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Benefits of CRISPR-Cas9 for drug discovery research and target identification


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. 
 
CRISPR-Cas9 provides a very useful tool for target-specific gene modifications and allows for the identification of drug targets or for drug validation or target optimization. In combination with recent advances in sequencing technology, e.g. NGS (next generation sequencing) and single-cell sequencing, researchers can exploit these tools to aim for whole-genome analysis and the identification of disease-specific targets has made big advancements over the last couple of years.

Advances in genome editing - CRISPR library screens



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 gene alterations
  3. Reliable method to introduce CRISPR RNPs (ribonucleoproteins) 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 loss or gain of function mutations 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 RNPs into primary cells was cumbersome and time-consuming, when using viral libraries. Transfection methods as lipofection and other chemical transfection methods have their challenges when it comes to transfecting primary cells or regarding reproducibility. Electroporation is 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.

Selected references for CRISPR screening approaches in 96-well or 384-well format

Dickson et al. (2023) Highly scalable arrayed CRISPR mediated gene silencing in primary lung small airway epithelial cells SLAS Discovery 28(2):29-35

Yu et al. (2022) Genome-wide CRISPR Screening to Identify Drivers of TGF-β-Induced Liver Fibrosis in Human Hepatic Stellate Cells Chem Biol 17(4):918-929

Wang et al. (2021) Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses Cell 184(1):106-119

Gordon et al. (2020) Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms Science 370(6521):eabe9403

Roth et al. (2020) Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies Cell 181(3):728-744.e21

Schumann et al. (2020) Functional CRISPR dissection of gene networks controlling human regulatory T cell identity Nat Immunol 21(11):1456-1466

Roth et al. (2018) Reprogramming human T cell function and specificity with non-viral genome targeting Nature 559(7714):405-409

Seki and Rutz (2018) Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells J Exp Med 215(3):985-997

Hultquist et al. (2016) A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T Cells Cell Reports 17:1438–1452

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Benefits of Nucleofector® Technology for CRISPR Cas9 screening

One method – as proven by many publications – to successfully deliver RNPs into primary cells is the Nucleofector® 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 various primary cells, e.g. primary human CD34+ T cells and both mouse and human T cells.
 

Alongside with high transfection efficiencies in primary cells, the Nucleofector® Technology shows high reproducibility and flexibility regarding cell numbers and substrate versatility. Using Nucleofector® Technology you can easily deliver various substrates, such as DNA, mRNA, RNPs or proteins, into primary cells or cell lines, e.g. supporting target identification and target validation approaches.

 

Find further peer-reviewed publications using Nucleofector® Technology for Genome Editing approaches including CRISPR in our Genome Editing Citation.

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How it Works - HT Nucleofector® System (Video Tutorial)

Workflow overview for using the HT Nucleofector® System

With the 4D-Nucleofector® 96-well Unit and the 384-well Nucleofector® System the Nucleofector® Technology offers two medium- to high-throughput platforms which are both suited to combine the potent CRISPR technology with a screening format. Both platforms can be integrated into LHS (liquid handling systems) e.g. from Tecan, Beckman, or Hamilton, thus facilitating the automation of CRISPR library screens with less hands-on time. Watch the video to see the 384-well HT Nucleofector® System in an automated setup.

Additional benefits of the Nucleofector® Technology for CRISPR screening and functional genomics

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

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® Technology provides a tool that supports arrayed crispr screening and facilitates target identification or validation in less time. 

CRISPR in drug discovery
Virtual event
Watch our Virtual Event CRISPR in Drug Discovery and learn how researchers set up CRISPR Screens
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Genome editing using Nucleofector® Technology

Technical Reference Guide

Containing a brief introduction to genome editing tools and technical tips for ZFN, TALEN or CRISPR delivery using the Nucleofector® Technology

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384-well Nucleofector® System
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4D-Nucleofector® 96-well Unit
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Important note: The user bears the sole responsibility for determining the existence of any third party rights, as well as obtaining any necessary licenses.