A New Era in Functional Genomics Using CRISPR/Cas9 Knockout Screening

In this commentary, we discussed the new exciting progress in CRISPR based screening technology field and highlight recent developments in the area of CRISPR-based functional genomics. High-throughput functional genomics using CRISPR-Cas9 revolutionized our ability to decipher cellular function in health and disease. Despite its limitations, the simplicity and effectiveness of CRISPR/Cas9 based screening, makes an enormous impact on genomic screening and thus scientific discovery.


Introduction
Genetic screening has been a powerful tool to identify gene function, in particular through studying cellular phenotypes arising from genome-wide perturbations. The main method for genomewide loss-of-function screening is using short hairpin (sh) RNA or siRNA libraries in order to knock down mRNA transcript levels. More recently developed techniques utilizing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing have significantly improved gain-or loss-of-function studies. It is now possible to make much more precise changes to endogenous genes and completely knock out their expression in vitro and in vivo [1][2][3]. As a powerful genetic tool, CRISPR/Cas9 has been used to study and potentially treat single gene disorders (e.g. sickle cell anemia and βthalassemia), cardiovascular diseases (e.g. coronary heart disease due to higher LDL cholesterol levels) and HIV infection (e.g. inactivating HIV co-receptors CCR5 and CXCR4) [4,5].

Discussion
In 2014, two seminal publications in Science first demonstrated that CRISPR/Cas9 system can be used as a screening tool for genetic studies [6,7]. They developed genome-scale lentiviral pooled libraries targeting approximately 17,000 and 18,000 human genes (with 5 -6 gRNAs/gene), respectively. Both positive and negative selection screening was successfully carried out with CRISPR pooled library in mammalian cells. Importantly, the CRISPR based screening was demonstrated superior to an shRNA screening because of its ability to knock out the genes efficiently. We have recently taken advantage of the genome-scale CRISPR-Cas9 knockout (GeCKO) library developed by the Broad Institute to study the mechanisms underlying FLT3 inhibitor resistance in acute myeloid leukemia (AML) [8]. In our screen, we identified SPRY3, an intracellular inhibitor of FGF signaling, and GSK3, a canonical Wnt signaling antagonist, and demonstrated that re-activation of downstream FGF/Ras/ERK and Wnt signaling as major mechanisms of resistance to the FLT3 inhibitor. In the last four years, numerous CRISPR based pooled genetic screens were performed to study various biological or pathological processes, uncovered mediators of drug resistance, pathogen toxicity, tumor growth/metastasis as well as defined cell-essential genes of the human genome and new roadblocks in reprogramming mouse embryonic fibroblasts etc. A genome-wide CRISPR screen in a mouse model of tumor growth and metastasis was conducted by transducing a CRISPR library into a non-small-cell lung cancer cell line and transplanted cells subcutaneously into immunocompromised mice [9]. Enriched single guide RNAs (sgRNAs) in lung metastases and late stage primary tumors were identified to target a small set of genes, suggesting specific loss-of-function mutations drive tumor growth and metastasis. A similar approach was used to identify tumor suppressor mechanisms of hepatocellular carcinoma as well as new immunotherapy targets [10,11]. More recently, Chow et al. delivered an adeno-associated virus (AAV)-mediated CRISPR library directly into the mouse brain that conditionally expressed Cas9 through stereotaxic injection to identify functional suppressors in glioblastoma [12].
Conventional pooled CRISPR screenings are limited to analyses of cell-population behavior during the screening process. This limitation was recently overcome through the combination of CRISPR screen with single-cell RNA-seq. The studies described CROP-seq [13], Perturb-seq [14,15], and CRISPR-seq [16], CRISPR-UMI [17] use the CRISPR-Cas9 system to create up to thousands of genetic perturbations in parallel within a single sample, as with conventional pooled screens. But by using single-cell RNA-seq as readout, the approaches enable the gene knockout and phenotype of each cell to be examined simultaneously. These new methods have already been proved to be a powerful tool to study cellular signaling including the T-cell receptor signaling pathway in Jurkat cells, and mammalian unfolded protein response, the transcriptional program in the bone marrow-derived dendritic cells (BMDC) response to lipopolysaccharide (LPS), mouse embryonic fibroblasts reprogramming as well as regulatory circuits of innate immunity.

Conclusion
Although CRISPR based screening has been reported to perform better with low noise, minimal off-target effects and experimental consistency, compared to knock down approaches using CRISPRi and shRNA [18], the application of the approach has its own limitations. The Cas9/gRNA does not always lead to knockout as the indels could be in-frame mutations, thereby keeping the gene function intact. Additionally, several studies have shown that the correlation between cellular lethality and the number of DNA double strand breaks (DSBs) in a cell, independent of the gene being targeted. Thus, CRISPR knockout based screens can identify false-positive hits for highly amplified genomic regions, including non-expressed genes [19,20].
Representations of the in vitro and in vivo screenings up to date are summarized in Table 1. Taken together, high-throughput functional genomics using CRISPR-Cas9 revolutionized our ability to decipher cellular function in health and disease. Despite its limitations, the simplicity and effectiveness of CRISPR/Cas9 based screening, promise many exciting new applications in the coming years.