Table 1 Major therapeutic genome editing approaches
From: To cleave or not to cleave: therapeutic gene editing with and without programmable nucleases
Method | DNA size | Types of edit achievable | Recognition length (nucleotides) | Frequency of target sequences | Cleavage type | Companies | Key advantages | Potential disadvantages |
|---|---|---|---|---|---|---|---|---|
Protein programmable nucleases (ZFNs and TALENs) | ~1–3 kb coding region | • Knockout • Knockin • Deletions • Precision point edits | ~20–40 | ~30–200 base pairs depending on class of nuclease | Blunt or overhang | Thermo Fisher, Cellectis, Sangamo, Two Blades and Precision BioSciences | • Advanced versions with higher specificity • High editing efficiency • Targeted whole-gene (cDNA) replacement mode | • Off-target cleavage • Time to generate longer than CRISPR or exogenous editing oligonucleotides • Requires exogenous protein expression, which adds to complexity of clinical applications |
CRISPR–Cas9 (ribonucleoprotein) | ~3.5–4.5 kb | • Knockout • Knockin • Deletions • Precision point edits | 17–20 | ~22 base pairs | Blunt; alternative Cas9s with overhangs | DuPont, Caribou, Editas, CRISPR Therapeutics, Cellectis and ToolGen | • Extreme ease of design • Low cost as a reagent • High targetability and multiplexing • Targeted whole-gene (cDNA) replacement mode | • Off-target cleavage • Intellectual property disputes; potentially many licences required • Requires exogenous protein expression, which adds to complexity of clinical applications |
Editing by nucleobase modification | ~2–5 kb | Precision point edits | ~20–40 | From every base pair to 22 base pairs | No cleavage | None? | • No random indels • Potential for high-efficiency point changes | • Only point changes, cannot achieve indels • Different design for each type of sequence change (for example, deamination for A to I (G)) • Early stage • Requires exogenous protein expression, which adds to complexity of clinical applications |
Chemically modified editing oligonucleotide | Typically 20–70 nucleotides | Precision point edits (small indels) | 20–70 | Every base pair | No cleavage | ETAGEN Pharma | • Low off-target activity • Ease of design • Ease of delivery in vivo • Multiple treatments, cumulative editing • Established GMP manufacturing infrastructure | • Low efficiency per treatment in most cases • No targeted whole-gene (cDNA) replacement mode |
Recombinogenic AAV | ~5 kb genome | • Knockin • Deletions • Precision point edits | Up to several kb | Every base pair | No cleavage | • Universal Cells • LogicBIO • Homology Medicine | • No target cleavage • Ease of design • Whole-gene (cDNA) replacement mode | • Low efficiency for early formats • Requires a gene therapy vector, which adds to complexity of clinical applications |
