Pspcas9 bb 2a puro

The sgRNA-23b and sgRNA-27b were designed using the Benchling program (Supplementary Figure S7) and inserted into pSpCas9(BB)-2A-Puro (Addgene 62988) as described previously [22 (link)]. HCT-Oxa-c cells (15 × 10⁴ cells/well) were seeded in 24-well plates 24 h before transfection and constructs were transfected into HCT-Oxa-c cells using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacturer’s instructions. After 48 h of transfection, cells were incubated for 48–72 h with 2 µg/mL puromycin (Sigma). Then puromycin-resistant cells were isolated through serial dilutions, seeding one cell per well of 96-well plates. Cells were grown in an incubator for 1–2 weeks, when cultivated into 24-well plates. Some of the cells were collected into tubes and indels were detected as described previously using polyacrylamide gel electrophoresis [23 (link)]. PCR was carried out with the Phire Tissue Direct PCR master Mix (Thermo Fisher Scientific). The list of primers and single-guide RNA (sgRNA) sequences (Metabion) are provided in Supplementary Table S15.

HLA-G knockout JEG-3 cells were generated using CRISPR/Cas9-based gene editing technology (20 (link)). As described previously, small guide RNAs were synthesized by the Shanghai Sheng Gong Company (21 (link)) (Supplemental Table 1); they were then cloned into the sg-RNA expression vector pSpCas9(BB)-2A-Puro (Addgene, USA). For transfection, a NEPAGENE (Nepa Gene, Japan) electroporator with a poring pulse (voltage, 150 V; pulse length, 7.5 ms) was used. After transfection, JEG-3 cells were selected using puromycin (2 µg/ml). The efficiency of HLA-G knockout was validated by polymerase chain reaction (PCR) and western blot analysis.

A HEK293T cell line stably expressing GFP-LC3-RFP and Cas9 (HEK293T GFP-LC3-RFP cells) was generated as previously described (6 (link)). LentiCas9-Blast (52962 (37 (link)); Addgene), human GeCKO v2 Library (1000000049 (37 (link)); Addgene), lentiCRISPR v2 (52961 (37 (link)); Addgene), and pSp-Cas9(BB)-2A-Puro (48139 (64 (link)); Addgene) were provided by F. Zhang (Massachusetts Institute of Technology, Cambridge, MA). For CRISPR knockouts, sgRNAs were cloned into lentiCRISPR v2 and pSp-Cas9(BB)-2A-Puro, sequences of which are listed in Table S5. For stable expression, GFP-LC3-RFP reporter was inserted into pMRX-IP (65 (link)) and pMRX-IB (6 (link)), and PFAS (NM_012393) tagged with 3 × FLAG was inserted into pMRX-IB (6 (link)) (these plasmids were generated from pMXs (66 (link))).

sgRNAs targeting the goat pre-miR-24 genomic sequence were designed using the Guide RNA web tool (http://chopchop.cbu.uib.no/ (accessed on 11 November 2020)). Two sgRNAs targeting both ends of the pre-miR-24 genomic sequence were selected for the Cas9-sgRNA1-sgRNA2 vector construction. The plasmid pSpCas9 (BB)-2A-Puro (62988, Addgene, Cambridge, MA, USA) was chosen as the backbone and used as a control. The double-stranded sequences of sgRNA1 and sgRNA2 were annealed by two complementary single-strand oligonucleotides from Invitrogen corporation (Shanghai, China). Thereafter, the double-stranded fragments of sgRNA1 and sgRNA2 were inserted at the BbsI site into the plasmid pX459 to construct pX459-sgRNA1 and pX459-sgRNA2 plasmids. The U6-sgRNA2-tracRNA sequences from pX459-sgRNA2 were amplified by PCR using the following primers: forward 5′-CACCTCTAGAGAGGGCCTATTTCCCATGATTCCTTCATAT-3′ and reverse 5′-CACCGGTACAAAAAAGCACCGACTCGGTGCCACTTTTTC-3′. The PCR fragment was then cloned at XbaI-KpnI sites into the plasmid pX459-sgRNA1 to generate the Cas9-sgRNA1-sgRNA2 plasmid, which was used to select single-cell clones and subsequent functional verification.

Px459 plasmid [pSpCas9(BB)-2A-Puro; Addgene] (Ran et al., 2013 (link)) was modified by the insertion of annealed, phosphorylated oligonucleotides (Forward: 5′-CACCTCATCGTGCACTCCGAGAAG-3′ and Reverse: 5′-AAACCTTCTCGGAGTGCACGATGA-3′) containing the Ky target sequence into the site created by AgeI digestion of the plasmid. C2C12 myoblasts were transfected with the modified Px459 plasmid using GenJet In Vitro DNA Transfection reagent for C2C12 cells, following the manufacturer's instructions (SL100489-C2C12). To test construct mutagenic capacity, puromycin-resistant cells selected en masse were harvested, lysed and the target region amplified by PCR (F: 5′-GGGGCCATTTGCAGCCTA-3′ and R: 5′-CGGAGAGGTTCGGATTAGCC-3′). PCR products were incubated at 37°C for 1 h with T7 endonuclease I; cleavage at the target site indicated successful mutagenesis. Individual clones were isolated by dilution following another round of construct transfection and puromycin selection. Clones were tested for mutagenesis by screening for heteroduplex formation in annealed WT and clone PCR amplicons run on 15% PAGE (Zhu et al., 2014 (link)). Alleles were then characterised by TA-cloning of PCR products using a TA Cloning kit (Thermo Scientific) and Sanger sequencing. Clones predicted to have two disruptive alleles (D, I and K) were carried forward for analysis and validated by qPCR (Fig. S1).