commonly through a process termed NHEJ that introduces a small insertion or deletion into the target gene sequence that results in the expression of a nonfunctional protein. Through changing the sequence of the sgRNA, it is theoretically possible to target Cas9 to any site in the genome. In studies aimed at deletion of the gene of interest, the editing efficiencies seen with CRISPR/Cas9 can exceed 90% and are typically above 50% or more of transfected cells making this a highly efficient tool to study the consequences of a loss of gene function in cells and animal models of disease. Furthermore, due to the simplicity of the system, a single Cas9 enzyme can be introduced into a cell with two or more guide RNA to result in the deletion of large pieces of DNA at a single genetic loci, or the independent deletion of multiple genes in parallel, making CRISPR/Cas9 an highly valuable and flexible tool for the study of gene function in cell and animal models.
The introduction of single nucleotide changes, or the insertion of small sequences of heterologous DNA, into a gene can be mediated through the introduction into a cell of Cas9, a sgRNA and a donor DNA template that contains the new sequence. Following gene repair by HDR, the point mutation or additional gene sequence can be introduced the target gene. In contract to gene deletion which is a highly efficient process, gene editing or repair using HDR is a low‐efficiency process, with typically less than 5% of cells being edited, with much work ongoing to develop methods with improved efficiency. While this is a low‐efficiency process, CRISPR enables precise genetic changes to allow the study of the effect of single nucleotide changes and protein truncation on gene function, and the introduction of affinity or other epitope tags into proteins to allow the study of protein location in cells.
In further applications of the technology, variants of Cas9 have been created in which an enzymatically inactive Cas9, that is no longer able to cut the target DNA, is fused to a transcriptional activator or repressor protein (Gilbert et al. 2014; Kampmann 2018). When recruited to the promoter region of a target gene, these versions of Cas9 are able to mediate an activation or repression of gene expression. Base Editor technology has been developed to address the challenge of creating editing systems with increased efficiency for the introduction of precise genetic changes into genes (Rees and Liu 2018). Base Editors consist of a fusion protein between an enzymatically inactive (one site) Cas9nickase and adenosine or cytosine deaminase. These proteins when introduced into cells alongside a targeting sgRNA mediate the enzymatic modification of a specific nucleotide in the genome. Cytosine base editors mediate the transformation of Cytosine to Thytmidine whereas Adenosine Base Editors mediate the transition from Adenosine to Guanosine. In contrast to the introduction of random indels (insertion/deletions) into cells using Cas9, Base Editors mediate specific editing of the target nucleotide.
A further innovation in gene editing technology arose with the publication of Prime Editing (Anzalone et al. 2019). In this technique, a fusion protein is created between an SpCas9 “nickase,” that rather than creating a double‐stranded break in the genome acts to cut a single strand of the DNA, and a reverse transcriptase. When introduced into cells alongside a “prime editing guide” RNA (pegRNA), the pegRNA targets Cas9 to a precise position in the genome where it creates a single‐stranded break in the DNA strand. In contrast to a sgRNA, the pegRNA encodes both a sequence to target the nuclease to a specific site in the genome and a template RNA sequence to be introduced into the genome. The reverse transcriptase creates a DNA copy of the pegRNA which is then introduced into the genome using the cells’ DNA mismatch repair mechanism, resulting in the insertion of a short piece of DNA. This method has been used to introduce point mutations, new codons, and to insert larger DNA sequences into target genes. This method can again theoretically be used to target any sequence in the genome and in contrast to earlier editing methods can result in highly efficient genome modulation.
The huge interest and range of applications for CRISPR have led to the establishment of a series of new vendor companies able to supply CRISPR reagents, both guide RNAs and editing enzymes, for use by the laboratory scientist. This includes organizations such as Synthego and Horizon Discovery as well as the creation of capability in established reagent supply companies including Merck and Thermo Fisher. As well as supplying CRISPR reagents for use in the scientists’ laboratory, these companies also offer a variety of services including the creation of CRISPR‐edited cell lines and animal models and the completion of Functional Genomic screens. Through the work of these companies, CRISPR technology has become democratized for use by any laboratory competent in basic molecular and cell biology techniques. Some of these commercial reagents are discussed in Chapter 4 of this book.
1.4 Applications of CRISPR Cas9 in Drug Discovery
The ability to precisely edit genomes with CRISPR/Cas9 and other related editing systems has become integral to the identification of new drug targets and the creation of engineered cell and animal models of disease. The impact of CRISPR in drug discovery is discussed at length throughout this book and is briefly introduced here. The field of Functional Genomics has advanced with the generation of whole genome‐wide CRISPR libraries and other reagents that enable the parallel deletion, upregulation, or downregulation of every gene in the genome to ask the question “what is the effect of modulating this gene on the biology of interest?” (Doench 2018). These libraries can be prepared in micro‐titer plate format with each well of the plate containing a cocktail of guide RNAs designed to delete a single gene. When screened against cellular models of disease, it becomes possible to identify specific genes which when modulated affect the biology under study. In addition to use for the identification of new drug targets, Functional Genomic screens are being applied widely to address questions such as the identification of genes that when modulated enable an increase in recombinant protein expression or an increase in the productive uptake of lipid nanoparticles. A further application of Functional Genomic screens in Oncology is the screening of whole genome‐wide CRISPR libraries against multiple cancer cell lines in the presence of known cancer medicines to identify genes that mediate resistance or sensitization to that medicine. Many hundred such screens have been performed at Institutes such as the Broad Institute and the Wellcome Trust Sanger Centre to create public domain databases that describe so‐called sensitivity maps of cancer types to drug action (Behan et al. 2019; Cui et al. 2021). Such studies are enabling the targeting of new medicines to specific tumor types, the identification of likely resistance mechanisms to new medicines, and drug combination opportunities in the clinic.
CRISPR is widely applied to create cellular and animal models of disease, both for the identification of new drug targets and for understanding the efficacy of new drug candidates within a discovery program (Lundin et al. 2020). CRISPR is used to create specific mutations in genes to understand the effect of that mutation on gene function and to introduce molecular tags into genes to track gene expression. The latter approach has been widely adapted to characterize the efficacy of Proteolysis Targeting Chimeras (PROTACs) drugs. PROTACs are a recently discovered class of small‐molecule drugs that rather than inhibiting the function of a drug target, act to degrade the target protein. To understand the efficacy of PROTAC drugs in cellular models of disease, the drug target is typically tagged with a short protein sequence that enables the creation of assays that allow PROTAC‐mediated degradation of the target to be followed in real time in an immortalized cell line or animal model of disease. CRISPR has revolutionized the ability to generate transgenic animal models of disease, both reducing the timelines and number of animals required for the creation of an animal model through the ability to highly efficiently edit the genome of the single cell embryo, while again enabling the creation of complex models of disease not previously possible.
CRISPR is being widely applied in the field of CAR‐T cell therapy both to enable precise insertion of the CAR, but also to identify and delete other T‐cell genes to enable improved efficacy of the cell product (Liu et al. 2017). There is also huge interest in the potential of CRISPR as a medicine in its own right to correct gene mutations in rare and perhaps common diseases and a number of biotechnology companies have been established to bring CRISPR medicines to the clinic, including Editas, CRISPR Therapeutics, Beam Therapeutics, Verve Therapeutics and Intellia. The first clinical studies of medicines to treat β‐thalassemia
