*2.1. Animals*

Heterozygous (K42E/+) *Dhdds* knock-in (KI) mice were generated on a C57Bl/6J background by Applied StemCell (Milipitas, CA, USA). Briefly, CRISPR guide RNA (5-TCGCTATGCCAAGAAGTGTC-3 with PAM site AGG) was generated using in vitro transcription and was used to create a double strand break in the murine *Dhdds* locus to promote introduction of a single-stranded oligodeoxynucleotide (SSO) carrying the K42E mutation and a second silent DNA polymorphism to eliminate the PAM recognition site required for cleavage by CAS9 (5-ATTATCTGTTCTCTTCTACAGGCTGGCCCAGTACCCAAACATATCGCGTTCATAATGGACGGC AACCGTCGCTATGCCAAGGAGTGTCAAGTGGAGCGCCAGGAGGGCCACACACAGGGCTTCA ATAAGCTTGCTGAGGTGGGTGCGGGTGACAGAGCCTAGA-3). Mouse zygotes were injected with 100, 100, and 250 ng/μ<sup>L</sup> of Cas9 enzyme, guide RNA, and SSO, respectively, which were then transferred into pseudo pregnan<sup>t</sup> CD-1 females. Three potential founder (F0) pups were identified out of 13 mice tested, and an F0 founder was verified by DNA sequence analysis. Sequence-validated heterozygous (*Dhdds*K42E/+) mice were crossed to generate homozygous (*Dhdds*K42E/K42E) mice, as confirmed by PCR and DNA sequencing (see below). C57Bl/6J wild type (WT) mice, age- and sex-matched, were used as controls. All procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Alabama at Birmingham. All animals were maintained on a standard 12/12 h light/dark cycle (20–40 lux ambient room illumination), fed standard rodent chow, provided water ad libitum, and housed in plastic cages with standard rodent bedding.

### *2.2. PCR Genotyping and DNA Analysis*

PCR primers were designed that spanned the targeted region (forward primer, 5-TCTAGGCTCTGTCACCCGCA-3 and reverse primer 5-TCTAGGCTCTGTCACCCGCA-3) amplifying a 292 bp segmen<sup>t</sup> of DNA in both WT and *Dhdds*K42E/K42E mice. For initial verification of the knock-in, PCR products were sequenced in the UAB Heflin Center for Genomic Sciences. The presence

of the knock-in sequence was confirmed in subsequent generations by restriction enzyme digestion with StyI, which cleaves the knock-in allele only (data not shown). Knock-in alleles were independently verified by Transnetyx, Inc. (Cordova, TN, USA) using proprietary technology. While the analysis was set up to recognize and differentiate the knock-in mutation and the PAM site polymorphism, only the knock-in mutation was maintained in all subsequent breeding.

### *2.3. Spectral Domain Optical Coherence Tomography (SD-OCT)*

*In vivo* retinal imaging was performed as previously described in detail by DeRamus et al. [15], using a Bioptigen Model 840 Envisu Class-R high-resolution SD-OCT instrument (Bioptigen/Leica, Inc.; Durham, NC, USA). Data were collected from *Dhdds*K42E/K42E and WT mice at postnatal day (PN) 1 (KI, n = 5; WT, n = 9), 2 (KI, n = 4; WT, n = 8), 3 (KI, n = 5; WT n = 3), 8 (KI, n = 4; WT n = 5), and 12 months (mos) (KI, n = 3; WT n = 3) to assess retinal structure. Layer thicknesses were determined manually using Bioptigen InVivoVue® and Bioptigen Diver® V. 3.4.4 software and the data were analyzed and graphed using Microsoft Excel software.
