Dp304

Total RNA was obtained from cells with TRIzol (Invitrogen, United States), while the cDNA was reverse-transcribed via utilizing PrimeScript RT reagent Kit (TaKaRa) following the manufacturer’s instructions. The genome DNA was isolated by special extraction kit (DP304, TIANGEN, China). SYBR Green PCR Master Mix (TaKaRa) and Applied Bio-systems 7500 Fast Real-Time RCR System (Applied Biosystems, United States) were used for RT-qPCR analysis. Data were acquired from three independent experiments and normalized to GAPDH. All the primers were shown in Supplementary Table 2.

Seventy-five percent of the edited cells (~500,000 cells) were collected every two days from day 20 to day 38 after cell sorting and washed once in a PBS buffer solution. Genome extractions were carried out according to the instructions of the kit (DP304, Tiangen, Beijing, China) and eluted with 80 μL distilled water for downstream analysis. Amplification of edited loci was performed with the locus-specific primer pairs described in Table S2 (Supplementary Materials) using 2 × Q5 master mix (M0494L, New England Biolabs, Ipswich, MA, USA) and 200 ng of genomic DNA. The thermocycler was set for one cycle at 98 °C for 30 s, 35 cycles at 98 °C for 15 s, 60 °C for 15 s, and 72 °C for 10 s, respectively, and one cycle at 72 °C for 1 min, and held at 4 °C. PCR amplicons were run on a 1.5% agarose gel to verify the size and purity, and quantified by nanodrop. The resulting DNA was used for direct analysis or reamplified with primers containing Illumina adaptors.

Blood samples were collected from the subjects. According to the manufacturer’s instructions, blood genome DNA was extracted using a DNA extraction kit (TIANGEN, DP304). Subsequently, amplification was performed by touchdown PCR technology. Finally, sequencing analysis was conducted to obtain the ALDH2 genotype. DNA extraction, PCR amplification and sequencing were carried out by Jinan Bo Shang Biotechnology Co., LTD., China.

DNA was extracted from skin samples using a blood/cell/tissue genomic DNA extraction kit (DP304; Tiangen Biotech Co., Ltd., Beijing, China). DNA concentrations were all above 4 ng/μl.
Three sets of primers were used to amplify the target sequences of the control region (D-loop), Cytochrome b (Cyt b), and Cytochrome C Oxidase subunit I (COI) (Table 1).
PCR was performed in 50 µl reactions, with 25 µl of 2×Taq Mix (RN03001S, MonAmp™), 2 µl of template DNA, 2 µl of each 10 uM primer, and ultrapure water to 50 µl. The PCR conditions of D-loop as follows: pre-denaturation at 94 °C for 3 min; followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 51 °C for 30 s, extension at 72 °C for 60 s; and final extension at 72 °C for 3 min. The PCR conditions of Cyt b as follows: pre-denaturation at 94 °C for 80 s; followed by 30 cycles of denaturation at 94 °C for 42 s, annealing at 47 °C for 30 s, extension at 72 °C for 80 s; and final extension at 72 °C for 5 min. The PCR conditions of COI as follows: pre-denaturation at 94 °C for 80 s; followed by 30 cycles of denaturation at 94 °C for 42 s, annealing at 55 °C for 30 s, extension at 72 °C for 80 s, and final extension at 72 °C for 5 min. PCR products were checked with a 1% agarose gel and successful reactions purified and Sanger sequenced using PCR primers at Guangzhou Ige Biotechnology Co., Ltd.

Parasite quantification in tissues was performed following the methods by Santoro et al. [18 (link)]. Animal tissues were collected at 8 days post infection and stored in individual vials at − 20 °C before genomic DNA extraction. For each sample, 1 g of tissue was individually homogenized, and 200 µl of the homogenate was used to extract genomic DNA according to the manufacturer’s protocols (DP304, Tiangen, China). A standard curve was obtained by linear regression analysis of the threshold cycle (Ct) value (y-axis) versus the log of the initial copy number present in each sample dilution (x-axis). PCR efficiency (E) was calculated as E = 10 (1/slope) ⁻¹.

Total genome of jejunum was prepared using a genome extraction kit (TianGen, China, DP304). Four pairs of primers were used to amplify region 1611-5657, amplified with a forward primer (5′-AAAAGCAGCCACCAATAAAG-3′) and a reverse primer (5′-GAGAAATGATGGTGGTAGGA-3′), region 5636-9914, amplified with a forward primer (5′-ACTCCTACCACCATCATTTC-3′) and a reverse primer (5′-GAGAAGGCTATGGTGAGGTT-3′), region 9876-14278, amplified with a forward primer (5′-TATGCCATCTACCTTCTTCA-3′) and a reverse primer (5′-ATTTGGACTATTAGGCAGAC-3′) and region 14258-1611, amplified with a forward primer (5′-AGTCTGCCTAATAGTCCAAA-3′) and a reverse primer (5′-CTTTATTGGTGGCTGCTTTT-3′), which covers the whole mtDNA sequence.
PCR of mtDNA using the genome of jejunum as template was performed, and the four amplified fragments were mixed for sequencing. Briefly, 3 μg mixture of mtDNA was used to construct a library as described^{85 (link)} for a better homogeneity of sequencing depth. Then, the library was sent to Berry Genomics. Co., Ltd (Beijing, China) and sequenced on an Illumina Hiseq4000 platform using 150 bp paired end reads for 3 G flux. mtDNA mutations were analyzed as described^{15 (link)}.