA Targeted CRISPR Different, Cas13d Targeted RNA . Although there are reports T Cells in recognition Cas13d Subsequently, pro-inflammatory cytokines will be produced ( Tang Wait, 2022 ), but achieving transcriptomic perturbation without altering the genome still offers significant advantages:

01、 None DNA Fracture, theoretically safer;

02, Tieu Wait ( 2024 ) Utilizing multiple effects gRNA Array screening platform ( MEGA ), through CRISPR/Cas13d Discovered T A paired regulatory factor of cellular functions.   In the body CRISPR Screening: Release of physiological relevance

 

The maturation of the aforementioned novel bottom-up editing tools has not only broadened the scope of in vitro mechanistic studies but has also laid the technical groundwork for advancing toward a more physiologically relevant in vivo environment.

 

The results of in vitro screening often cannot be directly translated into in vivo biology, because the complexity of the in vivo microenvironment—such as immune cell–cell interactions, tissue‑specific signaling, and metabolic conditions—can profoundly influence gene function. In vivo CRISPR Thus, screening has become a critical bridge linking basic discoveries to clinical applications.

 


Challenges of In Vivo Screening

The core challenges facing in vivo screening are:

 


Successful cases of in vivo T-cell screening

Cas9 Knock-in mouse development ( Chu Wait, 2016； Platt Wait, 2014 ) is a critical infrastructure for in vivo screening. Combined with adoptive transfer models (which introduce antigen-specific… T The cells were transferred into recipient mice and then subjected to antigenic stimulation to induce robust expansion; researchers have already… CD8⁺ T Systematic identification in cells revealed:

Tumor infiltration Factors regulating T-cell accumulation and antitumor activity (Baxter et al., 2023; Belk et al., 2022; Chen et al., 2021; Dong et al., 2019, among others)
CD4⁺ T cell differentiation regulatory factors (Fu et al., 2021; Huang et al., 2022; Sugiura et al., 2022)
T-cell migration regulatory factor (Kendirli et al., 2023)

Latest update: Liu Wait ( 2025 , preprint) in primary human cells T The entire genome has been realized within the cell. CRISPR Screening, by using overexpression CD3 scFv tumor cells to promote humans T Efficient expansion and recovery of cells in immunodeficient mice enabled genome-wide screening with high cellular coverage.


Surpass T cells: NK cells, dendritic cells, and macrophages

      In the body CRISPR Screening is expanding to other immune cell types:

| NK Cell:

Genome-scale in vitro CRISPR Screening has been performed in primary human cells. NK Targets that enhance anti-tumor efficacy have been identified in cells ( Biederstadt Wait, 2025 ), In the body CRISPR Screening and discovery CALHM2 It is regulation. CAR-NK Key genetic checkpoints of cellular antitumor activity ( Peng Wait, 2024a ).

｜Dendritic cells:

Whole-genome in vivo CRISPR Screening (bone marrow-derived) DC (Transferred into the tumor) revealed that PDE5 It is regulation. DC Key regulatory factors that mediate migration and enhance anti-tumor functions ( Tang Wait, 2025a ).

｜Macrophages:

In vivo targeting of cytokine signaling pathways CRISPR Screening identified genes that regulate macrophage migration and polarization in a mouse model of multiple sclerosis. of the Rose Wait, 2026 ).


Direct in vivo delivery: CHIME and iMAP systems

For immune cell types that are difficult to manipulate ex vivo, direct in vivo CRISPR Delivery offers new avenues:

| CHIME (Chimeric Immune Editing) System:

  • Use Cas9 Expression of mouse bone marrow hematopoietic stem cells, enabling genetic deletion across both innate and adaptive immune cell lineages via bone marrow transplantation;
  • Tumor infiltration has been identified. Treg The cell’s master regulatory factor ( Obradovic Wait, 2023 ).

| X-CHIME Extended Platform:

  • L-CHIME : Lineage-specific gene deletion;
  • I-CHIME : Inducible temporal gene perturbation;
  • Further expanded the original CHIME Scope of application of the strategy ( LaFleur Wait, 2024 ).

| iMAP (Inducible Chimeric Animal Perturbation) System:

  • Simplified gRNA Preparation and delivery steps;
  • Allows the simultaneous evaluation of multiple genetic perturbations under steady-state conditions ( Liu Wait, 2022 ). 03  The biological significance of the key findings

Through these Bulk CRISPR Through screening, researchers have amassed a wealth of important findings on immune regulation:

 

01 T Cell Activation and Function

  • RASA2 : Negative regulation T Cell proliferation; its absence enhances antigen sensitivity and long-term functionality ( Carnival Wait, 2022 );
  • CD5、SOCS1： T Negative regulators of cell proliferation; targeting these genes can enhance effector functions.
  • TFAP4+BATF Combination: Co‑integration significantly enhances T Cellular adaptability.

02 Metabolic and Nutritional Signaling

Multiple in vivo screens have revealed that mTORC1 Signal, lysosomal, and mitochondrial nodes in T A central role in cell fate determination ( Long Wait, 2021； Huang Wait, 2021； Raynor Wait, 2024 ).

 

03 Epigenetic Regulation

  • SWI/SNF Chromatin remodeling complex ( BAF and PBAF ) Differential regulation of exhaustion CD8⁺ T Epigenetic reprogramming of cells ( Baxter Wait, 2023 );
  • cBAF Complex components and MYC In CD8⁺ T Early cooperative actions in cell fate determination ( Guo Wait, 2022 ). Practical Guidelines for Surgical Selection

 

Faced with such diversity CRISPR Tools: How to choose the method best suited to your research question?

① Clearly define the research objectives: Filter / Verification / Mechanism / Treatment ?

② Consider the gene type: coding gene / Non-coding RNA/ Splicing variant ?

③ Evaluation experimental system :   In vitro / Organoid / Animal / Clinical samples ?

 

Choose the most suitable one. CRISPR A suite of tools to enhance research efficiency and biological reliability. !

Over several years of development, Bulk CRISPR Screening has evolved from a single… Knockout Screening has revealed profound shifts in multimodal perturbations. Novel editing tools (base editing, prime editing, RNA Editor), gain function filtering strategy ( CRISPRa 、 ORF Library, AI library), as well as an in vivo screening system ( CHIME 、 iMAP The emergence of such technologies has enabled researchers to dissect immune regulatory networks with unprecedented precision and breadth.

 

However, Bulk The inherent limitations of screening methods in distinguishing differential responses among cellular subpopulations have prompted researchers to develop more robust single-cell approaches. CRISPR Screening technology. This will be the topic of our next article.

 

PREVIOUS:

An efficient IVF‑ET assisted reproductive technology platform, enabling rapid colony expansion in just 45 days and fully meeting the needs of research laboratories for mice of all strains.

NEXT:

[Guide to Avoiding Common Pitfalls] Always running into trouble with mouse genotyping? Read this guide and master mouse genotyping with ease!