Dna gel purification kit

All PCR products of the expected size were successfully purified using the DNA Gel Purification Kit (Sangon, Shanghai, China) and sent for a bi-directional Sanger sequencing analysis (performed by Sangon, Guangzhou, China). Sequencing was conducted using the BigDyeTerminator v3.1 Cycle Sequencing Kit (from Applied Bio systems, Carlsbad, CA, USA) on an ABI Prism 3730 XL DNA Analyzer. DNASTAR Lasergene EditSeq v7.1.0 (http://www.dnastar.com/) was employed for editing the generated sequences, while Clustal X v2.1 (http://www.clustal.org/) was utilized for aligning them with reference sequences obtained from GenBank.
The data analysis was conducted using SPSS version 22.0 (SPSS Inc., IL, USA). The chi-square test and 95% confidence intervals (CIs) were employed to compare the prevalence of Cryptosporidium between diarrheic and asymptomatic individuals, as well as between boys and girls. A P-value less than 0.05 was considered to be statistically significant.

P. pastoris GS115-Cas9 (his4::Cas9) constructed in previous studies was used as the parent strain [9 (link)]. Restriction enzymes and T4 DNA ligase were purchased from NEB (Ipswish, MA, UK). Phanta DNA polymerase and 2 × Taq PCR mix were purchased from Vazyme (Nanjing, China). DNA gel purification kit and plasmid extraction kit were purchased from Sangon Biotech (Shanghai, China). TAL standard was purchased from TCI (Shanghai, China). All chemicals were purchased from Sangon Biotech (Shanghai, China) unless stated otherwise.

The rational sgRNAs, corresponding to particular loci of target genes, were designed according to the previous protocol to obtain the gene-targeted strain (Jiao et al., 2019 (link)). The α-terpineol synthase (aTS) gene from Vitis vinifera (GenBank ID: AAS79352.1) was codon-optimized and synthesized by Synbio Technologies (Suzhou, China). This synthetic gene has been submitted to NCBI (Accession Number: OQ983656). The truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1) gene was amplified from the plasmid p424-tHMG1 (Zhou et al., 2012 (link)). ERG20 was amplified from the Saccharomyces cerevisiae genome and then mutated to ERG20^ww (F96W/N127W). All plasmids were constructed using the restriction-free cloning method (Unger et al., 2010 (link)). The plasmid extraction kit and DNA gel purification kit were purchased from Sangon Biotech Co., Ltd. (Shanghai, China), and polymerase chain reaction (PCR) enzymes were purchased from Takara Bio (Dalian, China). All the sequences of plasmids and primers are provided in Supplementary Tables S2, 3.