adeno associated virus production aav  (TaKaRa)

Journal: Cell

Article Title: Phage-assisted evolution and protein engineering yield compact, efficient prime editors

doi: 10.1016/j.cell.2023.07.039

Figure Lengend Snippet: Development of dual-AAV compatible RT variants for installing long, complex edits (A) Summary of evolution and engineering campaigns used to generate PE6c and PE6d. (B) Conserved mutations from M-MLV RT evolution. The structure of XMRV RT (PDB: 4HKQ), which is highly homologous to M-MLV shows PACE-evolved residues (blue) lie close to the enzyme active site (dark gray) and DNA/RNA duplex substrate (pink/purple). An incoming dNTP, modeled by alignment with PDB: 5TXP, is shown in yellow. Below, pink lines indicate locations in the M-MLV RT at which PACE-evolved mutations truncated the protein. (C) Fold-change in editing efficiency relative to PEmax for PEmaxΔRNaseH, PE6c, and PE6d in HEK293T cells. Individual replicates are plotted, with n = 3 biological replicates per edit. (D) Editing efficiencies of PEmaxΔRNaseH and PE6d at the HEK3 +1 loxP insertion edit (pink) and the HEK3 +1 FLAG insertion edit (orange) in HEK293T cells. The NUPACK-predicted structures of the RTT and PBS extensions for each edit is shown. (E) Results of a TdT assay on the HEK3 +1 loxP insertion edit in HEK293T cells. The y axis indicates the percentage of total RT products of a given length, and the x axis represents the length of the product in base pairs. PEmaxΔRNaseH is shown in gray, and PE6d is shown in blue. The lines are mean values from n = 3 biological replicates. The pink box indicates DNA bases templated by the structured portions of the pegRNA. (F) Editing efficiencies of PEmaxΔRNaseH (gray) and PE6d (blue) at an example engineered hairpin edit and its corresponding unpinned control in HEK293T cells. The sequence of the RTT is shown, with point mutations in the unpinned control shown in red. The NUPACK-predicted structures of the RTT and PBS extensions for each edit is shown. (G) Relationship between pegRNA RTT/PBS secondary structure and PE6d improvements. The y axis reflects the fold-improvement of PE6d over PEmaxΔRNaseH. The x axis is the absolute value of the free energy of pegRNA folding as measured by NUPACK. Each dot represents one edit in HEK293T cells that was calculated from the mean values from n = 3 biological replicates. See Figure S4 D for individual editing values and edit identities. (H) Comparison of evolved and engineered RTs to PEmaxΔRNaseH at typical twinPE edits in HEK293T cells. Solid bars indicate editing efficiency. Striped bars indicate indels. (I) TwinPE-mediated insertion of the 38-bp attB sequence into the Rosa26 locus in N2a cells. Indel-free editing is shown in yellow, and indels are shown in gray. (J) PE-mediated insertion of a 42-bp sequence containing loxP into the Dnmt1 locus in N2a cells. Indel-free editing is shown in yellow, and indels are shown in gray. For D, F, and H-J, bars reflect the mean of n = 3 independent replicates. Dots show individual replicate values. See also Figure S4 .

Article Snippet: AAV production was performed as previously described., HEK293T/17 cells (ATCC) were cultured in DMEM with 10% fetal bovine serum without antibiotics in 150-mm 2 dishes (Thermo Fisher Scientific) and passaged every 2–3 days at 37°C with 5% CO 2 .

Techniques: Control, Sequencing, Comparison

Journal: Cell

Article Title: Phage-assisted evolution and protein engineering yield compact, efficient prime editors

doi: 10.1016/j.cell.2023.07.039

Figure Lengend Snippet: PE6 variants enable longer and more complex prime edits in vivo (A) Schematic showing a dual-AAV delivery system for twinPE (v3em twinPE-AAV). In the N-terminal AAV, production of the N-terminal portion of Cas9 (yellow) fused to an N-terminal Npu split intein (orange) is regulated by the Cbh promoter (green) and the SV40 late polyA signal (tan). In the C-terminal AAV, the C-terminal Npu split intein (dark green) is fused to the remainder of the prime editor (Cas9, yellow and RT, purple). The SV40 late polyA signal (tan), two epegRNAs (light and dark blue), AAV ITRs (black) are also shown. (B) Injection route and twinPE editing efficiency of PEmaxΔRNaseH and PE6d viruses in the for the twinPE-mediated insertion of a 38-bp attB sequence at murine Rosa26 in the mouse cortex. N- and C- terminal twinPE viruses are administered via ICV injection (4x10 10 vg total) along with a GFP-KASH virus. Editing efficiencies (light and dark blue) and indel frequencies (black and gray) are shown to the right. Bars reflect the mean of n = 3–4 mice. Dots show individual mice. (C) Injection route and PE editing efficiency of PEmaxΔRNaseH and PE6d viruses for the installation of a 42-bp insertion containing loxP at the Dnmt1 locus in the mouse cortex. (Left) The C-terminal virus is modified to include one epegRNA and one nicking sgRNA to encode a PE edit as opposed to a twinPE edit. (Right) Editing efficiencies (light/dark pink) and indel rates (black/gray). Bars reflect the mean of n = 3 mice. Dots show individual mice. See also Figure S7 .

Article Snippet: AAV production was performed as previously described., HEK293T/17 cells (ATCC) were cultured in DMEM with 10% fetal bovine serum without antibiotics in 150-mm 2 dishes (Thermo Fisher Scientific) and passaged every 2–3 days at 37°C with 5% CO 2 .

Techniques: In Vivo, Injection, Sequencing, Virus, Modification

Journal: Cell

Article Title: Phage-assisted evolution and protein engineering yield compact, efficient prime editors

doi: 10.1016/j.cell.2023.07.039

Figure Lengend Snippet: Development and characterization of highly processive, dual AAV-compatible RTs, related to Figure 4 (A) Editing efficiencies of prime editors containing single M-MLV mutants in HEK293T cells. Prime editing efficiencies used are the frequency of the intended prime editing outcome with no indels or other changes at the target site. Lines reflect the mean of n = 2 independent replicates per edit. Dots show individual replicate values. (B) Overview of the terminal deoxynucleotidyl transferase (TdT) assay for directly sequencing newly reverse-transcribed DNA flaps that have not been incorporated into the genome. 24 h after treatment with a prime editor and pegRNA, cells are lysed, and DNA is purified to capture and sequence newly reverse-transcribed DNA before its incorporation into the genome. A terminal transferase enzyme (yellow) adds a polyG sequence to all DNA 3′ ends. PCR amplification for high-throughput DNA sequencing is performed using a locus-specific forward primer and a polyC reverse primer. (C) Results of a TdT assay on the HEK3 +1 FLAG insertion edit in HEK293T cells. The y axis indicates the percentage of total RT products of a given length, and the x axis represents the length of the product in base pairs. PEmaxΔRNaseH is shown in gray, and PE6d is shown in blue. The lines are mean values from n = 3 biological replicates. (D) Editing efficiencies of PE6b-d, PEmax, and PEmaxΔRNaseH for edits engineered to contain varying levels of secondary structure. “UC” indicates an unpinned control for a corresponding hairpin edit. These values were used to generate the free energy vs. fold improvement plot in Figure 4 G. All edits are in HEK293T cells. Individual replicates are shown, with n = 3 replicates per condition. (E) Editing efficiencies (left) and indel rates (right) of PE6d (blue) and PEmaxΔRNaseH (gray) for a series of prime edits that use short unstructured pegRNAs in HEK293T cells. Bars reflect the mean of n = 3 independent replicates. Dots show individual replicate values. (F) Results of a TdT assay on the RNF2 +5 G to T edit in HEK293T cells. Note that the x axis differs from other TdT plots shown in this study: instead of RTT-templated bases correctly installed, it quantifies the number of sgRNA scaffold-templated bases aberrantly installed (for example, x = 1 indicates the addition of one extra scaffold-templated base). The y axis indicates the percentage of edit-containing flaps that have a given number of scaffold-templated bases. For each prime editor, the line reflects the mean of n = 3 independent replicates. Pie charts indicate the percentages of edit-containing flaps that either have ≤2 bp (solid color) or >2 bp (striped) of scaffold-templated bases. Data shown are the mean of three independent biological replicates. (G) Unique molecular identifier (UMI) analysis of prime editing efficiencies for twinPE edits in N2a cells (left) and HEK293T cells (middle, right). UMI protocol was applied to remove PCR bias, and trends agree with the data shown in Figure 4 . Bars reflect the mean of n = 3 independent replicates. Dots show individual replicate values.

Article Snippet: AAV production was performed as previously described., HEK293T/17 cells (ATCC) were cultured in DMEM with 10% fetal bovine serum without antibiotics in 150-mm 2 dishes (Thermo Fisher Scientific) and passaged every 2–3 days at 37°C with 5% CO 2 .

Techniques: Sequencing, Reverse Transcription, Purification, Amplification, High Throughput Screening Assay, DNA Sequencing, Control

Journal: Cell

Article Title: Phage-assisted evolution and protein engineering yield compact, efficient prime editors

doi: 10.1016/j.cell.2023.07.039

Figure Lengend Snippet:

Article Snippet: AAV production was performed as previously described., HEK293T/17 cells (ATCC) were cultured in DMEM with 10% fetal bovine serum without antibiotics in 150-mm 2 dishes (Thermo Fisher Scientific) and passaged every 2–3 days at 37°C with 5% CO 2 .

Techniques: Virus, Recombinant, Multiplex Assay, Purification, Gel Extraction, Plasmid Preparation, Cell Isolation, Transfection, DNA Extraction, Amplification, Titration, Sequencing, Mutagenesis, Software