CRISPR Screening in Single Cells
The combination of single-cell RNA-seq and CRISPR allows for efficient interrogation of possibly any number of genes, only limited by the sequencing capability. Here we describe the current protocols for CRISPR screening in single cells, from cloning and
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Introduction One of the key advances in next generation sequencing is the ability to multiplex. For example, sequencing has been made easy to scale by pooling multiple barcoded libraries. The single-cell transcriptomics field has pushed library pooling technology to the limit, in the quest to minimize cost. If each cell can be made an individual experiment, this can be harnessed to perform large-scale knock-out or perturbation phenotyping experiments. To date, there have been six studies published for CRISPRscreening at the single cell level [1–6]. In one of these papers, the multiwell plate method MARS-seq is used as the basis [2]. This method is however not commonly used and because it relies on multiwell plates, it is unable to scale to large number of cells. SC-CRISPR will most likely be applied to millions of cells in the future. This method will thus not be discussed further. The other studies utilize droplet-based system for the screen. Examples are Drop-seq [7] and 10 Chromium droplet RNA-seq [8]. However, essentially any droplet system can be used here. An overview of a single-cell CRISPR screen is shown in Fig. 1a. The cells are each expressing Cas9 and one sgRNA. The challenge is to identify which sgRNA is expressed in each cell, allowing them to be separated. Unfortunately the sgRNA is not a polyadenylated RNA molecule and will not be captured by standard single-cell
Valentina Proserpio (ed.), Single Cell Methods: Sequencing and Proteomics, Methods in Molecular Biology, vol. 1979, https://doi.org/10.1007/978-1-4939-9240-9_23, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Fig. 1 Single-cell CRISPR screening. (a) Overview of the procedure. Targeted sgRNA viruses are cloned, individually or pooled. These are transfected to packaging cells that produce virus. Cells of interested, already expressing Cas9, are transduced by the virus. Single-cell libraries are produced and sequenced. Optionally, and ideally, sgRNA reads are amplified and sequenced separately. (b) A typical barcoded sgRNA-plasmid. The barcode is here attached the BFP molecule whose mRNA is polyadenylated and captured using standard scRNA-seq chemistry. The sgRNA is transcribed by Pol III using the U6 promoter. The barcode and the sgRNA are far apart and cloning is more demanding. (c) The CROP-seq system. Only the sgRNA need be cloned in. Upon viral integration the 30 LTR is copied to the 50 . One of these copies is expressed as part of the virus and polyadenylated, while other copy is transcribed by Pol III as required for CRISPR to function. (d) The distribution of number of viruses per cell (k) as a function of infection rate (λ), or MOI
chemistries (e.g., derivatives of CEL-Seq [9] or Smart-seq2 [10]). Two solutions have so far been used. In Perturb-seq and Mosaicseq [1, 6], a separate barcode is coded into the end of the BFP selection marker (Fig. 1b). It is thus at the 30 end which is typically captured using droplet RNA-seq methods. The disadvantage is that the barcode has to be
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