General methods<\/p>\n
DNA amplification was conducted by PCR using Phusion U Green Multiplex PCR Master Mix (ThermoFisher Scientific) or Q5 Hot Start High-Fidelity 2\u00d7 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<\/a>. 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).<\/p>\n General mammalian cell culture conditions<\/p>\n Cell lines with homozygous PTCs in TPP1, HEXA\u00a0and 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\u2019s Modified Eagle\u2019s 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\u2009\u00b0C with 5% CO2. Cell lines were authenticated by their respective suppliers and tested negative for mycoplasma.<\/p>\n Generation of cell lines<\/p>\n HEK293T cells were seeded at 100,000 cells per well on 24-well plates (Corning). 16\u201324\u2009h after\u00a0seeding, cells were transfected at approximately 60% confluency with 600\u2009ng, 200\u2009ng and 60\u2009ng of PEmax plasmid, epegRNA plasmid and ngRNA plasmid, respectively using Lipofectamine 3000 according to manufacturer\u2019s instructions (Thermo Fisher Scientific). 4 d after\u00a0transfection, 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.<\/p>\n Lentiviral production<\/p>\n 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<\/a>. Medium was changed 24\u2009h after transfection. Virus-containing supernatant was collected and filtered through a 0.45-\u03bcM filter 48\u2009h after\u00a0transfection. Virus was used immediately or stored at 4\u2009\u00b0C for up to 1 week before cell transduction.<\/p>\n General high-throughput lentiviral screening protocol<\/p>\n 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<\/a>. The minimum number of PCR cycles was used to amplify (typically between 11\u00a0and\u00a013 cycles) and double-stranded inserts were checked for size and\/or inappropriate products by TapeStation (Agilent). For each 20-\u03bcl reaction cloned into the pSEP0308, pSEP0309 or pSEP0310 lentiviral backbones with a 300-bp insert, 50\u2009ng of the appropriate lentiviral backbone was assembled with 10\u2009ng 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-\u03bcl reaction was set up. Reactions were incubated at 50\u2009\u00b0C for 2\u2009h and then pooled and purified using the QIAquick PCR purification kit (Qiagen 28104) according to manufacturer\u2019s instructions. Reaction products were eluted in a minimum of 20\u2009\u03bcl ddH2O, but otherwise were eluted in 2.5\u2009\u03bcl per original 20-\u03bcl reaction.<\/p>\n Isothermal assembly reactions were electroporated into NEB 10-beta Electrocompetent Escherichia coli\u00a0(NEB C3020) and plated onto LB plates aiming for at least 1,000\u00d7 coverage of each library element. 14\u2009h later, colonies were scraped and prepared using Qiagen Plasmid Plus kits (Qiagen 12945 and 12963) according to manufacturer\u2019s instructions. Pooled plasmids were transfected into HEK293Ts alongside lentiviral packaging plasmids using Lipofectamine 2000. Medium was changed 24\u2009h after transfection. Virus-containing supernatant was collected and filtered through a 0.45-\u03bcM filter 48\u2009h after transfection. Cells were transduced with virus aiming for a multiplicity of infection of 0.3. Cells were transduced to ensure \u22651,000\u00d7 coverage of transduced cells per element of the library. 2 d after\u00a0transduction, cells were passaged with 1\u2009\u03bcg\u2009ml\u22121 puromycin for 3\u20134 days to enrich for transduced cells. For screens requiring FACS isolation of GFP-positive cells, we calculated approximate coverage by multiplying the per cent of GFP-positive cells by the number of elements in the library and aiming for 1,000\u00d7 coverage of that number. For example, if 10% of cells were GFP-positive for a 500 element library, we sorted at least 50,000 cells per replicate. Cells were pelleted and flash frozen on dry ice. Genomic DNA was isolated using QIAamp DNA Micro kits (Qiagen 56304) or QIAamp Mini kits (Qiagen 51304) depending on the cell number isolated.<\/p>\n All genomic DNA for each replicate was input into an initial set of PCR reactions using Q5 Hot Start High-Fidelity 2X Master Mix (NEB M0494L) to amplify the integrated lentiviral cassette and to add sequencing adapters, with a maximum of 5\u2009\u03bcg genomic DNA per 50-\u03bcl reaction. Primers used are indicated in each screening section and are also listed in Supplementary Table 1<\/a>. PCR reactions were purified using the QIAquick PCR purification kit (Qiagen 28104) according to manufacturer\u2019s instructions and 2\u2009\u03bcl of PCR product was input into a second PCR reaction to add unique sample indices and flow cell adapters to each amplicon. Final PCR reactions were bead purified with a ratio of 0.7\u00d7 using SPRI beads, quality controlled using a TapeStation (Agilent) and quantified using a Qubit prior to high-throughput sequencing on an Illumina MiSeq, Illumina NextSeq, or Element Biosciences AVITI instrument. Sequencing conditions and downstream analyses are specific to each screen and are indicated in each separate screening section.<\/p>\n PE2 screening<\/p>\n To convert each endogenous tRNA into a sup-tRNA, we identified 418 high-confidence tRNA genes and identified the two closest 20-bp spacers with an NGG protospacer-adjacent motif upstream and downstream of the anticodon. We allowed prime editing to target multiple tRNAs in the case of identical mature tRNA sequences. We systematically varied the PBS lengths from 11\u2009bp to 14\u2009bp, and varied the RTT homology lengths 3\u2032 of the last edited nucleotide from 6\u2009bp to 10\u2009bp, where feasible. We designed three prime editing screens, each with RTTs that replaced the anticodon of each endogenous tRNA with one of three sup-tRNA anticodons (CUA, UCA or UUA). To enhance prime editing efficiency, we appended a structured RNA motif, tevopreQ1, to the 3\u2032 end of each pegRNA, creating epegRNAs29<\/a>. In total, we designed 17,579 epegRNAs to convert endogenous tRNAs to sup-tRNAs for each of the three possible sup-tRNA anticodons. Each pool also included 420 control epegRNAs targeting serine and arginine tRNAs, swapping their anticodons for those of other serine and arginine anticodons, which are not expected to give signal in a readthrough-based screen (Supplementary Table 2<\/a>). We included a unique barcode sequence 3\u2032 of the polyT terminator sequence for each epegRNA to improve our ability to assign each sequencing read to the correct element. The final oligonucleotides had the following design: a common 5\u2032 end for isothermal assembly (5\u2032-tatcttgtggaaaggacgaaacacc-3\u2032), a unique epegRNA sequence followed by a polyT for Pol III termination, an adjustable length linker used to ensure that each element of the library was the same length, a unique 25\u2009bp barcode, and a common 3\u2032 end for isothermal assembly (5\u2032-ctcgagtactaggatccattaggcg-3\u2032). Oligonucleotides were amplified using oSEP0114 and oSEP0115 prior to isothermal assembly into a linearized pSEP0308 with a hU6 promoter for driving expression of the epegRNAs. Final libraries for next generation sequencing were sequenced on an Illumina MiSeq instrument using oSEP0213 as a custom Read1 primer with 300 cycles. For analysis, the first 25 cycles of the read were matched exactly to the barcode for each element of the screening pool, allowing for no mismatches. Reads were normalized on the basis of\u00a0sequencing depth and sorted samples were compared with the plasmid pool representation of each element. Oligonucleotides and results for this screen are listed in Supplementary Table 2<\/a>.<\/p>\n Lentiviral sup-tRNA screen<\/p>\n To evaluate a wide range of tRNA promoter variants in high-throughput, we took the 418 high-confidence tRNA sequences from the human genome and swapped their anticodon to CUA, UCA or UUA to target the stop codons TAG, TGA or TAA, respectively. A unique barcode was assigned to each tRNA variant to reduce the likelihood that sequencing errors would influence assignment of library members. In addition, a pre-integrated Nextera Read1 adapter was included so that Read1 sequences on an Illumina instrument will read straight into the highly complex barcode for defining clusters. We cloned this pool using isothermal assembly into three different lentiviral backbones to evaluate the effect of different upstream elements on sup-tRNA efficacy: pSEP0308, which has a hU6 promoter, pSEP0309, which has a minU6 promoter and pSEP0310, which contains no exogenous promoter. For analysis, the first 25 cycles of the read were matched exactly to the barcode for each element of the screening pool, allowing for no mismatches. Reads were normalized on the basis of\u00a0sequencing depth and sorted samples were compared with the plasmid pool representation of each element. Oligonucleotides and results for this screen are listed in Supplementary Table 3<\/a>.<\/p>\n Leader and terminator sequence screening<\/p>\n For the initial leader sequence screen, the 40\u2009bp upstream of each endogenous high-confidence tRNA in the human genome was determined and placed upstream of the mature sequence for tRNA-Leu-TAA-3-1 with its anticodon changed to CUA followed by a polyT termination sequence. As controls, the same leader sequences were placed upstream of the mature sequence for tRNA-Leu-TAA-3-1 with its native anticodon. Libraries were sequenced and exact matching was used for both the leader sequence and the anticodon region of the read to assign to the correct library element. Oligonucleotides and results for this screen are recorded in Supplementary Table 4<\/a>.<\/p>\n To next screen leader sequences across a wide range of sup-tRNAs, we generated a lentiviral library containing six leader sequences (two top-performing, two bottom-performing and two random sequences) paired with 418 human tRNAs with their anticodons switched to CUA, UCA or UAA. We also assessed the effect of each terminator sequence when placed downstream of sup-tRNAs paired with their endogenous leader sequences, with a total of 11,543 combinations of leader sequence, sup-tRNA and terminator. We included a unique 20-bp barcode sequence 3\u2032 of the terminator sequence for each tRNA to improve our ability to assign each sequencing read to the correct element. For analysis, the first 25 cycles of the read were matched exactly to the barcode for each element of the screening pool, allowing for no mismatches. For analysis, alignment was confirmed between tRNA elements and the 20-bp barcode region and then subsequently the 20-bp barcode was used to assign each read to the appropriate library element. Reads were normalized on the basis of\u00a0sequencing depth and sorted samples were compared with the plasmid pool representation of each element. Oligonucleotides and results for this screen are recorded in Supplementary Table 5<\/a>.<\/p>\n Saturation mutagenesis screening<\/p>\n To design variants for saturation mutagenesis screening, every sequence containing a SNV, paired substitution at all hairpin positions, and 1-bp deletion was generated computationally. Then, 25\u2009bp ends compatible with isothermal assembly were added to either side (see Supplementary Table 1<\/a>) and a unique barcode was assigned to each tRNA variant to reduce the likelihood that sequencing errors would influence assignment of library members. In addition, a pre-integrated Nextera Read1 adapter was included so that Read1 sequences on an Illumina instrument will read straight into the highly complex barcode for defining clusters. Variants were ordered as a Twist oligonucleotide pool for each individual tRNA for tRNA-Leu-TAA-4-1, tRNA-Arg-CCT-4-1, mouse tRNA-Leu-TAA-2-1 and tRNA-Tyr-GTA-2-1 (sequences in Supplementary Table 6<\/a>). A pooled library of all possible variants for tRNA-Leu-TAA-1-1, tRNA-Leu-TAA-2-1, tRNA-Leu-TAA-3-1, and tRNA-Leu-TAA-4-1 was ordered as a separate Leu-TAA focused library (sequences in Supplementary Table 7<\/a>).<\/p>\n For the tRNA-Arg-CCT-4-1, mouse tRNA-Leu-TAA-2-1, and tRNA-Tyr-GTA-2-1 screens, Twist oligonucleotides contained a leader sequence element for each tRNA and they were cloned by isothermal assembly into the pSEP0310 lentiviral backbone with no exogenous Pol III promoter. For the tRNA-Leu-TAA-4-1 and Leu-TAA focused library, Twist oligonucleotides did not contain a leader sequence element for each tRNA and they were cloned by isothermal assembly into the pSEP0308 lentiviral backbone with an exogenous hU6 promoter driving expression of the tRNAs. Libraries were sequenced on an Illumina instrument with 26 cycles for Read1 covering the barcode region and 86 cycles for Read2 covering the tRNA region. For analysis, alignment was confirmed between tRNA elements and the 25-bp barcode region and then subsequently the 25-bp barcode was used to assign each read to the appropriate library element.<\/p>\n epegRNA optimization with synthetic target-site screening<\/p>\n To optimize epegRNA architectures, we designed a lentiviral epegRNA library of 17,280 epegRNAs to test out five spacer variants, PBS lengths from 8 to 16\u2009nt, RTT lengths from 21 to 36\u2009nt, and combinations of each of the 19 mutation variants of interest alongside 720 control epegRNAs with RTTs that do not encode an anticodon edit. On the same oligonucleotide, we encoded an adjacent tRNA-Leu-TAA-1-1 synthetic target site directly adjacent to the lentiviral epegRNA to read out both the epegRNA and the outcome of editing in the same sequencing read. We transfected cells transduced with this library with a panel of prime editor proteins that\u00a0included PEmax and several engineered PE6 variants40<\/a> listed in Supplementary Data Tables 9<\/a> and 10<\/a>. We performed experiments in both MMR-deficient (HEK293T) cells and MMR-proficient (HeLa) cells and included conditions in each case in which MMR was transiently inhibited by co-transfection of a dominant-negative MMR protein (MLH1dn)38<\/a>. For experiments in HeLa cells, we collected genomic DNA 3 d after\u00a0transfection. For experiments in HEK293T cells, we collected genomic DNA at 3 d and 9 d after\u00a0transfection. We then performed high-throughput sequencing of the lentivirally integrated cassette. For analysis, sequencing reads were initially demultiplexed into individual fastq files by first aligning each read to a corresponding member of the epegRNA library using bowtie2\u00a0(ref.\u00a065<\/a>). Next, each individual fastq file was then trimmed to only include the full-length synthetic target site. Editing outcomes for each epegRNA sequence were quantified using CRISPResso2. Oligonucleotides and results for this screen are recorded in Supplementary Data Tables 9<\/a> and 10<\/a>.<\/p>\n RNA-seq and transcriptomic analysis<\/p>\n HEK293T cells were transfected with our optimized epegRNA sequence and ngRNA alongside PE6c or with an unrelated epegRNA and ngRNA pair targeting the HEK3 locus alongside PE6c (see Supplementary Table 1<\/a>). RNA was extracted 6 d after\u00a0transfection using the Qiagen RNeasy kit (Qiagen) according to manufacturer\u2019s instructions. RNA-seq libraries were generated using the SMART-Seq mRNA LP kit (Takara Bio) according to manufacturer\u2019s instructions and sequenced 2\u00d7\u200975-bp on an Element Biosciences AVITI instrument. Fastq reads were trimmed of adapter sequences using Trim Galore, aligned to the human genome using STAR, and differential expression analysis was performed using DESeq2 and custom R scripts.<\/p>\n Measurement of tRNA abundance<\/p>\n Total RNA, including small RNAs, was isolated from prime edited cells using miRNeasy kits (Qiagen) according to manufacturer\u2019s protocols. RNA was reversed transcribed with SuperScript IV (Thermo Fisher Scientific) using a primer specific to each tRNA gene family queried66<\/a> (Supplementary Table 1<\/a>). Next, quantitative PCR was performed using Power SYBR Green PCR master mix (Thermo Fisher Scientific) and primers specific to each tRNA gene family queried (Supplementary Table 1<\/a>). For tRNA-Leu-TAA-1-1, we performed targeted tRNA sequencing. Specifically, we measured the relative abundance of the desired tRNA edit versus unedited in a polyclonal edited population in both genomic DNA and total RNA. For targeted tRNA sequencing, we performed reverse transcription using a primer specific to tRNA-Leu-TAA-1-1 and the Induro RT enzyme that is tolerant to RNA modifications39<\/a>.<\/p>\n Sequence context screening<\/p>\n To generate a diverse PTC sequence context reporter library, 14,746 naturally occurring premature TAG stop codons in protein-coding genes were identified in ClinVar that were annotated as pathogenic, likely pathogenic, or of uncertain significance. Each TAG stop codon was flanked on either side by the 18 nucleotides present in the native mRNA sequence. Positive controls were generated that contained the same sequence context but a TTG Leucine codon instead of a TAG premature stop codon. For negative controls, 2,800 \u2018redundant stop\u2019 control library members were generated, which were a subset of the ClinVar variants and their TTG controls but with the codon following the TAG stop codon or TTG codon changed to a TAA stop codon followed by a +1 frameshift to prevent readthrough.<\/p>\n The pSEP0211 lentiviral backbone was first linearized using oSEP0163 and oSEP0164 and the oligonucleotide pool was amplified using oSEP0165 and oSEP0166 (Supplementary Table 1<\/a>). These sequences were cloned into the pSEP0211 lentiviral backbone between mCherry and GFP via Gibson assembly. Two days following transduction, we extracted both mRNA and genomic DNA from the transduced cell population, generated cDNA by reverse transcription, and sequenced the integrated reporter construct from both the cDNA and genomic DNA samples. For analysis, we calculated an \u2018RNA score\u2019 metric by quantifying the frequency of each element in the cDNA and dividing that value by the frequency of that element in the genomic DNA. To control for the impact each unique sequence context might have on transcript expression level independent of readthrough activity, we defined a \u2018readthrough score\u2019 for each ClinVar PTC by dividing the RNA score of each variant by the RNA score of its corresponding no-premature-stop equivalent. Oligonucleotides and results for this screen are recorded in Supplementary Table 15<\/a>.<\/p>\n Off-target prime editing screening<\/p>\n To identify candidate off-target prime editing sites, we used Cas-OFFinder67<\/a> to identify all human genomic sequences with up to six mismatches, or up to four mismatches combined with up to two bulges relative to our optimized epegRNA spacer sequence. To account for target-site binding and potential annealing of the full-length RTT product, we extracted the local sequence surrounding these sites to include 3\u2009bp upstream of the putative target site and 34\u2009bp downstream. We included 100 uniquely barcoded positive-control sequences corresponding to the endogenous tRNA-Leu-TAA-1-1 site and 694 negative-control sequences that shared no homology with the epegRNA spacer sequence. These sequences were cloned into the pSEP0310 lentiviral backbone and transduced into HEK293T cells after lentiviral production. These cells were then transfected with an epegRNA expression plasmid alone as a negative control, or with a PE6c prime editor expression plasmid alongside the epegRNA expression plasmid. 3 d after\u00a0transfection, genomic DNA was isolated and the lentiviral integrated target site was amplified for high-throughput sequencing. The amplified target-site was sequenced using 2\u00d7 115 paired-end high-throughput sequencing. For analysis, high-throughput sequencing reads were initially demultiplexed into individual fastq files by first aligning each read to a corresponding member of the target-site library using bowtie2\u00a0(ref.\u00a065<\/a>). Next, each individual fastq file was then trimmed to only include the full-length putative off-target site. Putative off-target editing events were characterized as previously described34<\/a>. In brief, the sequence encoded by the RTT was compared base-by-base to the nucleotide sequence 3\u2032 of the cas9 nick site of each potential off-target site with an \u2018off-target marker\u2019 sequence being identified as the minimal deviating sequence between the two. The presence of this off-target marker sequence was quantified using output from CRISPResso2 and was used as a proxy for off-target prime editing. Oligonucleotides and results for this screen are recorded in Supplementary Table 11<\/a>.<\/p>\n rhAmpSeq off-target-site amplification and analysis<\/p>\n For initial off-target analysis, we used Cas-OFFinder to identify all human genomic sequences with up to 4 mismatches relative to our optimized epegRNA spacer sequence. A pooled sequencing primer was generated for nominated human off-target sites using the rhAmpSeq design tool (IDT). Genomic DNA was extracted from editor-treated HEK293T cells and amplified with rhAmpSeq pooled sequencing primers according to the manufacturer\u2019s protocol. The amplified libraries were sequenced with 300-bp single-end reads with an Illumina MiSeq. Sequences for rhAmpSeq amplicons were extracted using the R Bioconductor BSGenome package (v.1.4.3) using the GRCh37\/hg19 (human) reference genomes. CRISPResso2 was used to align the rhAmpSeq reads to the amplicon reference sequences and quantify the number of reads with each possible edit.<\/p>\n Flow cytometry<\/p>\n Cells were trypsinized, resuspended in media containing 10% FBS, and the solution was filtered through a 45-\u03bcm cell strainer prior to flow cytometry or FACS isolation. Flow cytometry analysis was performed using the CytoFLEX LX Flow Cytometer (Beckman Coulter, C06779) at the Broad Institute Flow Cytometry Core, with CytExpert Acquisition and Analysis Software (v.2.4). FACS was performed on the SONY MA900 Cell Sorter (Sony Biotechnology) and cells were sorted into medium prior to being spun down.<\/p>\n General cloning<\/p>\n Plasmid vectors for mammalian expression of epegRNAs or ngRNAs were cloned via isothermal assembly as previously described67<\/a>. In brief, a human U6 promoter vector was linearized via polymerase chain reaction and incubated with IDT eBlocks encoding the full-length epegRNA or ngRNA sequence flanked by sequences necessary for isothermal assembly using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs). A list of epegRNA and ngRNA sequences used in this study are provided in Supplementary Table 1<\/a>.<\/p>\n AAV vector genomes were cloned via isothermal assembly as previously described50<\/a>. In brief, the v3em vector genome construct (Addgene, #198735) was linearized by restriction digest and new epegRNA and ngRNA sequences, encoded across two IDT eBlocks, were inserted via isothermal assembly using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs). The PE6d and PE6e v3em vectors were assembled via isothermal assembly using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs). The sequences of interest were PCR amplified out of existing vectors (Addgene, #207854 and 207855) and assembled into the corresponding v3em vector (Addgene, #198735 and #193734).<\/p>\n Arrayed genome-editing experiments<\/p>\n For all experiments performed in HEK293T or HeLa readthrough reporter polyclonal cell lines and experiments performed in Neuro-2a cells, cells were seeded at 12,000 cells per well into 96-well plates (Corning) and transfected the following day with Lipofectamine 3000 (Thermo Fisher Scientific). A total of 200\u2009ng, 66\u2009ng and 22\u2009ng of prime editor plasmid, epegRNA plasmid and ngRNA plasmid (where indicated), respectively, were transfected per well. 3 d after\u00a0transfection, cells were lysed and genomic DNA was collected by incubation in a lysis buffer containing 10\u2009mM Tris-HCl, pH 8.0, 0.05% SDS and 800 units per \u03bcl of proteinase K (New England BioLabs) at 37\u2009\u00b0C for 1\u2009h, followed by enzyme inactivation at 80\u2009\u00b0C for 30\u2009min.<\/p>\n For rescue experiments performed in the HEK293T disease models, cells were initially seeded at 200,000 cells per well in 12-well plates (Corning). The following day, cells were transfected with 700\u2009ng of PE6c prime editor plasmid, 200\u2009ng of epegRNA plasmid and 75\u2009ng of ngRNA plasmid using Lipofectamine 3000 (Thermo Fisher Scientific). 3 d after\u00a0transfection, genomic DNA was extracted as described above.<\/p>\n Protein isolation and enzymatic activity assays<\/p>\n