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

ISSN: 1747-0862

Journal of Molecular and Genetic Medicine

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

Wenzhi Cai1,5#, Weijing Li2,5#, Dan Yang3, Huafeng Xie4 and Jian Huang5*
1Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, PR China
2Hematology and Oncology Laboratory, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, PR China
3Wuhan Institute of Design and Sciences, Wuhan, PR China
4Institute for Life Sciences, South China University of Technology, Guangzhou, PR China
5Department of Pathology and Laboratory Medicine, Lewis Katz School of Medicine, Temple University, Philadelphia, Pennsylvania, USA
#Contributed equally to this work
*Corresponding Author: Dr. Jian Huang, Department of Pathology and Laboratory Medicine, Lewis Katz School of Medicine, Temple University, Philadelphia Pennsylvania, 19140, USA, Tel: 215-707-2647, Email: [email protected]

Received Date: Mar 23, 2018 / Accepted Date: Apr 05, 2018 / Published Date: Apr 09, 2018

Abstract

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.

Keywords: Genome-editing; CRISPR based screening; Genetic screening

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 [1-3]. As a powerful genetic tool, CRISPR/Cas9 has been used to study and potentially treat single gene disorders (e.g. sickle cell anemia and β- thalassemia), 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.

          In vitro           Representative study reference Cas9 Protein gRNAlibrary Cells Methods Scientific Implications
Wang, et al. 2014, Science Dox-inducible WTcas9 7,114 genes, 73,151 gRNAs Human leukemia cell lines (KBM7 and HL60) Positive and negative selection Established CRISPR/Cas9 screens as a powerful tool for systematic genetic analysis in mammalian cells
Shalem, et al. 2014, Science ConstituitiveWTcas9 Human GeCKO Human melanoma cell line (A375)  andstem cell line (HUES62) Positive and negative selection Demonstrated feasisbility and advantages of CRISPR/Cas9 system for pooled genome-scale functional screening
Hou, et al. 2017, Cancer Res ConstituitiveWTcas9 Human GeCKO Human leukemia cell line (MV4-11) Positive selection Identification of genes whose loss confer resistance to drug in AML
Zhou, et al. 2014, Science WTcas9 291 genes, 869 gRNAs Human cervical carcinoma cell line (Hela) Positive selection Identification of genes essential for cell intoxication
Park, et al. 2016, Nature Genetics WTcas9 18,543 genes, 187,536 gRNAs Human CD4+ T cell line (CCRF-CEM) Positive selection Identification of host genes important in facililtating virus infection
Hart, et al. 2015, Cell WTcas9 90K, TKO library Human colorectal carcinoma cell line (HCT116), colorectal carcinoma cell line (DLD1), glioblastoma cell line (GBM), immortalized retinal epithelial cell line (RPE1), melanoma cell line (A375) negative selection Expansion of the catalog of human cell line fitness genes and identification of genetic vulnerabilities and therapeutic targets
Tzelepis, et al. 2016, Cell Reports WTcas9 18,010 genes, 90,709 gRNAs Human AML cell lines (MOLM-13, MV4-11, HL-60, OCI-AML2, OCI-AML3) negative selection Identification of genetic vulnerabilities and therapeutic targets
Arroyo, et al. 2016, Cell Metabolism WTcas9 18,335 genes, 74,687 gRNAs Human CML cell line (K562) Death screening Genetic analysis using dead cells
          In vivo Chen, et al. 2015, Cell WTcas9 Mouse GeCKO Mouse lung cancer cell line (KPD) Mutated cells were subcutaneously injected into immunocompromised Nu/Nu mice Providing a road map for in vivo screening
Song, et al. 2017, Gastroenterology WTcas9 Mouse GeCKO Mouse embryonic liver progenitor cell Mutated cells were subcutaneously injected into immunocompromised Nu/Nu mice In vivoCRISPR-based genetic screening in tumor models
Manguso, et al. 2017, Nature WTcas9 Mouse TSG Mouse melanoma cell line (B16) Mutated cells were subcutaneously injected into mice treated with immunotherapy In vivoCRISPR-based genetic screening in tumor models
Chow, et al. 2017, Nat Neurosci Conditional WTcas9 expression 56 genes, 288 gRNAs Mouse primary astrocyte Mutated cells were sterotaxically injected into the mice brain In vivoCRISPR-based genetic screening in tumor models
            Single-Cell CRISPR Screening Dixit, et al. 2016, Cell WTcas9 24 transcription factors (67 gRNAs) Mouse bone marrow derived dendritic cells Cells were stimulated with LPS in 7 days after infection Dissecting the transcriptional program in the BMDC response  to LPS
WTcas9 10 transcription factors (46 gRNAs) Human CML cell line (K562) stably expressing Cas9 Cells were stimulated with LPS in 7 days after infection Global transcriptional modules predict individual TF functions
Adamson, et al. 2016, Cell dCas9 9 three-guide vectors, 91 sgRNAs Human CML cell line (K562) stably expressing dCas9-KRAB Treatment of 4mg/mL tunicamycin for 6 hrs Revealing bifurcated UPR within a population and allows unbiased discovery of UPR-controlled genes
  Jaitin, et al. 2016, Cell   WTcas9-GFP 57 gRNAs targeting 22 genes CD11c+ myeloid cells sorted from Cas9-GFP transgenic mice Cells were treated with lipopolysaccharide (LPS) Rewiring of regulatory circuits in myeloid cells
A pool of Cebpb, Irf8, Rela, Stat1, Stat2 and two control gRNAs Mouse CD11c+ myeloid cells sorted from Cas9-GFP transgenic mice Cells were treated with LPS after 7 days following transplantation Uncovering the complexity of myeloid regulatory circuits in immune niches in vivo
Datlinger, et al. 2017, Nat Methods WTcas9 TCR-related 87 gRNAs (29 genes) Human T-ALL cell line (jurkat) stably expressing Cas9 T-cell -receptor induction Facilitating large screens by providing a vector that makes the guide RNA itself readable
Non-targeting 20 gRNAs
Essential 9 gRNAs
Michils, et al. 2017, Nat Methods Dox-inducible Cas9 26,514 guides targeting 6,560 genes Mouse embryonic fibroblasts (MEFs) Positive selection screen Identifying new roadblocks of cellular reprogramming
Note: Dox: Doxycycline; GeCKO: Genome-scale CRISPR-Cas9knockOut library; sgRNA: Single guide RNA; LPS: Lipopolysaccharide

Table 1: Representative CRISPR based screenings In vitro and In vivo and combined with single cell RNA-seq.

References

Citation: Cai W, Li W, Yang D, Xie H, Huang J (2018) A New Era in Functional Genomics Using CRISPR/Cas9 Knockout Screening. J Mol Genet Med 12: 345 DOI: 10.4172/1747-0862.1000345

Copyright: © 2018 Cai W, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

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