The Evolution of CRISPR-Cas9 Screening

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July 23, 2019

The identification of gene function is a fundamental goal of functional genomics, with scientists aiming to link genotype to phenotype to determine cellular processes, understand the causative agents of disease, and identify targets for drug discovery. Functional genomic screening has been given a boost with the development of CRISPR-Cas9 gene-editing technology, providing scientists with the means of performing genome-wide perturbation of gene expression within the technical capabilities of most laboratories, and CRISPR-Cas9 is now a popular choice for administering large-scale pooled screens. However, recent developments have seen CRISPR-Cas9 screening evolving beyond simple growth-based proliferation assays. Orthogonal screening is now possible with CRISPRi and CRISPRa and complex analyses such as high-throughput FACS, high-content microscopy, and even single-cell RNASeq analysis can be incorporated into the screening workflow.

This article will discuss the evolution of CRISPR-Cas9 technology for functional genomics and the screening opportunities now available.

Functional genomic screening with CRISPR-Cas9

A high-throughput pooled library screen with CRISPR-Cas9 aims to systematically perturb the function of every gene in the genome, and separate those cells based on the required phenotype. Those sgRNA that are enriched or depleted are then identified by massively parallel sequencing to determine the genes responsible for the observed phenotype. The simplest, and to date the most popular means of phenotypic selection are proliferation-based screening methodologies, which allow both positive and negative approaches. In both cases, cells transduced with the lentiviral library are subjected to the required selection (i.e. growth assays, treatment with a drug or toxin) and the sgRNA remaining in the screened population compared to the untreated control.

The purpose of a negative screen, also known as a drop-out screen, is to identify those sgRNA that are absent compared to control following selection—and therefore affect cell viability or proliferation to identify essential genes or genetic dependencies of cancer cells, as well as synthetic lethal interactions.

In contrast, positive screens involve the application of a strong selection pressure so that there is a low probability of cell survival without a genetic perturbation. Positive screens have been utilized to identify drivers of resistance to drugs and toxins and are generally the easiest screens to perform, as hits are enriched in the screened population and so sgRNA barcodes are easy to pull out against background. This differs from a negative screen, where identification of depleted sgRNAs against a background of often tens of thousands of remaining sgRNAs can be more challenging—although background noise is significantly reduced relative to orthologous technologies such as RNAi.

Alternative selection and complex readouts with CRISPR-Cas9 screening

The early adopters of CRISPR-Cas9 screening were for the most part those working in cancer biology or investigating disease where pathophysiology was marked by a survival signal. However, to expand upon the biological processes that can be studied, and allow screening in short-term primary cell cultures, alternative phenotype selection other than lethality is required, such as changes in protein expression. In these screens, cellular targets are tagged with endogenous fluorescent proteins or labeled with highly specific antibodies, and cells displaying the phenotype of interest are isolated by fluorescent-activity cell sorting (FACS).

This approach allows for comprehensive interrogation of genetic components based on protein expression in a cellular context and was utilized by Parnas et al. to identify regulatory networks in innate immune cells.¹ By studying TNF expression in response to lipopolysaccharide stimulation, the authors were able to isolate cells by FACS based upon their TNF expression, and then identify regulatory markers that mediate the immune response. Similarly, DeJesus et al. used a neuroglioma H4 cell line that stably expressed GFP-tagged SQSTM1, a key component of the NF-kB signaling cascade, along with a genome-wide CRISPR-ko to identify genes involved in the regulation of SQSTM1 based on GFP-fluorescence.²

Readouts from CRISPR screens using quantification of the abundance of sgRNA following proliferation or marker-based selection is relatively straightforward but does not necessarily provide information about the cell state, or allow high-throughput assaying of complex phenotypes. Recent advances in single-cell analysis have been coupled with CRISPR screening to provide transcriptomic analysis at the single-cell level. Several techniques have been developed, such as Perturb-Seq, CROP-Seq, CRISP-Seq, and Mosaic-Seq that combine a pooled CRISPR screening workflow with single-cell sequencing methods to facilitate high-dimensional phenotyping without having to resort to array-based screening methods.^3-6 While this approach is technically challenging, it has shown great promise when screening a small subset of genes and in the dissection of complex cellular pathways.

“Single-cell approaches will be increasingly important as we move to more complex cell systems, where being able to understand highly complex phenotypic signatures simultaneously with tissue heterogeneity is going to be key.”

Dr. Benedict Cross, Head of Functional Genomic Screening at Horizon Discovery

Combination screening with orthogonal Cas9

Loss of function studies targeting single genes are a powerful tool in mapping gene interactions, but they fail to address the directional dependencies of genetic information. Boettcher et al. combined two Cas9 proteins from different species to develop an orthogonal screening platform—where two different genes can be activated and repressed in a single cell.⁷ Using a K562 cell line expressing the S.pyogenes-based SunTag CRISPRa system and Cas9 from S.aureus, the authors were able to study 87 genes and identify directional interactions of cancer relevant genes in a myeloid leukemia cell model. This orthogonal approach allows for elucidation of genetic interactions, identifying whether the activated gene is functionally dependent upon the deleted gene, or whether compensation can occur. Understanding these interactions is crucial for precision medicine, target identification for drug discovery, and therapeutic interventions.

Screening in animal models and primary cells

The application of CRISPR-Cas9 screening has now been extended for use in primary cells and whole animal models enabling better recapitulation of cellular events with increased biological relevance. Shifrut et al. have developed SLICE, a new method to overcome the challenges of performing CRISPR-based screening in human T cells.⁸ By combining SLICE with single-cell analysis, the regulatory pathways involved in T cell responses can be characterized, enabling unbiased interrogation pf human T-cell biology. A platform to perform CRISPR-based screening in human iPSC-derived neurons has also been developed by Kampman et al.—using both CRISPRi and CRISPRa screening to provide complementary biological insight and FACS analysis, the authors hope to better understand cellular mechanisms involved in neurodegenerative disease and identify potential therapeutic targets.⁹

Whole animal screens commonly involve xenograft models, where cells transduced with the CRISPR library are transplanted into animal models. This approach was first demonstrated by Chen et al. where a non-metastatic cancer cell line was transduced with a sgRNA library, and the sgRNA present in lung metastases and late-stage primary tumors sequenced to determine which loss-of-function mutations drive tumor growth and metastasis.¹⁰ Similarly, Song et al. used this approach to identify liver tumor suppressors—this time performing the genome-wide CRISPR screen on p53 null mouse hepatocytes that were subsequently transplanted subcutaneously into nude mice.¹¹

The CRISPR evolution

From the outset, it has been apparent that CRISPR-Cas9 as a tool for functional genomic screening is tremendously powerful. It overcomes some of the limitations of RNAi—such as lower off-target effects and background noise, while retaining some of the key benefits, namely the ability to screen every gene in the genome in a single experiment. The approach, however, was not without limitations, in particular the ability to only screen loss of function mutations, and initially only to work in cell lines that can be grown over an extended period of time.

Now the expanded CRISPR toolkit enables screening of both loss or gain of function mutations with CRISPRi and CRISPRa, and by combining the technology with other recent developments, such as single-cell analysis, even greater depths of discovery are now possible.

Pooled library screening with CRISPR-Cas9
CRISPR-Cas9, a recently repurposed bacterial adaptive immune system, requires only the delivery of a sgRNA complementary to the target site with the Cas9 endonuclease to facilitate robust and targeted gene knockout (CRISPR-ko)
Other genetic perturbations are also possible with the expansion of the CRISPR-Cas9 toolkit—by tethering transcriptional repressors or activator domains to a catalytically inactive version of Cas9 (dCas9), transcriptional modulation can also be induced with CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa)
Libraries of sgRNA targeting every gene in the genome are delivered via lentiviral vector, with viral titer carefully controlled so there is only a single editing event per cell
Delivery by lentiviral vector results in the stable integration of the sgRNA into the genome, therefore providing each cell with a molecular tag or barcode
Cells are then separated according to the phenotype of interest and those sgRNA enriched or depleted can be easily identified from the pool by massively parallel sequencing to determine those genes responsible for the observed phenotype