CRISPR-Cas Screening for Cell Viability
Key Points
- Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a method of gene editing that uses the Cas9 protein and specific guide RNAs to either delete host genes or insert specific gene sequences into the host genome.
- CRISPR screening is used to find a small number of important genes or genetic sequences within a massive number of genetic sequences such as the entire genome.
- There are three main types of CRISPR screening assays which include viability-based negative selection screens, viability-based positive selection screens and marker-based selection screens
Content
Background
-What is CRISPR/Cas
- CRISPR/Cas9 Mechanism of Action
CRISPR Screening
- What are CRISPR Screening Assays?
- How Do CRISPR Screening Assays Work?
- What are the Types of CRISPR Screening Assays?
Final Notes
Background
What is CRISPR-Cas?
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a method of gene editing that uses the Cas9 protein and specific guide RNAs to either delete host genes or insert specific gene sequences into the host genome. CRISPR was originally found in bacteria and was identified as its adaptive immune system in order to protect itself from viruses. It is now being used at the forefront of genetic engineering (The Genetic Resources Core Facility, 2020).
It is a naturally occurring RNA-guided endonuclease which can cleave double-stranded DNA, and allows researchers to easily alter DNA sequences and change gene function (Vidyasagar, 2020). CRISPR-Cas is a cheaper, faster, more accurate and efficient way to carry out genome editing in comparison to other existing genome editing methods such zinc-finger nucleases and TALENs (Genetic Home Reference, 2020).
CRISPR consists of repeated sequences of 29 nucleotides in length interspaced with variable sequence fragments of 32 nucleotides called spacers. These variable sequences were found to have very similar sequence homology to viral gene sequences. These CRISPR elements are adjacent to multiple well-conserved genes called CRISPR-associated (Cas). These Cas genes code for an endonuclease which cleaves double stranded DNA at specific sites. The most common and widely used Cas protein is the Cas9 enzyme (Rodríguez‑Rodríguez et al., 2019).
CRISPR-Cas9 Mechanism of Action
CRISPR/Cas is an RNA-guided precision genome editor. Many CRISPR systems have been identified; however, the type II CRISPR enzyme Cas9 is the most widely used for genomic editing and regulation among Cas proteins (Wang, La Russa and Qi, 2016). Type II CRISPR systems requires one RNA-guided (gRNA) endonuclease whereas other CRISPR system types require a large complex of several effector proteins. Cas9 is brought to the target cleavage site by a single-guide RNA (sgRNA). This sgRNA recognises the target site by base pairing with the DNA, making the system highly specific.
The sgRNA must base pair with the target site, however this interaction alone is not sufficient for Cas9 activity to occur. To activate Cas9 and interaction must occur between the nuclease and a protospacer-adjacent motif (PAM) sequence at the target site, further adding to the specificity to the system. The target site must be positioned directly 5’ of a PAM sequence to allow cleavage to occur. Cas9 proteins from other bacterial species may recognise alternative PAM sites, allowing alternative targeting specificities depending on the locus being targeted (Sander and Joung, 2014). The absence of this PAM sequence will prevent cleavage by Cas9. This process allows the permanent modification of the genomic target sequence whilst also repairing any damage caused to the DNA (Rodríguez‑Rodríguez et al., 2019).
Figure 1: CRISPR/Cas9 cleavage of DNA. The presence of a non-coding trans-activating CRISPR RNA, tracrRNA, is fused to a crRNA to create the guide RNA. The 20 nucleotide region of the crRNA will guide the Cas9 enzyme to the target site allowing cleavage of the double-stranded DNA. Adapted from Corrigan-Curay et al., 2015.
CRISPR Screening
What are CRISPR Screening Assays?
The CRISPR-Cas9 system allows one to edit the genome through the precise knockout of target genes via the introduction of insertions or deletions within the targeted DNA. This provides the tools to observe phenotypes of cells that require complete loss of protein function (Grassian et al., 2015). CRISPR screening is used to identify specific genes within a genome and determine their function (Synthego, 2020).
It enables thousands of genes to be modified and their function assessed in a single experiment. The cutting-edge CRISPR technology can aid in the identification and validation of novel drug targets or the study of the underlying causes of disease (Horizon Discovery, 2020). It is also being used to determine the effects of genetic mutation on drug resistance and susceptibility in patients (Horizon Discovery, 2020). identifies essential genes in the human genome, the genes responsible for driving cancer metastasis and the roles played by noncoding genes or genetic sequences in the entire genome (Salzman, 2019).
The mechanism of CRISPR Screening Assays
Most CRISPR screening is carried out in cell culture. The main idea of CRISPR screening is to knockout every gene that is deemed important in the cell culture but only knockout one gene per cell (Spencer, 2019). The outcome of this process is a population of cells with different genes knocked out in different cells in each well. Certain cells in the culture will die due to essential genes being knocked out, whilst others will survive and possibly thrive due to the knockout of certain genes (Grassian et al., 2015).
After the cells that have survived are selected and are grown and then next-generation sequencing (NGS) is carried on the entire population of cells to determine which genetic sequences are present or absent (Spencer, 2019). The presence or absence of the sequences correlates to the specific genes that are essential or not for survival of the cells (Spencer, 2019). Once the genes necessary for survival of cells in normal conditions is established, the identification of genes that allow cells to survive under artificial physiological conditions can be determined.
Once the physiological condition is chosen, such as subjecting cells to a certain drug to determine which are susceptible or resistant, a screening of the genomes of the population of cells is carried out in order to generate a list of CRISPR targets. These targets are usually around 20 bases in length in the genome and are adjacent to PAM motifs (Thorne, 2020). CRISPR screening requires all genes to be knocked out, therefore to increase the probability of cutting in the genome, several target sites within each of the genes must be selected.
Once the target sequences are identified, a pool of oligos are designed. The oligos will be used to make lentiviruses which contain DNA to encode the single-guide RNA (sgRNA) including the target sequence (Shalem et al., 2013). Each oligo must also have specific sites upstream and downstream to allow cloning into lentiviral gene-containing plasmids (Joung et al., 2017, Merten ; Hebben and Bovolenta, 2016)
Each virus thus codes for a specific CRISPR target sequence to be recognised by the Cas enzyme.
When synthesising the oligos, it is important to identify the most promising sgRNAs that will work efficiently with the Cas enzyme of choice. S.pyogenes sgRNA paired with the Cas9 enzyme is a very effective system that rarely causes off-target cutting. The CRISPR classic sgRNA Synthesis Kit or the express sgRNA synthesis kit are effective tools for accurately carrying out a CRISPR-Cas experiment.
A cell line that naturally expresses a Cas enzyme is often easier to use, however the pool of lentiviruses used to incorporate the sgRNAs into the cells can also encode the desired Cas enzyme (Ludwig et al., 2018).
After the cells have been transfected with the lentivirus, the cells are incubated for a period of time to allow for phenotypic CRISPR-mediated changes to occur. This measures the viability of cells after a period of time based on the essentiality of genes. Then, the drug or treatment is performed on the cells and the genomic DNA from the control and treated group are sequenced using NGS. This allows the identification of genes necessary for drug or treatment resistance or susceptibility (Spencer, 2019).
Types of CRISPR Screening Assays
Viability-Based Negative Selection Screens
The purpose of a negative selection screen is to identify the factors that affect the survival or proliferation of cells, which cause affected cells to be depleted during selection. Selection in negative screening is often treating the cells with a target drug. In negative selection screens, two sets of cell populations are transduced with lentiviruses but only one set is subjected to selection whilst the other serves as a control. The genomes of both populations are then sequenced and analysed in order to identify sequences that have been depleted due to selection (Wang, La Russa and Qi, 2016)
Viability-Based Positive Selection Screens
In a positive selection screen, the main aim is to identify genes whose mutation, silencing or overexpression, gives a positive advantage over a given selective pressure such as drug treatment. Positive selection screens are often used to study the mechanisms of drug resistance (Sanson et al., 2018).
Marker-Based Selection Screens
In marker-based selection screens, the mutations that change marker gene protein expression are identified. In this type of screen, the marker gene is either endogenously tagged with a fluorescent protein such as GFP or labelled with highly specific antibodies. The gRNAs that target genes whose perturbations contribute to the expression of the marker genes are isolated using technology such as fluorescence-activity cell sorting (FACS) approaches (Sharma and Petsalaki, 2018).
A type of marker-based selection screening assay used to determine cell viability is a luminescent cell viability assay. This assay determines the number of viable cells in culture based on the amount of ATP present, which is an indicator of metabolically active cells. The quantity of ATP is directly linked to the number of viable cells present in the culture.
Final Notes
CRISPR-Cas technology is an extremely useful tool for genetic engineering as it is cheap, quick, easy to use and scalable. CRISPR screening has revolutionised genome sequencing as it facilitates the discovery the functions of multiple key genes or genetic sequences in huge quantities of cells simultaneously. This technology can be used for identifying and prioritizing drug targets, finding the genes that confer drug sensitivity and resistance, guiding patient selection, and identifying targets and pathways for potential combination therapies.
References
Corrigan-Curay, J., O'Reilly, M., Kohn, D., Cannon, P., Bao, G., Bushman, F., Carroll, D., Cathomen, T., Joung, J., Roth, D., Sadelain, M., Scharenberg, A., von Kalle, C., Zhang, F., Jambou, R., Rosenthal, E., Hassani, M., Singh, A. and Porteus, M. (2015). Genome Editing Technologies: Defining a Path to Clinic. Molecular Therapy, 23(5), pp.796-806.
Genetic Home Reference (2020). What are genome editing and CRISPR-Cas9?. [online] Genetics Home Reference. Available at: https://ghr.nlm.nih.gov/primer/genomicresearch/genomeediting [Accessed 12 Feb. 2020].
The Genetic Resources Core Facility (2020). CRISPR Basics – Genetic Resources Core Facility, Johns Hopkins University, School of Medicine. [online] Grcf.jhmi.edu. Available at: https://grcf.jhmi.edu/products/crisprs/crispr-basics/ [Accessed 12 Feb. 2020].
Grassian, A., Scales, T., Knutson, S., Kuntz, K., McCarthy, N., Lowe, C., Moore, J., Copeland, R., Keilhack, H., Smith, J., Wickenden, J. and Ribich, S. (2015). A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality. Biological Procedures Online, 17(1), p.15.
Horizon Discovery (2020). CRISPR screening service drives drug discovery. [online] Nature.com. Available at: https://www.nature.com/articles/d42473-018-00344-1 [Accessed 12 Feb. 2020].
Joung, J., Konermann, S., Gootenberg, J., Abudayyeh, O., Platt, R., Brigham, M., Sanjana, N. and Zhang, F. (2017). Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nature Protocols, 12(4), pp.828-863.
Ludwig, M., Michmerhuizen, N., Hoesli, R., Mann, J., Devenport, S., Kulkarni, A., Birkeland, A. and Brenner, J. (2018). Generation and Utilization of CRISPR/Cas9 Screening Libraries in Mammalian Cells. Genome Editing and Engineering, pp.223-234.
Merten, O., Hebben, M. and Bovolenta, C. (2016). Production of lentiviral vectors. Molecular Therapy - Methods & Clinical Development, 3, p.16017.
Rodríguez‑Rodríguez, D., Ramírez‑Solís, R., Garza‑Elizondo, M., Garza‑Rodríguez, M. and Barrera‑Saldaña, H. (2019). Genome editing: A perspective on the application of CRISPR/Cas9 to study human diseases (Review). International Journal of Molecular Medicine, 4(43), pp.1559–1574.
Salzman, S. (2019). How CRISPR Is Revolutionizing Screening Technology. [online] GEN - Genetic Engineering and Biotechnology News. Available at: https://www.genengnews.com/insights/how-crispr-is-revolutionizing-screening-technology/ [Accessed 13 Feb. 2020].
Sander, J. and Joung, J. (2014). CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology, 32(4), pp.347-355.
Sanson, K., Hanna, R., Hegde, M., Donovan, K., Strand, C., Sullender, M., Vaimberg, E., Goodale, A., Root, D., Piccioni, F. and Doench, J. (2018). Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nature Communications, 9(1), p.5416.
Sharma, S. and Petsalaki, E. (2018). Application of CRISPR-Cas9 Based Genome-Wide Screening Approaches to Study Cellular Signalling Mechanisms. International Journal of Molecular Sciences, 19(4), p.933.
Spencer, N. (2019). Overview: What is CRISPR Screening?. [online] Integrated DNA Technologies. Available at: https://www.idtdna.com/pages/education/decoded/article/overview-what-is-crispr-screening [Accessed 13 Feb. 2020].
Synthego (2020). Synthego | Full Stack Genome Engineering. [online] Synthego.com. Available at: https://www.synthego.com/products/crispr-kits/screening-libraries?utm_term=%2Bcrispr%20%2Bscreen&utm_campaign=2018-06+Library&utm_source=adwords&utm_medium=ppc&hsa_tgt=kwd-320468959416&hsa_grp=59041322569&hsa_src=g&hsa_net=adwords&hsa_mt=b&hsa_ver=3&hsa_ad=276174409347&hsa_acc=6964378581&hsa_kw=%2Bcrispr%20%2Bscreen&hsa_cam=1440025400&gclid=EAIaIQobChMI58vng9fL5wIVRbTtCh3xRAWEEAAYBCAAEgI_QPD_BwE [Accessed 12 Feb. 2020].
Vidyasagar, A. (2020). What Is CRISPR?. [online] livescience.com. Available at: https://www.livescience.com/58790-crispr-explained.html [Accessed 12 Feb. 2020].
Wang, H., La Russa, M. and Qi, L. (2016). CRISPR/Cas9 in Genome Editing and Beyond. Annual Review of Biochemistry, 85(1), pp.227-264.
Recent Posts
-
Biological Role of GLP-1
Glucagon-like peptide-1 (GLP-1) is a critical hormone in the regulation of glucose met …20th Jun 2024 -
Th17 Cell Differentiation: Insights into Immunological Dynamics
Th17 cells, a subset of T helper cells characterized by their production of interleukin-17 (IL- …25th May 2024 -
Assay Genie New Asian Distributor May 2024
Dublin, Ireland — May 20th 2024 — Assay Genie, a leading supplier of ELISA Kits, Ant …19th May 2024