General methods
DNA amplification was conducted by PCR using Phusion U Green Multiplex PCR Master Mix (ThermoFisher Scientific) or Q5 Hot Start High-Fidelity 2× Master Mix (New England BioLabs) unless otherwise noted. DNA oligonucleotides were obtained from Integrated DNA Technologies. Plasmids expressing epegRNAs were constructed by Gibson assembly using a custom acceptor plasmid. Sequences of sgRNA and epegRNA constructs used in this work are listed in Supplementary Table 1. All vectors for mammalian cell experiments were purified using Plasmid Plus Midiprep kits (Qiagen) or PureYield plasmid miniprep kits (Promega), which include endotoxin removal steps. All experiments using live animals were approved by the Broad Institute Institutional and Animal Care and Use Committees. Wild-type C57BL/6 mice were obtained from Charles River (027).
General mammalian cell culture conditions
Cell lines with homozygous PTCs in TPP1, HEXA and NPC1were generated using prime editing. HEK293T (ATCC CRL-3216), Neuro-2a (ATCC CCL-131) and HeLa (CCL-2) cells were purchased from ATCC and cultured and passaged in Dulbecco’s Modified Eagle’s Medium (DMEM) plus GlutaMAX (ThermoFisher Scientific), supplemented with 10% (v/v) fetal bovine serum (Gibco, qualified). All cell types were incubated, maintained, and cultured at 37 °C with 5% CO 2 . Cell lines were authenticated by their respective suppliers and tested negative for mycoplasma.
Generation of cell lines
HEK293T cells were seeded at 100,000 cells per well on 24-well plates (Corning). 16–24 h after seeding, cells were transfected at approximately 60% confluency with 600 ng, 200 ng and 60 ng of PEmax plasmid, epegRNA plasmid and ngRNA plasmid, respectively using Lipofectamine 3000 according to manufacturer’s instructions (Thermo Fisher Scientific). 4 d after transfection, single cell clones were isolated by limiting dilution cloning and expanded over a 2-week period. The resulting colonies were further expanded and genotyped by high-throughput sequencing of the targeted locus and those found to be homozygous for the expected edit were retained for downstream experiments.
Lentiviral production
HEK293Ts (ATCC CRL-3216) were transfected with pMD2.G (Addgene #12259) and delta8.2 (Addgene Plasmid #8455) packaging plasmids alongside the appropriate lentiviral backbone using Lipofectamine 2000. Lentiviral backbone sequences used in this work are listed in Supplementary Table 1. Medium was changed 24 h after transfection. Virus-containing supernatant was collected and filtered through a 0.45-μM filter 48 h after transfection. Virus was used immediately or stored at 4 °C for up to 1 week before cell transduction.
General high-throughput lentiviral screening protocol
All oligonucleotides for high-throughput screening were ordered from Twist Biosciences as single-stranded oligonucleotide pools. Oligonucleotide pools were amplified using Q5 Hot Start High-Fidelity 2X Master Mix (NEB M0494L) to create double-stranded inserts for isothermal assembly. Primers used are indicated in each specific screening section of the Methods. The minimum number of PCR cycles was used to amplify (typically between 11 and 13 cycles) and double-stranded inserts were checked for size and/or inappropriate products by TapeStation (Agilent). For each 20-μl reaction cloned into the pSEP0308, pSEP0309 or pSEP0310 lentiviral backbones with a 300-bp insert, 50 ng of the appropriate lentiviral backbone was assembled with 10 ng of the appropriate double-stranded insert using NEBuilder HiFi DNA Assembly Master Mix (NEB E2621L). Reactions were scaled according to the number of elements in the library. For each 1,000 elements, an additional 20-μl reaction was set up. Reactions were incubated at 50 °C for 2 h and then pooled and purified using the QIAquick PCR purification kit (Qiagen 28104) according to manufacturer’s instructions. Reaction products were eluted in a minimum of 20 μl ddH2O, but otherwise were eluted in 2.5 μl per original 20-μl reaction.
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