Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV production
- Wyss Institute for Biologically Inspired Engineering, United States
- Department of Genetics, Harvard Medical School, United States
Figures
Figure 1 with 1 supplement
Comprehensive mutagenesis library design and production assay.
(A) Organization of the AAV2 genome and Rep protein domains. Top: single-stranded DNA genome, middle: RNA transcripts, bottom: Rep proteins. Dotted lines indicate mutated regions. (B) Density plot …
Figure 1—figure supplement 1
Comprehensive mutagenesis library design and production assay results for WT AAV2 format library.
(A) Density plot of barcode counts in the WT AAV2 plasmid library. (B) Amino acid level production fitness values from replicate transfections for the WT AAV2 library. Pearson R correlation …
Figure 2 with 7 supplements
Effects of all single amino acid substitutions and deletions in the Rep78/68 proteins on AAV2 production.
Amino acid level production fitness values from the pCMV-Rep78/68 production assay were calculated as in Figure 1C by summing barcode counts for synonymous mutations. Rectangles are colored by …
Figure 2—figure supplement 1
Effects of all single amino acid substitutions and deletions in Rep78, Rep68, Rep52, and Rep40 on AAV2 production.
Amino acid level production fitness values from the WT AAV2 library production assay were calculated as in Figure 1C by summing barcode counts for synonymous variants. Rectangles are colored by …
Figure 2—figure supplement 2
Mutations to AAV2 rep have similar effects on AAV2 production in pCMV-Rep78/68 and WT AAV2 format libraries.
(A) Production fitness values for library production assay with pCMV-Rep78/68 and WT AAV2 format libraries, Pearson R correlation coefficient calculated after log transformation, (B) DNase-resistant …
Figure 2—figure supplement 3
Comparison of Rep variant and wild-type production fitness values.
For each amino acid variant, mean production fitness values were calculated by averaging the production fitness values for all uniquely barcoded variants coding for the relevant amino acid change. …
Figure 2—figure supplement 4
Codon level production fitness values for the pCMV-Rep78/68 format library.
Codon level production fitness values were calculated as in Figure 1C by summing counts for all barcodes corresponding to a given codon variant. Rectangles are colored by mutational effect on the …
Figure 2—figure supplement 5
Codon level production fitness values for the WT AAV2 format library.
Codon level production fitness values were calculated as in Figure 1C by summing counts for all barcodes corresponding to a given codon variant. Rectangles are colored by mutational effect on the …
Figure 2—figure supplement 6
The distribution of production fitness values is narrower for synonymous variants than for nonsynonymous variants.
(A) Positional mean-centered fitness values for all codon variants in the WT AAV2 library and (B) synonymous codon mean-centered fitness values for all codon variants in the WT AAV2 library. For …
Figure 2—figure supplement 7
Inclusion of synonymous codon variants enables interrogation of nucleotide-level effects.
Analysis of pCMV-Rep78/68 variants with premature stop codons in +1 (A) and +2 (B) reading frames does not identify any frameshifted open-reading frames. Pink dots: average production fitness for …
Figure 3 with 1 supplement
Beneficial substitutions cluster in DNA-interacting residues.
(A) Average production fitness values for all substitutions at each position mapped onto the structure of the origin-binding domain in complex with the AAVS1 Rep-binding site (PDB 4ZQ9). (B) Closeup …
Figure 3—figure supplement 1
Average production fitness values from the pCMV-Rep78/68 library production assay mapped onto (A) the structure of the origin-binding domain active site (PDB 5DCX) and (B) the structure of the helicase domain (PDB 1S9H).
H90, H92, and the active site nucleophile, Y156, are shown as sticks in (A). Residues are colored by mutability, with red indicating higher mutability and black indicating lower mutability.
Figure 4
Comparison of comprehensive mutagenesis measurements to variation in nature.
(A) Distribution of wild-type normalized production fitness values for conserved variants (blue) and variants found only in the library (gray). (B) Total number of variants and number of conserved …
Figure 5 with 1 supplement
Validation of AAV2 library production assay results.
(A) DNase-resistant particle titers for 14 single amino acid pCMV-Rep78/68-inverted terminal repeat variants produced individually. Titers for previously characterized variants are plotted to the …
Figure 5—figure supplement 1
Effect of single amino acid Rep substitutions on the viral genome titer, physical particle titer, and relative transduction efficiency of affinity purified rAAV2.
(A) Capsid (gray) and viral genome (black) titers for pAAV-EF1a-FLuc-WPRE-HGHpA rAAV2 produced with the indicated Rep variant and affinity purified with AVB Sepharose. For each variant, samples A …
Figure 6 with 5 supplements
Mutations to AAV2 rep have similar effects on AAV2, AAV5, and AAV9 capsid production.
Wild-type normalized production fitness values from the library production assay with (A) AAV5 and AAV2 capsids and (B) AAV9 and AAV2 capsids. Pearson R correlation coefficient calculated after log …
Figure 6—figure supplement 1
Effects of all single amino acid substitutions and deletions in the Rep78/68 proteins on AAV5 capsid production.
Amino acid level production fitness values from the pCMV-Rep78/68 production assay were calculated as in Figure 1C by summing barcode counts for synonymous mutations. Rectangles are colored by …
Figure 6—figure supplement 2
Effects of all single amino acid substitutions and deletions in the Rep78/68 proteins on AAV9 capsid production.
Amino acid level production fitness values from the pCMV-Rep78/68 production assay were calculated as in Figure 1C by summing barcode counts for synonymous mutations. Rectangles are colored by …
Figure 6—figure supplement 3
Codon level production fitness values for the AAV5 capsid production assay.
Codon level production fitness values were calculated as in Figure 1C by summing counts for all barcodes corresponding to a given codon variant. Rectangles are colored by mutational effect on the …
Figure 6—figure supplement 4
Codon level production fitness values for the AAV9 capsid production assay.
Codon level production fitness values were calculated as in Figure 1C by summing counts for all barcodes corresponding to a given codon variant. Rectangles are colored by mutational effect on the …
Figure 6—figure supplement 5
DNase-resistant particle titers for AAV2, AAV5, and AAV9 capsids produced individually with the indicated Rep variants.
Tables
Table 1
Percent of expected variants sequenced in plasmid and viral libraries.
| pCMV-Rep78/68 | WT AAV2 | |||
|---|---|---|---|---|
| Plasmid (%) | Viral (%) | Plasmid (%) | Viral (%) | |
| Barcodes | 99.8 | 98.0 | 98.3 | 97.6 |
| Codon variants | 100 | 99.9 | 99.7 | 99.7 |
| Amino acid variants | 100 | 100 | 99.9 | 99.9 |
Table 2
Viral genome and physical particle titers for rAAV2 produced with Rep variants.
| Rep variant | Replicate | qPCR titer (vg/mL) | qPCR SD (vg/mL) | ELISA titer (capsids/mL) | ELISA titer SD (capsids/mL) | Viral genome:capsid titer ratio |
|---|---|---|---|---|---|---|
| WT | A | 6.18E+11 | 5.77E+10 | 7.82E+11 | 1.04E+11 | 0.79 |
| B | 3.00E+11 | 3.85E+10 | 1.77E+11 | 8.95E+10 | 1.70 | |
| Q439T | A | 7.01E+11 | 6.47E+10 | 8.14E+11 | 4.27E+10 | 0.86 |
| B | 4.55E+11 | 2.33E+10 | 3.61E+11 | 1.21E+11 | 1.26 | |
| S110R | A | 1.45E+12 | 3.24E+10 | 1.73E+12 | 1.56E+11 | 0.84 |
| B | 1.04E+12 | 6.81E+10 | 1.43E+12 | 1.31E+11 | 0.73 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HEK293T | ATCC | ATCC:CRL-3216 | |
| Recombinant DNA reagent | pCMV-Rep78/68 plasmid library | This paper | RRID:Addgene_198050 | Complete list of plasmid sequences included in library can be found in the GitHub repo linked below |
| Recombinant DNA reagent | pEf1a-Luc-WPRE (ssAAV) plasmid | Addgene | RRID:Addgene_87951 | |
| Recombinant DNA reagent | pCAG-Luc (ssAAV) plasmid | Addgene | RRID:Addgene_83281 | |
| Recombinant DNA reagent | pCAG-GFP (scAAV) plasmid | Addgene | RRID:Addgene_83279 | |
| Sequence-based reagent | 5′-GAGCATCTGCCCGGCATTTC-3′ | This paper | Forward primer, binds to 5′ region of AAV2 rep gene | |
| Sequence-based reagent | 5′-ATCTGGCGGCAACTCCCATT-3′ | This paper | Reverse primer, binds to 5′ region of AAV2 rep gene | |
| Sequence-based reagent | 5′-HEX-ACAGCTTTG-ZEN- TGAACTGGGTGGCCGA-3IABkFQ-3′ | This paper | HEX-based probe, binds to 5′ region of AAV2 rep gene | |
| Sequence-based reagent | 5′-GAACCGCATCGAGCTGAA-3′ | This paper | Forward primer, binds to GFP sequence | |
| Sequence-based reagent | 5′-TGCTTGTCGGCCATGATATAG-3′ | This paper | Reverse primer, binds to GFP sequence | |
| Sequence-based reagent | 5′-FAM-ATCGACTTC-ZEN- AAGGAGGACGGCAAC-3IABKFQ-3′ | This paper | FAM-based probe, binds to GFP sequence | |
| Sequence-based reagent | 5′-CATGTACCGCTTCGAGGAG-3′ | This paper | Forward primer, binds to luciferase sequence | |
| Sequence-based reagent | 5′-GAAGCTAAATAGTGTGGGCACC3′ | This paper | Reverse primer, binds to luciferase sequence | |
| Sequence-based reagent | 5′-FAM-CTTGCGCAG-ZEN- CTTGCAAGACTATAAGATTC-3IABKFQ-3′ | This paper | FAM-based probe, binds to luciferase sequence | |
| Antibody | Anti-Rep 303.9 (mouse monoclonal) | American Research Products | American Research Products: 03-61069; RRID:AB_1540388 | 1:100 |
| Antibody | B1 anti-VP (mouse monoclonal) | American Research Products | American Research Products: 03-61058; RRID:AB_1540385 | 1:250 |
| Antibody | Anti-β-actin (rabbit polyclonal) | Abcam | Abcam: ab8227; RRID:AB_2305186 | 1:20,000 |
| Antibody | Anti-mouse IgG IRDye 800CW (goat monoclonal) | LI-COR | LI-COR: 925-32210; RRID:AB_2687825 | 1:10,000 |
| Antibody | Anti-rabbit IgG IRDye 680RD (donkey monoclonal) | LI-COR | LI-COR: 925-68073; RRID:AB_2716687 | 1:10,000 |
| Commercial assay or kit | Promega One-Glo EX Luciferase Assay System | Promega | Promega:E8110 | |
| Commercial assay or kit | Capsid ELISA | Progen | Progen:PRATV | |
| Software, algorithm | Python scripts for library design and data analysis | This paper | https://github.com/churchlab/aav_rep_scan; churchlab, 2023 |
