CRISPR Screening and its Applications in Drug Discovery

Site-directed gene modifications with high precision are a desired methodology to investigate diseases. Hence, it is no surprise that CRISPR-Cas9 became the method of choice when it comes to research in Drug Discovery. Researchers in the pharmaceutical industry and academia are concerned with choosing the right targets for potential drug candidates and often are confronted with cumbersome procedures to effectively knockout genes to study their function as part of functional genomics approaches. 

To make use of CRISPR technology in CRISPR screenings some prerequisites need to be fulfilled:

1. Availability of CRISPR libraries for the respective gene family or disease pathway
2. Relevant cell types to investigate consequences of LOF (Loss of function) or gain of function gene alterations
3. Reliable method to introduce CRISPR RNPs into the cells and produce gene knockouts with high efficiency
4. Options to automate screening applications dealing with knockouts of several hundreds or even thousands of genes

The commercial availability of CRISPR screening libraries from many sources enables scientists to make use of this technology in the field of functional genomics. A certain gene family or pathway can be analyzed by creating specific gene knockouts and investigating the phenotypic alterations as a result of the knockout. CRISPR screens were initially performed mostly by transduction of cell lines with pooled or arrayed lentiviral of retroviral libraries. Viral transduction however, has its limitations when it comes to fast generation of libraries, using a different cell type for studying LOF/GOF in functional genomics and costs for maintaining and monitoring a BSL-2 (or greater) laboratory, if live viruses are being used.

Taking advantage of the biological relevance of primary cells in contrast to cell lines is a hurdle for host specific viral transduction often leading to inefficient transduction of the primary cells. Primary cells however are superior to cell lines when it comes to investigation of cell signaling pathways, representing a biologically more relevant model.

In this light, introducing Cas9-sgRNA ribonucleotide protein complexes (RNPs) into primary cells was cumbersome and time-consuming, when using viral libraries. Transfection methods as lipofection and chemical transfection methods have their challenges when it comes to transfecting primary cells or regarding reproducibility. Electroporation as another option regarding transfection efficiencies and reproducibility of transfection results in primary cells. However, the option for multi-well transfection and automation integration is lacking for most electroporation systems, making this option labor-intensive and time consuming.

One method – as proven by many publications – to successfully deliver RNPs into primary cells is the Nucleofector^TM Technology, an improved electroporation technology. This concept has been used by many renown researchers for transfecting the Cas9 along with gRNAs (as plasmid, mRNA or RNP), and in part is responsible for the high editing efficiencies that were achieved in e.g. primary human CD34+ T cells and both mouse and human T cells.

Alongside with high transfection efficiencies in primary cells, the Nucleofector^TM Technology shows high reproducibility and flexibility regarding cell numbers and substrate versatility, and Nucleofector^TM Technology is largely agnostic with respect to the substrates that can be transfected, and can easily deliver:   

• Plasmid DNA
• mRNA
• oligonucleotides
• siRNAs
• miRNAs
• peptides
• proteins
• RNP
• morpholinos
• Q-Dots
• small molecules
• different combinations of these substrates into relevant cell types.

With the 96-well Shuttle^TM Device and the 384-well Nucleofector^TM System the Nucleofector^TM Platform offers two Medium- to High-Throughput platforms which are both suited to combine the potent CRISPR technology with a screening format. Both platforms come with the option for integration into LHS (liquid handling systems) e.g. from Tecan, Beckman, or Hamilton facilitating the automation of CRISPR library screens with less hands-on time.

Additional benefits of the Nucleofector^TM Technology for CRISPR screening and functional genomics include:

• Transfection of a whole plate in about one minute
• Efficient transfection of biologically relevant primary cells and cell lines
• Enabling potent CRISPR-mediated gene knockout by efficient delivery of CRISPR substrates like RNPs
• Reliable technology proven by CRIPSR publications in high ranking journals
• Automation-compliant 96-well and 384-well platforms

CRISPR screening applications are a major breakthrough for the field of functional genomics and became a versatile and useful tool over the last decades. Especially for Medium- to High-Throughput approaches, the Nucleofector^TM Technology provides a tool that supports these applications and facilitates target identification in less time.