Miseq 2 300 platform

AF were stored at −80°C immediately after collected. Total bacterial DNA was extracted from AF samples using QIAamp DNA Mini Kit (Qiagen, Valencia, CA) according to manufacture's instruction. Bacterial 16S rRNA V3-V4 region was amplified using the 343F/798R primer set (343F 5′-TACGGRAGGCAGCAG-3′, 798R 5′-AGGGTATCTAATCCT-3′). PCR reaction was performed using phusion high-fidelity PCR Mastermix (Invitrogen, Carlsbad, CA, USA) with the following condition: 95°C for 3 min (1 cycle), 95°C for 30 s /55°C for 30s /72°C for 30s (35 cycles), 72°C for 10 min. PCR product was purified using Agencourt AMPure XP beads (Beckman coulter, Brea, CA) according to manufacture's protocol. Pyrosequencing was conducted on an Illumina Miseq 2^*300 platform according to protocols.

Microbial DNA was isolated from rumen samples using a repeated bead beating plus column method [15 (link)]. The V1–V3 region of the bacterial 16S rRNA gene was PCR-amplified using the 27F forward [16 (link)] and 519R reverse [17 (link)] primer pair. PCR reactions were performed with the Phusion Taq DNA polymerase (Thermo Scientific, Waltham, MA, USA) under the following conditions: Hot start (4 min, 98 °C), followed by 35 cycles of denaturation (10 s, 98 °C), annealing (30 s, 50 °C) and extension (30 s, 72 °C), then ending with a final extension period (10 min, 72 °C). PCR products were separated by agarose gel electrophoresis, and amplicons of the expected size (~500 bp) were excised for gel purification using the QiaexII Gel extraction kit (Qiagen, Hilden, Germany). For each sample, approximately 400 ng of amplified DNA were submitted to Molecular Research DNA (MRDNA, Shallowater, TX, USA) for sequencing with the Illumina MiSeq 2 × 300 platform to generate overlapping paired end reads. Briefly, libraries for 16S rRNA amplicons were prepared using overhanging primers targeting the 27F and 519R recognition sequences; the primer overhangs encoded barcodes for sample indexing as well as adapter sequences. MiSeq 2 × 300 sequencing was performed using the MiSeq Reagent Kit v3 following the manufacturers specifications.

The total DNA from vaginal swabs was extracted using the modified protocol MoBio Powersoil modified Method #3 as previously published (Mattei et al., 2019 (link)). DNA concentration was measured using Nanodrop.
The V1-V3 regions of the 16S rDNA were amplified using forward primers: 27F with 12 bp golay barcodes containing a specific Illumina 5’ adapter for each sample and a common reverse primer 515 R (Mattei et al., 2019 (link)). In brief, PCR was performed in triplicate in a 50 μl reaction mixture containing 10 ng of template DNA and 2x Phusion HotStart Ready Mix. The following thermal cycling conditions were used: 5 min of initial denaturation at 94°C; 25 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 30 s, and elongation at 72°C for 30 s; and the last step at 72°C for 10 min. The amplified PCR products of approximately 650 bp in size from each sample were pooled in equimolar concentrations. This pooled PCR product was purified using AgenCourt AMPure XP magnetic beads. High throughput sequencing was performed on an Illumina MiSeq 2 × 300 platform (Illumina, Inc. San Diego) in accordance with the manufacturer’s instructions. Image analysis and base calling were carried out directly on the MiSeq.

HEK-293T cells were transfected using jetPRIME (Polyplus-transfection, Strasbourg, Alsace, France) in accordance with the manufacturer’s instructions. When the confluency of cells seeded on six-well plates reached approximately 70%, the cells were transfected with 1500 ng of pCMV-PE2 plasmid and 500 ng of pegRNA. In this experiment, 12 plasmids of pegRNAs were transfected with different lengths of PBS and RT templates: six plasmids with nicking sgRNA (1319N, 1324N, 1327N, 1519N, 1524N, and 1527N) and six plasmids without nicking sgRNA (1319, 1324, 1327, 1519, 1524, and 1527). Genomic DNA was extracted from the transfected cells 72 h after transfection. The region encompassing the targeted locus was PCR amplified using Phanta^® Max SuperFidelity DNA polymerase (Vazyme, Nanjing, China) and detected by Sanger sequencing and targeted deep sequencing. The primers used for PCR and targeted deep sequencing are listed in Supplementary Table S1. PCR amplicons were sequenced using an Illumina Miseq (2 × 300) platform at Sangon Biotec, Shanghai, China. The reads with targeted-deletion (sequence: GTAAGTTTATG) and lengths more than 200 nt were counted. The editing efficiency (Figure 1D) was calculated as the ratio of the summation reads to the total reads.

Fresh feces samples were collected in a sterile polypropylene container, frozen, and kept at −70 °C until processing. Later, bacterial DNA was obtained by DNA stool kit (QIAGEN), following the instructions of the manufacturer. A spectrophotometer was employed to determine DNA concentration. Specific primers were used for the sequencing of the V3–V4 hypervariable region. DNA libraries were sequenced at the Unidad de Secuenciación at INMEGEN by an Illumina Miseq 2 × 300 platform (Illumina, San Diego, CA, USA). Processing of the Illumina FASTQ reads was performed using the quantitative insights into microbial ecology (QIIME 1.8) software package [14 (link)]. The UCHIME algorithm was used for detection and removal of chimeric sequences [14 (link)]. An alignment to the Greengenes database was performed using some representative sequences (sequences are available in https://www.ncbi.nlm.nih.gov/sra/PRJNA649973).