The doors CRISPR libraries have and will open in phenotypic drug screening 

Steve Wowk, VP, Business Unit and General Management of Integrated DNA Technologies, shared with DDW the value of CRISPR-Cas9 in drug discovery. 

Some of the most important breakthroughs in pharmaceutical research were adapted from naturally occurring phenomena. The CRISPR-Cas9 system, awarded the Nobel Prize in Chemistry in 2020 for its applications for gene editing, was initially encountered in the bacterial immune system. In an effort to defend themselves against viral infection, bacterial cells capture and copy DNA fragments of bacteriophages into their genome. In the event of an attack from similar bacteriophages, the bacteria can recognise these segments and use this to discern and cleave the viral DNA, which disrupts the viral gene function. 

Today, scientists leverage this defence mechanism to learn more about the genomic alterations in diseased states as well as the impact of drug treatments on the human genome. Bacterial RNA is replaced by a synthetic guide RNA (gRNA) that can recognise a specific target DNA sequence. The gRNA is accompanied by the Cas9 enzyme responsible for cutting DNA. This is then followed by DNA repair conducted by the cells’ own repair machinery, mainly non-homologous end joining (NHEJ), which introduces random mutations that can alter gene function. These modifications lead to alterations in the function of the proteins expressed by these genes, manifesting in cellular processes dependent on those proteins. 

Role of CRISPR-Cas9 in drug discovery 

The unique properties of the CRISPR-Cas9 system have massive potential to revolutionise drug discovery. Scientists can now target, activate, or silence any genes of interest to perturb the phenotype of cell populations. This helps them unearth the contribution of these genes to various diseases, which is driving the discovery of novel targets. For example, when investigating the tumour formation mechanism in a specific cancer type, researchers can apply CRISPR to their cell model to identify the set of genes necessary for tumour growth. This indicates that an efficient anti-cancer therapy must inhibit the activity of the proteins expressed by these genes. 

CRISPR-induced knockout can also be used to identify genes that confer resistance to current drug treatments, especially in cancer. One can induce further mutations in the tumour genome through CRISPR and detect genes that could possibly help the cancer cell escape apoptosis. Because many cancer types frequently undergo adaptations, the ability to predict possible drivers of drug resistance will become an indispensable part of drug discovery. 

CRISPR libraries: Why do we need them? 

There is an increased complexity from genes to proteins encoded by these genes and to the perplexing disease pathways involving those proteins. The underlying disease mechanism often involves perturbations in a subset of genes in massive genomes. Therefore, the CRISPR-Cas9 approach must be tailored to run genome-wide knockout screens in a systematic manner. This can be achieved by CRISPR screening. This high-throughput screening method involves CRISPR-target libraries to knock out a large number of genes and elicit their relationship to the resulting cell phenotype. 

Pooled CRISPR screening and concerns with lentiviral libraries 

Conventionally, pooled CRISPR screens are used to identify the genes of interest within large gene populations. This requires a cell model comprising a large cell population (approximately 1.67×108 cells1). The library is introduced to the cells so that only one gene knockout occurs per cell. This generates a diverse hub of cells, each with distinct knockouts, hence the different phenotypes. These cells are pooled together in the same dish to monitor cell behavior—mainly, their viability. Some cells die while others thrive. Through next generation sequencing, scientists can determine the prevalent genes as well as the depleted ones in the population. When applied to a cell population in the presence of a drug molecule, researchers can identify the set of genes necessary for cell survival. These genes constitute novel co-targets driving cell survival and resistance to the drug used during the screening. 

The delivery of gRNAs is crucial for precise DNA truncation by Cas9. Pooled CRISPR screens usually employ lentiviral libraries comprising gRNA-containing plasmids packaged into lentiviral particles. The lentiviruses in the library contain gRNAs that target genes generated from the complete list of genes in the genome. It is important to note that several target sites are often selected per gene to ensure complete knockout. 

In pooled CRISPR screening, CRISPR-Cas9 editing does not commence immediately after infection with the lentivirus. Instead, the lentiviral RNA is reverse-transcribed to DNA that integrates into the host cell genome, and the gRNA is synthesized from this DNA fragment within the cell. This mechanism introduces a significant pitfall in pooled CRISPR screening, as the integration is random, which can cause gene alterations around the insertion point. In addition, continuous expression from integrating DNA transcribed from lentiviral RNA can trigger off-target editing and dramatically impact the subsequent phenotypic cell readouts. Another drawback of pooled screening is that it is challenging to link phenotype to genotype, so it requires advanced data analysis and deconvolution through next generation sequencing. 

Arrayed CRISPR libraries and the future of CRISPR-based drug discovery 

The off-target editing risks and biosafety concerns in lentiviral libraries prompted researchers to seek CRISPR screening alternative methods. Arrayed CRISPR screening emerged as a means to perform a more sophisticated elucidation of target genes after determining a shortlist of candidate genes from initial genome-wide screening. Instead of lentiviruses as vehicles, arrayed screens employ synthetic gRNA libraries, where guides are distributed among separate wells in an array format in a multi-well plate. Each well contains either a single gRNA or a pool of gRNAs targeting a single gene, with the latter method being the common one so that only one gene knockout per well occurs. The gRNAs are transfected into the cells in wells and the cells are monitored for resulting changes. 

Using synthetic gRNA libraries in arrayed formats offers various advantages in CRISPR screening. Because the target sites are already well-characterised, one to three sites per gene can suffice for the complete knockout as opposed to six to eight sites per gene in pooled screening2. Furthermore, the synthetic gRNA can be complexed not only with the Cas enzyme but also with additional conjugates such as fluorophores. This introduces a few upgrades to the scope of CRISPR screening. First, it enables screening in cell types that are less abundant and cannot synthesize Cas9, such as primary cells and neurons. In addition, the ability to customise the gRNAs means a researcher can screen for more sophisticated phenotypic readouts that can be viewed via microscopy, such as protein expression levels, instead of conventional live/dead cell monitoring. From this perspective, arrayed CRISPR screening also eliminates the need for labor-intensive next generation sequencing. 

As previously mentioned, the synthetic gRNA approach in arrayed screening mitigates the biosafety concerns encountered in lentiviral libraries. Unlike lentiviral RNA, synthetic gRNA is discarded immediately after knock-out, so the cell genome is no longer susceptible to off-target editing. 

Considered collectively, the advantages of arrayed CRISPR screening and synthetic gRNA libraries can transcend the applications of CRISPR libraries beyond their current scope. For example, scientists still rely on pooled screenings of immortalised cancer cells that can proliferate and multiply over several generations. However, despite their ease of cultivation, these cell lines are manipulated and mutated to such an extent that they do not necessarily represent biologically relevant tumour environments. Arrayed screening provides researchers with the opportunity to work with cell populations that are harder to extract and grow in large numbers, yet necessary to address, such as stem cells. Because stem cell differentiation plays an incredibly significant role in relapse and resistance mechanisms in cancer, arrayed CRISPR screening can help get to the root causes of the most malignant and treatment-resistance cancer types.  

Another key advantage is that arrayed screening allows the flexibility to use other Cas proteins of interest and not have to rely on Cas9. With the recent development of other Cas proteins, such as Cas12a and Cas13, which rely on different PAM sequences, available target space can be expanded. Unlike Cas9, which requires both CRISPR RNA and a transactivating RNA sequence for activation, these proteins require only the CRISPR RNA. 

Biography  

Steve Wowk is VP, Business Unit and General Management of Integrated DNA Technologies. He leads IDT’s four business units—custom oligos and qPCR, synthetic biology, CRISPR and functional genomics, and next generation sequencing. Wowk earned his bachelor’s degree from University of Dayton, Ohio, and a Master of Science from John Carroll University. He completed an Executive MBA from the University of Texas, Austin. 

 

References  

  1. Joung J, Konermann S, Gootenberg JS, et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening [published correction appears in Nat Protoc. 2019 Jul;14(7):2259]. Nat Protoc. 2017;12(4):828-863. 
  2. Canver MC, Haeussler M, Bauer DE, et al. Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments. Nat Protoc. 2018;13(5):946-986. 

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