Neb5α competent e coli cells

After verifying the Dicer1e sequence, the gene was custom synthesized by GenScript (Piscataway, NJ) from the start to stop codons with NotI and ApaI restriction sites added 5′ and 3′ relative to each codon, respectively. The full-length gene was then cloned by GenScript into the EcoRV site within the pUC57 plasmid vector, after which it was subsequently subcloned in-frame into pGen2.1, a mammalian N-terminal FLAG-tag gene expression vector, using the NotI and ApaI restriction sites. All constructs were verified by DNA sequencing. Upon receiving the the pGen2.1-Dicer1e plasmid construct, the contruct was transformed into NEB 5-α competent E. coli cells (New England BioLabs, Ipswich, MA) and purified for transfection purposes using the EndoFree^® Plasmid Maxi kit (Qiagen).

All expressed ORFs used in these studies—including wild-type human disease-associated ORFs, human ORFs with constructed alleles, and the GFP control—were transferred into the destination vector pCM188- URA [55 (link)] by Gateway LR reactions using the All Gateway LR Clonase enzyme kit from Life Technologies. The destination vector pCM188-URA was obtained from ATCC, and subsequently altered to be Gateway compatible following the procedure applied in Sun et al (Genome Research 2016) to vectors pHYCDest-LEU2 and pHYCDest-NatMX. Plasmids generated by Gateway LR cloning were transformed into NEB5α competent E. coli cells (New England Biolabs) and selected on LB Agar plates with 100μg/mL Ampicillin. All plasmid DNA samples were isolated and purified using the NucleoSpin 96 Plasmid toolkit (Ref: 740625.24) and confirmed by Sanger sequencing. Plasmids carrying expressed ORFs were then transformed into the corresponding yeast temperature-sensitive strains.

A Gateway cloning destination vector was constructed from the pHiDest-DB (CEN/ARS-based, ADH1 promoter, and LEU2 marker). Two-step modification of the original pHiDest-DB resulted in two Gateway-compatible destination vectors with different selection markers. First, the entire GAL4 DNA-binding domain was deleted from the pHiDest-DB resulting in pHYCDest-LEU2 (for use in strains for which the ts allele is linked to NatMX). Next, pHYCDest-natMX (for use in strains for which the ts allele is linked to KanMX) was constructed by replacing LEU2 with natMX in pHYC-LEU2. These modifications were achieved by separate PCR amplification of the pHiDest-DB backbone and natMX4 cassette followed by homologous recombination in yeast. Sequences of primers used are listed in Supplemental Table S5.
The wild-type or mutated disease-associated ORFs and the GFP gene were transferred into the pHYCDest by Gateway LR reactions followed by transformation into NEB5α competent E. coli cells (New England Biolabs) and selection for ampicillin resistance. After confirmation of ORF identity and expected mutations by Sanger sequencing, plasmids expressing wild-type ORFs, mutated ORFs, and GFP were further transformed into the corresponding yeast ts or haploid-convertible heterozygous diploid knockout mutants.

Inserts of the fusion constructs were prepared by overlap-extension PCR. The expression constructs were cloned into pNIC28-Bsa4 plasmids (Addgene plasmid # 26103) using SLIC restriction free cloning method^{44 (link)}. The insert encoding TNKS2 ARC4 was cloned into pNIC28-MBP. We prepared pNIC28-MBP by insertion of the sequence for E. coli maltose binding protein (UniProt ID: P0AEX9, MBP_27-392) between His₆-tag and TEV protease cleavage site of pNIC28-Bsa4. Briefly, the pNIC28-Bsa4 or pNIC28-MBP plasmids were linearized and 100 ng of linearized plasmids were mixed in a 1:3 molar ratio with PCR products of the expression constructs (Table S1) and incubated with T4 polymerase for 2.5 min at room temperature and for 10 min at 4 °C. The mixture was used to transform NEB 5α competent E. coli cells (New England BioLabs) according to manufacturer’s instructions. Colonies were grown on LB agar containing 5% sucrose using the SacB-based negative selection marker^{45 (link),46 (link)}. All constructs were verified by sequencing of the insert regions.

The sense and antisense guide RNA oligonucleotides were synthesized (Table 1) with overhangs (F overhang is CACC and R overhang is CAAC). Each pair of oligonucleotides was annealed and phosphorylated with T4 PNK (NEB, USA) in a thermocycler using the following parameters: 37 °C for 30 min, 95 °C for 5 min and then ramped down to 25 °C at 5 °C/min. The modified guide expression backbones were digested with Bbs I (NEB, USA) for 30 min at 37 °C. Subsequently, the phosphorylated and annealed oligo duplex was ligated into the corresponding Bbs I-digested guide expression backbone by Quick Ligase (NEB, USA) at room temperature for 10 min. Then, NEB 5α competent E. coli cells (NEB, USA) were transformed using 2 μl of the assembled product. The plasmids were extracted from overnight culture using a Plasmid Miniprep Kit (MN, Germany) and verified by sequencing. Finally, a mixture of the constructed guide RNA backbone and Cas13b destination plasmid was treated with BsmB I (NEB, USA) and T4 DNA ligase (NEB, USA) in a thermocycler with the following program: 30 cycles of 5 min at 37 °C and 5 min at 16 °C followed by 5 min at 60 °C. After transformation into One Shot Stbl 3 chemically-competent E. coli cells (Transgene, China), the plasmids were prepared by a Plasmid Midiprep Kit (Qiagen, Germany).