GoKawiilSingle guide RNA design and cloning
The gRNA used for SGE was designed using Benchling’s CRISPR design tool to search the RNU4-2 locus, including upstream and downstream regions of low sequence homology to RNU4-1 and pseudogenes, identifying a candidate with high on-target and low off-target scores. The selected gRNA was not predicted to target RNU4-1, owing to eight mismatches occurring in the protospacer and PAM. The gRNA spacer sequence was ligated into the pX459 backbone as previously described33. In brief, complementary primers containing the spacer were ordered from IDT (Supplementary Table 3), phosphorylated, hybridized and ligated into the pX459 linearized backbone followed by PlasmidSafe DNase (Lucigen) digestion. Next, 2 µl of the ligation reaction were transformed in NEB Stable Competent Escherichia coli cells using the high-efficiency transformation protocol and 75 µl of transformant was plated on ampicillin-resistant plates and cultured overnight at 30 °C. Three colonies were then picked and grown overnight at 37 °C in 7 ml of Luria–Bertani medium supplemented with carbenicillin (100 µg ml−1). Plasmid DNA was extracted using the QIAprep Spin Miniprep kit (Qiagen) and verified using Plasmidsaurus whole-plasmid sequencing. The selected clone was then grown in 100 ml of Luria–Bertani medium at 37 °C in a shaking incubator supplemented with carbenicillin. The cells were then pelleted and the plasmid was extracted using a ZymoPure Maxiprep kit (Zymo Research), endotoxins were removed using EndoZero columns (Zymo Research) and the product was quantified with the Qubit double-stranded DNA (dsDNA) BR assay kit (Invitrogen).
HDR library cloning
An oligonucleotide library comprising RNU4-2 variants was manufactured by Twist Bioscience and subsequently cloned into a vector containing homology arms for RNU4-2 to make the HDR library for SGE.
To generate the vector with homology arms, a nested PCR was performed on genomic DNA (gDNA) extracted from HAP1 cells10 using primers designed to generate homology arms of 700–800 base pairs (bp) flanking RNU4-2 (Supplementary Table 3). The PCR was performed using the Kapa HiFi HotStart ReadyMix (Roche). The product was purified using AmpureXP (Beckman Coulter) magnetic beads at 1.2× volume and eluted in 12 µl of nuclease-free water. The amplicon containing RNU4-2 homology arms was then inserted in the linearized pUC19 backbone using In-Fusion HD cloning (Takara) and 2 µl of cloning reaction was transformed into NEB Stable cells following the manufacturer’s 5-min transformation protocol. Cells were plated on agar plates containing ampicillin and incubated at 30 °C overnight. The pUC19 plasmid containing RNU4-2 homology arms (pUC19-RNU4-2-HA) was purified and sequence-verified from a successfully transformed clone. pUC19-RNU4-2-HA was then diluted to 8.7 pg in a 50-µl PCR reaction and amplified with Kapa HiFi to obtain a linearized product with 17–18 bp complementarity to the RNU4-2 oligo library. A PAM-blocking mutation was introduced 27 nt upstream of the RNU4-2 sequence (chromosome 12:120291930-C-G) by means of primer overhang extension during PCR. The location of the PAM-disrupting edit was selected to minimize recutting by Cas9, converting a 5′-G G G PAM sequence to 5′-G C G. The PAM-disrupting edit had a CADD score of 4.20 (Phred) and a 100 vertebrates PhyloP score of 0.11. The reaction was treated with 1 µl of DpnI (NEB) for 30 min at 37 °C, gel extracted and quantified. Then, the RNU4-2 oligo library was amplified using Kapa HiFi and purified using AmpureXP (1.2×). The amplified library and linearized pUC19-RNU4-2-HA plasmid were then assembled using the In-Fusion HD cloning kit, and the product was transformed into NEB Stable cells using the high-efficiency transformation protocol. To quantify efficiency, 1% of cells in the transformation reaction were plated and the remainder were cultured in 100 ml of Luria–Bertani medium with carbenicillin overnight at 37 °C. Cells were then pelleted by centrifugation and the final RNU4-2 HDR library was extracted using the ZymoPure Maxiprep kit (Zymo Research) with endotoxin removal. The isolated HDR library was quantified with a Qubit dsDNA BR assay kit and sequence-verified by Plasmidsaurus.
HAP1 cell culture
HAP1 cells used for SGE (the HAP1-LIG4-KO line; herein referred to as ‘HAP1’) show increased rates of editing by HDR due to a frameshifting mutation in LIG4 (ref. 10). Frozen HAP1 cells were thawed at 37 °C in a water bath, then supplemented with 10 ml of prewarmed Iscove’s Modified Dulbecco’s Medium (IMDM) containing l-glutamine, 25 nM HEPES (Gibco), 10% FBS (Gibco), 1% penicillin–streptomycin (Gibco) and 2.5 μM 10-deacetyl-baccatin-III (DAB, Stratech), herein referenced to as IMDMc. Cells were centrifuged at 300g for 3 min. The supernatant was then aspirated and the cells were resuspended in fresh media, plated on a 10-cm dish and cultured at 37 °C with 5% CO 2 . The next day, the IMDMc media was replaced, and cells were cultured routinely from that point forward.
The HAP1 subculture routine included a 1:5 split every 48 h or 1:10 split every 72 h to prevent cells from exceeding 80% confluency. To split cells, the media was aspirated and the dish washed with 10 ml of room-temperature Dulbecco’s PBS (Gibco). Following Dulbecco’s PBS aspiration, the cells were treated with 1 ml of 0.25% trypsin–EDTA (Gibco) and incubated for 3 min at 37 °C. Next 14 ml of prewarmed IMDMc was then added and cells were collected and centrifuged at 300g for 5 min. Cells were then resuspended in 10 ml of IMDMc, counted and seeded on a 10-cm dish.
Generation of diploid HAP1 cells
Parental HAP1 cells were cultured for 9 days after thawing in IMDMc without DAB supplementation to allow for the spontaneous occurrence of diploid cells. On day 10, cells were stained with 5 µg ml−1 Hoechst working solution (Thermo Fisher Scientific) for 1 h at 37 °C, followed by fluorescence-activated cell sorting to select diploid cells using a BD FACSAria Fusion Flow Cytometer. Diploid cells were sorted on the basis of their G2/M peak (4n), with gates established using a monoclonal diploid HAP1 control population. Sorted diploid HAP1 cells were then expanded for 10 days in IMDMc without DAB supplementation before the subsequent SGE experiment.
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