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May 10, 2022 / Oncology / Immunology / Neuroscience / Developmental Biology

Answering your questions about Single Cell 5’ CRISPR Screening

Olivia Habern

At our recent launch webinar, we shared details about our new Single Cell 5’ CRISPR Screening assay, which provides comprehensive multiomic readouts of perturbation effects in single cells—including direct capture of guide RNAs along with whole transcriptome gene expression and surface protein expression—from existing CRISPR-Cas9 libraries. If you’re new to single cell CRISPR screening, or familiar with this approach but want to learn more about why we developed the 5’ assay, these highlights from our live Q&A following the launch webinar may be helpful for you. 

Keep reading to find answers to questions about the value of single cell CRISPR screens compared to traditional pooled or arrayed screens, when someone should use the 5’ assay versus single cell 3’ CRISPR screening, and more. Then, check out our on-demand webinar to get all the details on Single Cell 5’ CRISPR Screening. 

Why single cell CRISPR screens 

When should I think about using a single cell CRISPR screen versus other types of screening methods?

Although pooled CRISPR screens offer a powerful method for identifying genes involved in complex biological mechanisms, such as cell proliferation, cell survival, and drug or toxin resistance, these screening approaches provide limited insights into a gene’s impact on other genetic pathways. Understanding how a gene affects the expression of other genes and the overall cellular phenotype can reveal important information about its underlying functional role. This necessitates a single cell transcriptomic readout for the clearest insights into cell type–specific gene function and pathways analysis. 

Traditional CRISPR screening techniques can force a trade-off between depth of characterization and scale. Single cell CRISPR screening provides an effective alternative, allowing researchers to obtain whole transcriptome readouts for a perturbation while also allowing for screening up to thousands of guides in a single experiment. If your research question calls for assessment of many gene edits, or even a genome-wide functional screen, it may be most efficient to leverage a high-throughput single cell CRISPR screening approach. 

These two factors, efficiency and high-resolution content, were some of the main drivers behind the development of single cell CRISPR screening technology. In collaboration with members of 10x Genomics R&D, Joseph Replogle, MD-PhD student in Jonathan Weissman's lab at the Whitehead Institute for Biomedical Research, MIT, developed the foundational direct capture Perturb-seq method described in this Nature Biotechnology paper. Replogle shared more thoughts on the limitations of traditional screening platforms in a Biotechniques interview, noting they “suffered from one of two problems. They either: (i) required lentivirus to be prepared in an arrayed format (i.e. one well per guide RNA in the screen) or, (ii) were not compatible with studying genetic interactions, where a targeted set of multiple genetic perturbations were made in each cell."

Replogle noted it’s also important to consider the biological factors that determine if a research question can be addressed with single cell CRISPR screening. 

"The first question I ask is whether the cellular behavior of interest has a transcriptional phenotype. There are some cellular processes, such as cell death, where transcription is not a very useful or interpretable readout. The second question I ask is whether the cellular behavior is cell-autonomous, meaning that the cell’s genetic perturbation primarily affects its phenotype rather than the phenotype of interacting cells.

Assuming these criteria are met, a final question is whether there is likely to be complexity in the transcriptional response that could not be dissected with a simple, one-dimensional reporter. Oftentimes, this is a difficult question to answer a priori and I have been surprised many times. For instance, I was recently looking at hematopoietic differentiation, which I expected to be quite stereotyped, and I was surprised to see the number of non-canonical states that could be produced with genetic perturbations."

Taken together, these considerations can help you decide if single cell CRISPR screening is a good fit for your research questions and sample types. 

3’ versus 5’ CRISPR screening

10x Genomics offers two high-throughput and scalable single cell CRISPR screening products that enable single cell gene expression analysis coupled with Feature Barcode technology for CRISPR screening. 

Chromium Single Cell Immune Profiling with Feature Barcode technology for CRISPR Screening (also referred to as Single Cell 5' CRISPR Screening) provides a multiomic approach to simultaneously detect gene expression, CRISPR guides, cell surface proteins, and/or immune cell clonotype frequencies from the same single cell. The Single Cell 5' CRISPR Screening approach is compatible with most Cas9 CRISPR vectors and does not require integration of specific capture sequences into the sgRNAs for compatibility. This allows researchers to leverage their existing CRISPR-Cas9 libraries for the 5’ assay, enabling a streamlined workflow.

Chromium Single Cell 3' Gene Expression with Feature Barcode technology for CRISPR Screening (also referred to as Single Cell 3' CRISPR Screening) allows for the capture of gene expression profiles along with CRISPR-mediated perturbation phenotypes in the same single cell. Single Cell 3' CRISPR Screening is enabled by engineering sgRNAs with one of two possible capture sequences that are required for assay compatibility.

Explore more of the differences between both assays in our Experimental Planning Guide

How does the 5’ system compare head to head with the 3’ system? When do you recommend one kit over the other?

Sensitivity is comparable between the 3’ and 5’ CRISPR screening assays, both in terms of gene expression and guide capture. In the majority of cases, customers should consider the 5’ CRISPR assay. The following criteria can help you decide which assay best fits your experiment. 

Customers should select the 5’ CRISPR assay if they meet the following criteria:

  • They already have a pre-built, compatible guide RNA library they want to use in the single cell CRISPR screening workflow. 
  • They are new to single cell CRISPR screening.
  • They are experienced with single cell CRISPR screening, but do not wish to perform additional steps to modify guide libraries with assay-specific sequences. 

Customers should select the 3’ CRISPR assay if they meet the following criteria:

  • They want to compare their single cell CRISPR experiment to data from previous experiments that also used the 3’ assay. The same CRISPR assay should be used in order to minimize batch effects.

Can the approach used for 5' CRISPR screening be applied to 3' chemistry, so that the 3' method does not need a capture sequence to detect the guide?

Due to the directionality of the reverse transcription enzyme, this is not feasible. However, the two assays have very similar workflows and comparable sensitivity, so there’s no downside to selecting the 5’ chemistry. 

That being said, if you have a strong preference for the 3’ assay, our compatible partner MilliporeSigma offers plasmids that are compatible with our 3’ assay. They provide more guidance for that process in this technical article

How can you scale up to thousands of genes using Single Cell 5' CRISPR Screening?

For homogenous sample types, we recommend planning for around 100 cells per gene to have enough statistical power for your experiment. Therefore, to scale up the experiment to thousands of genes tested in a single project, you simply need to scale up the number of cells that you run. For example, a single high-throughput experimental run using our 5’ assay can analyze 320,000 cells, which is enough for 3,200 genes assayed. 

Exploring other CRISPR innovations

Are there advantages to using this method over CROP-seq or Perturb-seq?

Perturb-seq involves indirect capture of the guide RNA data by detection of a separate guide barcode sequence on the cassette, which is polyadenylated. Since the guide barcodes are often several kilobases from their corresponding sgRNAs, viral recombination–mediated swapping of barcodes and sgRNAs is a possibility and can lead to less accurate data. The 10x Genomics method involves direct capture of the guide RNA sequence, rather than inferral, so this is not a concern using our assay. 

CROP-seq involves capture of the guide sequence by polyadenylation, and the guide sequence is, therefore, captured in the gene expression library. This means that CROP-seq requires sequencing the gene expression library to a sufficient depth in order to detect the guide sequences. The 10x Genomics method has a separate CRISPR library, which reduces the sequencing burden. 

To learn more about our new Single Cell 5' CRISPR Screening assay, please review the on-demand introductory webinar

And find further details on our product page →