Fig. 4.

Mutated loxp sites with decreased homology mitigate spontaneous recombination and leak expression of sensitive transgenes from an ORF-disrupted AAV design. (A) Schematic of the directionally biased Cre-dependent recombination reaction when using mutant lox sites. Asterisks denote locations of mutations. (B) Candidate canonical and mutant lox sites with known compatibility and decreased homology. Nonhomologous bases within pairs are shown in red. (C) The frequency of spontaneous recombination in the pUC19-inv_LacZ test when using canonical and mutant loxp sequences as measured by qPCR. Circles indicate individual measurements, mean shown by a vertical line, and horizontal lines signify SD. n = 7. For a full list of tested sequences and recombination rates, see SI Appendix, Fig. S3. (D) Sequence homology, as measured by 1 minus the normalized Levenshtein distance between within-pair sequences, plotted against mean recombinant frequency as measured by qPCR in the pUC19_inv_LacZ test. Line of best fit was calculated using loxp derivatives (blue crosses). R² = 0.9632. NHS condition shown for comparison (pink cross). (E) Images of brain tissue slices following injections of equal quantities of AAV8 nef-DIO-FlpO (Left column) and AAV8 nef-CIAO-Flp (Right column) into primary visual cortex of RCF-tdTomato x Pvalb^Cre (Top) and RCF-tdTomato (Bottom). Asterisk in AAV model denotes removal of ATG start codon from Flp ORF. Flp-dependent tdTomato expression is in red and parvalbumin antibody in green. (Scale bars, 100 um.) (F) Quantification of total tdTomato⁺ neurons in RCF-tdTomato mice injected with AAV8 nef-DIO-FlpO (n = 5) and AAV8 nef-CIAO-Flp (n = 6). Error bars indicate SD; P = 0.0225, 2-tailed independent t test (asterisk).