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The seminal publications in 2013 describing the ability of CRISPR/Cas9 to edit mammalian genomes have led to an explosion in the ability to make precise genetic changes within mammalian cells and animal models using a variety of CRISPR‐based editing methods (Cong et al. 2013; Mali et al. 2013). Since these publications, there has been a dramatic increase in the efficiency and variety of genome editing methods available with these methods now in widespread use in the pharmaceutical industry and academia for target and drug discovery, alongside the discovery and characterization of a number of new editing enzymes and the development of forms of Cas9 with improved editing activity (Gilbert et al. 2014; Kampmann 2018).

CRISPR/Cas is part of the bacterial immune response system where its natural role is to recognize and destroy non‐host nucleic acid sequences as part of the host immune defense system (Wiedenheft et al. 2012). The commonly used laboratory CRISPR system uses the Cas9 nuclease cloned from Streptococcus pyrogenes (SpCas9) although additional Cas9 enzymes have been cloned and characterized (Acharya et al. 2019). In contrast to TALENs and Zinc Finger technology, CRISPR/Cas9 is simple to use in any laboratory. It does not require protein engineering to create a nuclease able to recognize a specific site within the genome, rather targeting of the Cas9 nuclease to specific sites within the genome is mediated through the design of a specific synthetic guide RNA (sgRNA), complementary in sequence to the region of the genome to be targeted, that positions Cas9 at the target site in the genome to result in the creation of a double‐stranded DNA break. Guide RNA design is very straightforward, indeed a number of public‐domain and commercial design tools have become available for the immediate design of highly specific sgRNAs that can be ordered through the Internet and delivered to the laboratory within days. When the sgRNA is introduced into cells alongside SpCas9, the sgRNA recruits the Cas9 nuclease to a specific site in the genome at which a double‐stranded DNA break is introduced into the genome. This is then repaired using the cells’ endogenous DNA repair machinery, 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.