Which platform has lowest off-target mutation rate?
Off-target mutations determine safety for therapeutic genome editing. CRISPR tolerates mismatches distal to PAM generating relatively higher off-target cleavage. ZFNs and TALENs show lower tolerance due to longer protein-DNA interface and requirement for dimerization, yet still recognize near cognate sites. Homing endonucleases or meganucleases like I-SceI recognize 18-24 base pair asymmetric sequences, often exceeding 20 base pairs of specificity, resulting in rarity of cognate site in complex genome and extremely low off-target rate. Their long recognition site accounts for highest specificity among editing nucleases.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.