Single-cell CRISPR (scCRISPR) screens have quickly become a game-changer in genomics. scCRISPR screening enables researchers to delve deeply into the functional consequences of gene perturbations at the single-cell level. While single-cell RNA sequencing (scRNA-seq) has long been the gold standard for capturing transcriptomic data from individual cells, it can be limiting when faced with the challenge of targeting thousands of genes. Constructing scRNA libraries for each knockout becomes increasingly complex and resource-intensive.
This is where Perturb-seq (Dixit et al., 2016) steps in. Perturb-seq offers a scalable and cost-effective solution for high-throughput genetic screens, providing a comprehensive view of gene function across diverse cell populations.
This blog will discuss the basics, different types of scCRISPR screens, and key factors to consider when designing them. If you are interested in how our services can help your CRISPR experiments, contact our sales team for further information.
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Single-cell CRISPR Fundamentals
CRISPR/Cas9 system
CRISPR/Cas9 system CRISPR is a powerful tool that allows one to perturb a target region of the genome readily and easily. At the heart of this technology is Cas9, an endonuclease enzyme that can be directed to a specific gene by a single guide RNA (sgRNA).
The sgRNA is a crucial component in the CRISPR/Cas9 system. sgRNA can be designed for any genomic region, provided that a short, three-base pair Protospacer Adjacent Motif (PAM) sequence is located adjacent to the target DNA sequence. The PAM sequence is essential because it is recognized by Cas9, guiding the enzyme to the correct location on the DNA strand.
Researchers can implement the CRISPR/Cas9 system using either a single-vector or a two-vector approach. In a single-vector system, the same plasmid expresses both the Cas9 enzyme and the sgRNA. This method simplifies the experimental setup but can pose challenges in achieving long-term stability.
Alternatively, the two-vector system involves using separate plasmids for the Cas9 enzyme and the sgRNA. This method is preferred because it allows researchers to create a stable cell line. This cell line expresses Cas9 without needing sgRNA expression. Separating these components helps maintain a consistent Cas9 enzyme. This allows for changes to the sgRNA to target different genomic areas or adjust experimental conditions.
Bulk CRISPR screen
A traditional CRISPR screen, often referred to as a bulk CRISPR screen, allows researchers to assess thousands of targets simultaneously. This works by introducing a library of sgRNA to your cell line expressing the Cas9 system. Each sgRNA in the library targets a different gene, creating a diverse pool of gene knockouts within the cell population.
After introducing the sgRNA library and allowing time for the CRISPR-Cas9 system to act, the cells are subjected to a particular condition or treatment. Subsequently, the sample is collected, and the sgRNAs present in the cell population are sequenced. This sequencing step is crucial as it helps determine which sgRNAs are still abundant and which have diminished in number.
Interpreting sgRNA Abundance:
sgRNA Drop-off: If a sgRNA that targets a specific gene is significantly reduced or absent after treatment, it suggests that the knockout of this gene has a negative effect on cell survival or fitness. In other words, the targeted gene is essential for the cells’ ability to thrive under the given conditions
More sgRNA: If a sgRNA targeting a gene increases after treatment, it may mean the gene is helpful for the cell in the experiment. If the gene targeted by the sgRNA is an oncogene, knocking it out could slow down or stop the cell's growth. This could lead to more cells with the sgRNA targeting this gene.
Single-cell CRISPR screen
Screening with single-cell technology leverages CRISPR screening to its highest potential by adding a transcriptomic readout. Through single-cell sequencing, we can directly detect both the transcriptome and the sgRNA present in each cell. Thus, each cell tells us how a given perturbation affects gene expression on a transcriptome-wide level. One sample can allow you to interrogate dozens to hundreds of genes simultaneously, dramatically increasing throughput.
CRISPR screening at scale
Learn about our 1 million-cell whole-genome CRISPR screen, conducted in collaboration with Illumina and Scale Biosciences. We discussed how Scale Biosciences' kits were instrumental in this project and how we can support your research.
Types of single-cell CRISPR screens
CRISPR
The traditional screen utilizes the canonical function of Cas9 to knock out a target gene. Cas9 is recruited to a gene body, and, through its endonuclease activity, the DNA is cut. Researchers prefer this method when knockout is necessary and typically use it in bulk screens.
DNA repair mechanisms can make this method less effective. It does not guarantee that an indel or larger deletion will successfully disable the gene. Examples for knockout based CRISPR screens include:
- Target transcription factors in dendritic cells exposed to lipopolysaccharide (Dixit et al., 2016).
- In vivo for cortical assessment (Zheng et al., 2023)
CRISPR interfernce (CRISPRi)
Traditional CRISPR has limitations because it is unreliable for targeting regulatory elements such as enhancers. These non-coding elements primarily function as transcription factor binding sites. As a result, cleavage may not guarantee prevention of factor binding or may not significantly affect the element's function. In these cases, researchers prefer CRISPRi.
This utilizes a catalytically dead Cas9 (dCas9) that is fused to a chromatin modifier. The Kruppel-associated-box (KRAB) domain is a popular fusion protein partner which results in broadly repression of the target region. While this only induces a knockdown, it is a reliable method that works well across promoters and enhancers. Examples include:
- Targeting cardiac enhancers in stem cell to cardiomyocyte differentiation (Armendariz et al., 2023)
- Optimization of direct sgRNA detection (Replogle et al., 2020)
CRISPR activation (CRISPRa)
While the previous two examples have shown the power of CRISPR for repression, activation is also possible. CRISPRa is similar to CRISPRi. It uses a dCas9 protein fused to a transcriptional activator domain. This helps recruit other factors or makes the target site more accessible.
Popular modifiers include VP64, VPR, and p300. Activation works best at promoter elements, where a significantly higher effect size can be observed, while functional at enhancers. Example includes:
- Activating enhancers in differentiated neurons (Chardon et al., 2024)
Beyond
Ultimately, the true advantage of Cas9 is not in its cleavage ability. The CRISPR system allows one to recruit any factor to a given loci in an easy-to-use assay through fusion with dCas9. Most approaches will be compatible with single-cell sequencing because they directly detect the sgRNA in most cases.
How single-cell CRISPR screens work
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sgRNA structure
An sgRNA contains a 17-20bp sequence that complements the target site and a scaffold usually between 100-300bp long. Researchers modify this scaffold based on the assay's requirements and optimizations. The U6 promoter drives sgRNA expression, which RNA Pol III transcribes, resulting in a non-polyadenylated product. Detecting the sgRNA is essential for scCRISPR screens because the sgRNA determines the cell's perturbation.
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Guide Detection: CROP-seq
CROP-seq (Datlinger et al., 2017) is a method that makes sgRNA detectable in single-cell assays. It works by producing two transcripts from the CROP-seq vector. The first transcript, driven by the U6 promoter, expresses the sgRNA. The second transcript, driven by the EF1a promoter, includes both a selection cassette and the sgRNA cassette by reading through the U6 promoter.
The polyadenylation of this long transcript allows for its detection in a single-cell assay. CROP-seq is widely used because it works with any single-cell RNA sequencing method that captures poly-A tails and doesn't require altering the sgRNA structure.
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Guide Detection: Feature Barcode technology
Recent advancements have made feature barcodes the preferred method for detecting sgRNA. In most assays, including those from 10x Genomics, feature barcode technology involves adding a specific capture sequence to the sgRNA scaffold. This allows the single-cell assay to directly capture the sgRNA.
Considerations
To achieve an ideal single-cell CRISPR screen experiment, careful planning and quality control are crucial before proceeding to the single-cell assay. Below are key considerations that can enhance the perturbation's effect size and ensure compatibility with single-cell assays.
Design
The design of a single-cell CRISPR screen depends on the target types and whether activation or repression is desired. This process involves selecting the appropriate CRISPR system, choosing a method for sgRNA detection, and designing the sgRNA sequences.
Quality Control (QC)
Optimizing the screen is just as important as the initial design. Key QC steps include verifying the perturbation effect size in bulk, establishing an effective CRISPR line, and testing the sgRNA capture efficiency.
Consultation
If you are interested in how our services can help your CRISPR experiments, please contact our sales team. When your samples are ready for processing, we can perform all the steps for you - from generating the single-cell library to sequencing and data analysis.
Watch webinar
CRISPR screening at scale
In this webinar, you will learn:
- Why CRISPR screens require large number of cells
- How researchers are approaching these demands with the Scale Biosciences CRISPR Guide Enrichment Kit.
- How to achieve high throughput single-cell sequencing for projects that demand it
