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When a cell population is transduced with a Cas nuclease and a pooled CRISPR sgRNA library, the knockout (KO) of each different gene alters the biology of the host cell depending on that particular gene's function. As a result, the function of a gene can be inferred, provided we have a way to detect the changes induced by its KO. With bulk CRISPR screens, whole cell populations can be analyzed for general phenotypic changes, such as cell viability or activation of specific reporters, to identify essential genes or genes required to activate particular pathways.
However, instead of bulk analysis, CRISPR-library transduced cell populations can also be studied using single-cell transcriptomic (scRNA) analysis, which makes it possible to measure gene expression changes in hundreds to thousands of cells simultaneously. As a result, changes in gene expression induced by the knockout or other perturbation of a gene can be detected, characterized, and linked to the disruption or altered expression of the CRISPR target gene.
This technique using scRNA analysis on cells transduced with pooled CRISPR libraries, referred to asPerturb-Seq, CROP-Seq or CRISPR-Seq, greatly expands the utility of using CRISPR libraries in analyzing gene function. Traditional CRISPR screens are limited to identifying genes whose function is related to cell viability or activation of a specific pathway or reporter. With Perturb-Seq screens, however, it is possible to detect any significant transcriptome changes associated with a particular gene disruption.
Perturb-Seq screens identify genes driving changes in gene expression and pathway regulation that produce phenotypic changes in cells. As a result, the approach can be used to study a wide range of biological processes, including developmental processes, cell fate decisions, and responses to environmental stimuli. It has been used to identify new regulatory mechanisms, to characterize the functions of non-coding RNA molecules, and to study the roles of specific transcription factors in gene regulation.
Running Perturb-Seq screens, however, can be challenging. With current single-cell platforms, there is a limit on the number of cells that can be analyzed in one run. For example, with the 10X Chromium platform, cDNA from only ~60-80K cells can be captured and analyzed in a single reaction. As a result, it is not practical to screen a genome-wide sgRNA library, which typically has ~80,000 sgRNA constructs, at 4 sgRNAs per gene. Of course, multiple batches can be run but, based on studies that Cellecta has conducted, the best results are obtained when at least ~100 cells are analyzed for each sgRNA (see table below). To analyze this number of cells for each of the ~80,000 guides in a genome-wide library would require running ~800 reactions, with each of these also needing to be sequenced.
Given the throughput limitations discussed above, CRISPR sgRNA libraries used for Perturb-seq analysis need to be relatively small and targeted. A reasonable size for a library analyzed using the 10X Chromium System described above would target ~50 to 300 genes with 3-4 guides per gene. Libraries larger than this may require a large number of runs to analyze comprehensively. As a result, almost all Perturb-Seq libraries need to be custom-made to target a small number of genes most relevant to the experiment.
In addition to the library size, it is also critical to make sure the CRISPR library that is transduced into the cell population is designed so that each transduced guide is detected on a cell-by-cell basis with the single-cell analysis platform and protocol being used for analysis. The typical platform for single cells transcriptomic analysis is the 10X Single Cell Chromium System. There are a few ways to design CRISPR sgRNA libraries when using the 10X Chromium System, depending on which Chromium protocol will be used to capture the scRNA.
(1) 10X 3’-Chromium capture beads include unique “feature barcode” capture sequences. sgRNA sequencing modified with complementary sequences in the tracr sequence can be captured together with the poly-A tailed mRNA. Libraries with this design can be used with the standard 10X 3’-Chromium protocol and analyzed using the standard Cell Ranger software.
(2) Libraries can be made in our pScribe vectors the “CROP-Seq” Design, the sgRNA expression cassette is expressed on the 3’-UTR of the selection marker for the library vector. A separate amplification of the barcodes from the cDNA produced by the single-cell RNA platform allows these to be detected.
(3) Finally, any standard library that expresses the sgRNA from a U6 promoter can be used with the 10X 5’-Chromium protocol. No special modifications are needed, but a separate amplification is required to amplify the guides from the rest of the cDNA for sequencing.
As part of our custom library design service, Cellecta can provide any variation of the above sgRNA libraries or other customized designs for alternate scRNA analysis.
In addition, Cellecta offers a Single-Cell CRISPR Screening Service -- end-to-end workflowincluding construction and packaging of custom pooled sgRNA libraries, single-cell RNA-Seq on 10X Chromium, sequencing and aligning reads to identify hits, along with post-screening analysis.
Fig. 1
HEK293-Cas9 cells transduced with pooled sgRNA library (88 sgRNAs), followed by TNFα treatment/mock treatment. Single-cell partitioning using the 10X Chromium System was performed on 4000 cells, 7,500 cells, and 15,000 cells. The right column shows that scRNA analysis of 47 cells on average for each sgRNA in the library results in the loss of data for ~35% of the library. To see the whole study, refer to the poster here.
More information about Cellecta's Single-Cell CRISPR Screening Service is available here.
