Ultrapure water

TQ5500 triple quadrupole mass spectrometer (AB Sciex, Framingham, MA, USA); Acquity (Waters, Milford, MA, USA) and Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm, Waters, MA, USA); AL204-IC electronic analytical balance (METTLER TOLEDO, Zürich, Switzerland); Talboys digital display vortex oscillator (Anpu, Shanghai, China); high-speed desktop centrifuge (Sorvall ST 16R, Thermo Scientific, Waltham, MA, USA); ultra-pure water (18.2 MΩ·cm, Merck, Darmstadt, Germany).

Twenty-six ovarian genome samples (containing 18 validation samples) of wild-type and mutant FecB of small-tailed Han sheep were collected from the Hengshui test sheep farm in Hebei Province and cryopreserved. The animal study protocol was approved by the institutional review board of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (protocol code IAS2024-180 and date 16 March 2024). Among them, BB represents mutant type, ++ represents wild-type, and B+ represents heterozygous type. Based on the Cas12a pronuclease spacer adjacent sequence (PAM) site, a crRNA was constructed to correspond to FecB mutant sequence and then synthesized by Integrated DNA Technologies (Coralville, IA, USA) and cryopreserved. Cas12a nuclease was obtained from Beijing Zeping Technology Company (Beijing, China). TCEP and ethanol were sourced from Shanghai Maclin Biochemical Technology Company (Shanghai, China), while Tris-HCl buffer with varying pH values was purchased from Leaf Biology (Shanghai, China). Sodium acetate was provided by Sigma-Aldrich (Beijing, China), and acetic acid (CH₃COOH) was procured from Shanghai Aladdin Company (Shanghai, China). Reagents and kits included a magnetic bead-based animal genomic DNA extraction kit and an EZ-10 column DNA gel recovery kit, both purchased from Bioengineering (Shanghai, China) Company. Additional materials, including 2× SanTaq PCR Mix premix, DNA molecular weight standard marker (100–2000 bp), 50× TAE buffer, 10,000× 4S GelRed nucleic acid dye, 6× glycerol gel loading buffer VII, primers, and fluorescent reporter genes, were obtained from Shenggong Bioengineering (Shanghai, China) Company. The specific sequences are listed in Table 1. All experiments used ultra-pure water (Millipore, Burlington, MA, USA), and all chemical reagents were of analytical grade, requiring no further purification.

Sulfuric acid (H₂SO₄) (95–97%, Sigma-Aldrich),
sodium nitrite (NaNO₂, ISOLAB), sodium chloride (NaCl)
(>99.8%, Sigma-Aldrich), anhydrous
sodium sulfate (Na₂SO₄) (Sigma-Aldrich), diethyl
ether (99.5%, Sigma-Aldrich), hexane (95%, Sigma-Aldrich), l-Valine (l-2-amino-3-methylbutanoic acid) (98%, Sigma-Aldrich), l-Leucine (l-2-amino-4-methylpentanoic acid) (99%,
Sigma-Aldrich), p-toluene sulfonic acid monohydrate (PTSA) (≥98%,
Sigma-Aldrich), and toluene (99.5%, ISOLAB) were used for monomer
synthesis. Sn(Oct)₂ (94.5%, Sigma-Aldrich), benzyl alcohol
(>99%, TCI), glycolide (94.5%, Sigma), calcium hydride (CaH₂) (93%, Acros Organics), dichloromethane (DCM) (≥99%,
ISOLAB),
and methanol (≥99.8%, ISOLAB) were employed in the synthesis
and purification of copolymers. DCM was distilled over CaH₂, and methanol was dried over magnesium and iodine. l-DIBG, l-DIPG, and glycolide were dried by azeotropic distillation
at 35 °C in dry toluene, which was previously distilled over
sodium metal in the presence of a benzophenone indicator, prior to
the copolymerization reactions. The benzyl alcohol initiator was dried
by vacuum distillation over CaH₂. Tetrahydrofuran (THF,
99.9%, HPLC grade) was supplied from Sigma-Aldrich and employed as
a mobile phase in the GPC measurements. Ultrapure water, ethylene
glycol (EG), formamide, α-bromonaphthalene, methylene iodide,
and hexadecane were purchased from Merck. Glass slides (76 ×
26 mm, ISOLAB, Türkiye) were employed as substrates.