I. Background Knowledge

CRISPR/Cas9 has become a popular gene-editing tool due to its ease of use and fewer off-target effects compared to other gene regulation tools, such as siRNA technology.

CRISPR/Cas9 knockout screening libraries mainly exist in two forms: pooled libraries and arrayed libraries. CRISPR pooled and arrayed screens are powerful tools in gene research, but they are applied in different contexts due to their unique characteristics [1].

While pooled CRISPR/Cas9 screens are generally higher throughput and less time-consuming, arrayed CRISPR/Cas9 screens offer greater technical flexibility in endpoint functional assays.

 

Figure 1: Differences in the workflow of two different library screens

 

II. Scope of Application

 

	Lentiviral pooled library screening	CRISPR arrayed library screening
Application Use	1. Suitable for phenotypes that can be altered by selective pressure, such as cell viability/proliferation assays or FACS analysis 2. Identification of drug resistance genes 3. Suitable for cells that are difficult to transfect	1. Correlation between gene and phenotype 2. Complex screening analysis, such as morphological analysis using microscopy 3. Suitable for multi-parameter screening experiments
Advantages Advantages	1. Relatively consistent settings, regardless of library size 2. Requires less automation or specialized equipment 3. Requires less experimental manipulation 4. Suitable for custom libraries 5. Suitable for longer time points	1. One gene per well, potential hits are easily identified 2. Can perform very complex readings 3. Multi-parameter readings are possible 4. Suitable for custom libraries 5. Suitable for 3D and co-culture studies 6. Cells do not need to actively divide and grow
Special Requirements Requirements	1. Need to select/isolate cells with specific phenotypes from the population 2. Large starting cell number, screening needs to be maintained in large culture flasks 3. Cells must generally actively divide and grow throughout the screen 4. Requires virus treatment 5. Requires NGS and extensive bioinformatics to find hit genes	1. Cells must be suitable for transfection or electroporation 2. Requires automated liquid handling equipment 3. Requires automated consumables, high cost 4. Potential inter-plate or inter-well variations 5. Requires sufficient cryopreservation space 6. Shorter detection time points

III. Application Cases

 

3.1 Pooled screen

1. Gene function research

Genome-wide CRISPR pooled screens play a key role in gene function screening. By systematically knocking out genes and observing their effects on cell phenotypes, the function of genes and their roles in various biological processes can be revealed.

Researchers have identified a variety of positive regulators that promote T cell function through genome-wide large-scale screening [2]. These genes increase the proliferation and activation of human CD4+ and CD8+ T cells and the secretion of key cytokines, providing new strategies for optimizing and improving T cell therapy.

 

2. Drug target discovery

Genome-wide CRISPR pooled screening can simultaneously knock out tens of thousands of genes, helping to understand drug resistance mechanisms and discover new drug targets, providing new ideas and methods for overcoming drug resistance.

1) Finding drug resistance targets

Researchers have identified genes that cause drug resistance and new drug resistance targets through genome-wide CRISPR/Cas9 knockout screening of two SHP2i-sensitive cell lines [3].

2) Studying drug resistance mechanisms

Through genome-wide screening of liver cancer cells (HepG2) using the CRISPR/Cas9 system [4], the core gene KEAP1 for sorafenib resistance in liver cancer was identified, revealing the regulatory role of the KEAP1-NRF2-FGF21 axis in sorafenib resistance in hepatocellular carcinoma.

3. Studying interactions

Large-scale CRISPR pooled screening can systematically reveal the interactions and functional relationships between genes, expanding the gene interaction map.

1) Gene interactions

Researchers have used combinatorial screening to identify the synthetic lethal interaction of WDR45B-PIK3R4 and the proliferation-enhancing interaction of ATG7-KEAP1 [5].

2) Drug synergy

Studies have found synergistic effects between donafenib and GSK-J4 in hepatocellular carcinoma by combining drug library screening with CRISPR library screening [6]. This study has discovered a new synergistic lethal combination in hepatocellular carcinoma through indiscriminate screening, which plays a positive role in exploring new treatment methods for hepatocellular carcinoma.

Liman Bio provides CRISPR pooled library screening services for the above-mentioned research, assisting in research on gene function, drug resistance, and gene interaction.

3.2 Arrayed screen

Arrayed libraries typically allow for individual screening of each target gene or specific genomic region, enabling the assessment of gene function at high resolution. This single-gene screening approach helps accurately identify the function of specific genes.

1. Genotype-phenotype relationship

In primary human monocyte-derived dendritic cells, an arrayed sgRNA library targeting approximately 300 genes was used to screen for specific regulators of TNF and IL-10 expression under LPS stimulation, revealing the mechanisms of inter-individual variation [7].

 

2. Complex screening analysis

The development of arrayed CRISPR libraries has broadened the utility of reverse genetic screening to phenotypic readouts, including fluorescence/luminescence and image-based methods.

1) Multi-parameter screening

A multi-parameter arrayed in vitro CRISPR screen of protein kinases in activated primary mouse CD8+ T cells revealed [8] that MAPK14 (also known as p38) is a key negative regulator of T cell clonal expansion and CD62L expression, and a positive regulator of reactive oxygen species production and genomic stress.

2) Cell interaction

Arrayed CRISPR screening in primary cDC1s [9] was performed to determine how different gene perturbations affect the antigen-dependent proliferation of CD8 T cells. Transduced cDC1 cells were co-cultured with CD8+ T cells in separate wells to directly measure how each perturbation affects FACS-measured cDC1-dependent CD8 T cell proliferation and activation. Ultimately, WDFY4, which regulates intracellular vesicle transport, was identified as a positive regulator of cDC1 cross-presentation.

Liman Bio provides the CRISPR arrayed library-related services involved in the above research. Our arrayed libraries offer higher accuracy, simplified data interpretation, strong experimental reproducibility, and broad adaptability to research needs.

 

IV. References

[1] Przybyla, L., Gilbert, L.A. A new era in functional genomics screens. Nat Rev Genet ,23, 89-103 (2022).

[2] Legut, M., Gajic, Z.,et al.A genome-scale screen for synthetic drivers of T cell proliferation. Nature 603, 728–735 (2022).

[3] Wei W, Geer MJ,et al. Genome-wide CRISPR/Cas9 screens reveal shared and cell-specific mechanisms of resistance to SHP2 inhibition.J Exp Med. 2023 May 1;220(5):e20221563.

[4] Chen J, Jiang S,et al.CRISPR-Cas9-based genome-wide screening identified novel targets for treating sorafenib-resistant hepatocellular carcinoma: a cross-talk between FGF21 and the NRF2 pathway. Sci China Life Sci. 2022 Oct;65(10):1998-2016.

[5] Diehl V, Wegner M, et al.Minimized combinatorial CRISPR screens identify genetic interactions in autophagy. Nucleic Acids Res. 2021 Jun 4;49(10):5684-5704.

[6] Zheng C, Zhang B,et al. Donafenib and GSK-J4 Synergistically Induce Ferroptosis in Liver Cancer by Upregulating HMOX1 Expression.Adv Sci,2023 Aug;10(22):e2206798.

[7] Jost M, Jacobson AN, et al. CRISPR-based functional genomics in human dendritic cells. Elife. 2021 Apr 27;10:e65856.

[8] Gurusamy D, Henning AN, et al.Multi-phenotype CRISPR-Cas9 Screen Identifies p38 Kinase as a Target for Adoptive Immunotherapies. Cancer Cell. 2020 Jun 8;37(6):818-833.e9.

[9] Gurusamy D, Henning AN, et al.Multi-phenotype CRISPR-Cas9 Screen Identifies p38 Kinase as a Target for Adoptive Immunotherapies. Cancer Cell. 2020 Jun 8;37(6):818-833.e9.