Introduction to Genome Editing

The process of genome editing involves the use of custom engineered endonucleases, or “molecular scissors,” to accurately modify endogenous genomic DNA sequences. Engineered endonucleases most commonly consist of a customizable DNA binding domain (DBD) attached to a non-specific endonuclease, enabling double strand breaks (DSBs) to be introduced at specific genomic loci. DSBs are repaired by one of two cellular DNA repair mechanisms: non-homologous end joining (NHEJ) or homology-directed repair (HDR). Repair by NHEJ typically introduces small insertions or deletions (indels) potentially leading to a frameshift mutation and ultimately, a functional gene knockout. Precise nucleotide corrections, generation of reporter lines, and precise gene deletions or insertions are accomplished by co-delivery of an exogenous donor DNA template integrated by HDR (Choulika et al., 1995).

Development of the CRISPR/Cas9 (Clustered Regularly-Interspaced Short Palindromic Repeats/CRISPR Associated Protein 9) system has made genome editing more accessible to the wider scientific community (Mali et al., 2009). Originally derived from Streptococcus pyogenes, the system consists of Cas9 nuclease and a guide RNA (gRNA), containing either a single guide RNA (sgRNA) or the two-part CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The gRNA harbors a customizable sequence approximately 20 nt in length that guides Cas9 to the target sequence for the creation of a double-strand break. The relative ease of gRNA synthesis has established CRISPR/Cas9 as the genome editing tool of choice.

Off-target editing is currently a major concern within the genome editing field, with the specificity of the early systems in question. Modified versions of wild-type Cas9 are being developed to increase specificity. For example, a mutant form of Cas9 known as Cas9 nickase (Cas9D10A) can only create single-strand nicks at the specified target and does not initiate DNA repair (Chiang et al., 2016). When two complexes are targeted to opposite strands in close proximity, a double-strand break occurs, which initiates DNA repair. Cas9 nickase, as well as other improved methods, are paving the way for the use of CRISPR/Cas9 in future cell and gene therapies.

The relative ease of sgRNA synthesis has established CRISPR/Cas9 as the genome editing tool of choice, ahead of ZFNs and TALENs whose complex protein engineering has proved to be a stumbling block in achieving ease-of-use. However, specificity of the CRISPR/Cas9 system is frequently questioned. ZFNs and TALENs must dimerize in order for FokI nuclease activity to occur, making the DNA binding process highly specific. In contrast, Cas9 functions as a monomer, reducing sequence specificity and increasing the risk of encountering and cleaving “off-target” sequences in the genome. As a result, researchers have begun replacing Cas9 nuclease with Cas9 nickase which produces single strand breaks (SSBs) or ‘nicks’ rather than DSBs. When Cas9 nickase is employed as a pair, each cleaving one of two DNA strands at two adjacent sites, a DSB is introduced. This improved method for reducing off-target effects with the CRISPR/Cas9 system is paving the way for its use in future cell and gene therapies.

Key Publications

• Choulika et al. demonstrate induced homologous recombination in chromosomal DNA by the double-strand break repair mechanism in mammals using the mitochondrial intron-encoded endonuclease I-SceI of Saccharomyces cerevisiae.
  Choulika A et al (1995) Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol Cell Biol 15(4): 1968-73.
• Ding et al. compare the relative efficacies of CRISPRs and TALENs. They found that CRISPRs consistently and substantially outperformed TALENs in their system.
  Ding Q et al. (2013) Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell 12(4): 393-4.
• The authors report on the use of the CRISPR/Cas9 system for human genome engineering. The type II bacterial CRISPR system was modified to function with custom gRNA in human cells. This resulted in efficient and robust genome editing, as well as multiplex editing of target loci upon simultaneous addition of multiple gRNAs.
  Mali P et al. (2013) RNA-guided human genome engineering via Cas9. Science 339(6121): 823-6.
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• Ran et al. describe a highly efficient approach to generate isogenic gene knockouts and knock-ins with minimal off-target effects using a combination of catalytic mutant Cas9^D10A nickase and pairs of guide RNAs to introduce targeted double-strand breaks.
  Ran FA et al. (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154(6): 1380-9.
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