Journal: bioRxiv

Article Title: Systematic Development of Reprogrammed Modular Integrases Enables Precise Genomic Integration of Large DNA Sequences

doi: 10.1101/2024.05.09.593242

Figure Lengend Snippet: a. Performance of Zinc Finger (ZF)-targeted Serine Recombinases Gin and Tn3. The data shown in this graph is derived from separate experiments and is representative for similar work that has been performed over a period of more than two years. In contrast to Bxb1, both Gin and Tn3 fusion proteins can result in high levels of indels causing low product purity and hindering further improvement of target integration frequencies. We also observed indels within the assayed TI junction site. Data is derived from a PCR-based NGS. b. Performance of wild-type Bxb1 against natural attB and attP target sequences in human cells. We first established K562 landing pad cell lines by installing the natural attB or attP sequence in the human AAVS1 locus. We noticed improved performance of Bxb1 with a C-terminal NLS compared the construct with a N-terminal NLS. This guided future Bxb1 designs where all evolved variants presented in this study have a C-terminal NLS. We also noticed higher targeted integration into the attB landing pad. Notably, no or only minimal levels of indels were observed within the landing pad target sequences. Data is derived from a PCR-based NGS.

Article Snippet: All constructs were sequence confirmed using Sanger sequencing services provided by Elim Biopharm Inc., and whole plasmid sequences were verified using either the Nextera XT DNA library prep kit (Illumina, Catalog #FC-131-1096) or whole plasmid sequencing services (Plasmidsaurus Inc.; Elim Biopharma Inc.).

Techniques: Derivative Assay, Sequencing, Construct

Journal: bioRxiv

Article Title: Systematic Development of Reprogrammed Modular Integrases Enables Precise Genomic Integration of Large DNA Sequences

doi: 10.1101/2024.05.09.593242

Figure Lengend Snippet: a. Schematic of our two-plasmid directed evolution system; pRex encodes an integrase gene while pRep contains an antibiotic marker disrupted by a stuffer sequence flanked by modified attB and attP sequences such that recombination by an active integrase excises the stuffer sequence and restores the open reading frame of the antibiotic resistance marker. This system enables either screening of libraries of integrase variants against a single DNA target, or screening of a library of DNA targets against a single integrase. b. Libraries where the loop, helix or hairpin submotif has been randomized are transformed along with pRep plasmids where the corresponding positions of the attB and attP target sites have been changed c. Specificity assay for individual selected integrase variants, where DNA target libraries are tested against a single integrase variant, each target site in the library contains the same modification at both half-sites of the attB and attP sites. d. Example DNA specificity plots of selected helix variants. e. Example DNA specificity plots of selected hairpin variants. f. Example DNA specificity plots of selected loop variants.

Article Snippet: All constructs were sequence confirmed using Sanger sequencing services provided by Elim Biopharm Inc., and whole plasmid sequences were verified using either the Nextera XT DNA library prep kit (Illumina, Catalog #FC-131-1096) or whole plasmid sequencing services (Plasmidsaurus Inc.; Elim Biopharma Inc.).

Techniques: Plasmid Preparation, Marker, Sequencing, Modification, Transformation Assay, Variant Assay

Journal: bioRxiv

Article Title: Systematic Development of Reprogrammed Modular Integrases Enables Precise Genomic Integration of Large DNA Sequences

doi: 10.1101/2024.05.09.593242

Figure Lengend Snippet: a. Depicted is an attB pseudo-site on chromosome 3 and performance measurements of various Bxb1 helix domain variants against this target site using a PCR-based NGS assay. SATALKR is the wild-type helix peptide sequence and the target DNA sequence is shown as the reverse complement of the sequence in to make it easier to visualize the region we are targeting in this experiment. b. On-target performance of a Bxb1 variant with combined helix and hairpin variations, and data summary of a genome-wide specificity evaluation using a modified version of the assay described in (see ). Note that the indicated Bxb1 variant has to be mixed with wild-type Bxb1 in order to be active at the intended chr3 target site so the genome-wide specificity assay was performed with a mixture of the indicated Bxb1 variant and wild-type Bxb1. The top 3 off-targets as well as two others are also targets for wild-type Bxb1 and are likely caused by the presence of wild-type Bxb1 in the experiment. The experiment was also performed with a pool of donor constructs containing all 16 possible central dinucleotide sequences. The intended target on chromosome 3 is the only target on this list with a GA or TC central dinucleotide and thus the other sites presumably wouldn’t have been detected if only a single donor with a GA or TC dinucleotide had been used in the genome-wide specificity experiment. c. Sequence of four additional Bxb1 pseudo-sites in the human genome. d. Bxb1 peptide sequences of evolved Bxb1 variants that showed improved performance against the half-sites of pseudo-sites shown in and panel c. e. Screening data using synthetic DNA targets tested in human K562 cells that was used to identify the constructs shown in panel d . Activity is determined by the number of DNA sequence reads corresponding to recombined versions of each synthetic target; activity is normalized to the activity of wild-type Bxb1 alone against the same synthetic target site. f. Results from a PCR-based NGS assay demonstrating improved performance of evolved Bxb1 variants against their chromosomal endogenous targets in K562 cells. The presence of a wild-type Bxb1 expression construct is necessary to bind the wild-type attP sequence on the donor plasmid.

Article Snippet: All constructs were sequence confirmed using Sanger sequencing services provided by Elim Biopharm Inc., and whole plasmid sequences were verified using either the Nextera XT DNA library prep kit (Illumina, Catalog #FC-131-1096) or whole plasmid sequencing services (Plasmidsaurus Inc.; Elim Biopharma Inc.).

Techniques: Sequencing, Variant Assay, Genome Wide, Modification, Construct, Activity Assay, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Systematic Development of Reprogrammed Modular Integrases Enables Precise Genomic Integration of Large DNA Sequences

doi: 10.1101/2024.05.09.593242

Figure Lengend Snippet: a. Schematic of plasmid-based system for testing evolved Bxb1 variants against artificial DNA targets. Our LSR engineering strategy divides each endogenous target site into four “quarter-sites” where the left and right half-sites are each further divided into the portion recognized by the RD domain and the portion recognized by the ZD domain of Bxb1. Individual selected Bxb1 variants are screened against a library of plasmid targets that includes their intended quarter-sites. Successful RD and ZD variants are then combined and tested against the same library of plasmid targets that also includes the relevant half-sites. A single plasmid target library can contain quarter-site and half-site targets for numerous full endogenous target sites. Left and right site candidates derived from this assay can then be tested as pairs against the endogenous target site. See Extended Data Fig. 7b,c for additional details. b. Sequence of two Bxb1 pseudo-sites in the human genome. Both sites were identified experimentally using wild-type Bxb1. c. Bxb1 peptide sequences of evolved Bxb1 variants that showed improved performance against their corresponding half-sites. d. Results from a PCR-based assay demonstrating improved performance of evolved Bxb1 variants against their chromosomal endogenous targets in K562 cells. The presence of a wild-type Bxb1 expression construct is necessary to bind the wild-type attP sequence on the donor plasmid.

Article Snippet: All constructs were sequence confirmed using Sanger sequencing services provided by Elim Biopharm Inc., and whole plasmid sequences were verified using either the Nextera XT DNA library prep kit (Illumina, Catalog #FC-131-1096) or whole plasmid sequencing services (Plasmidsaurus Inc.; Elim Biopharma Inc.).

Techniques: Plasmid Preparation, Derivative Assay, Sequencing, Expressing, Construct

Journal: bioRxiv

Article Title: Systematic Development of Reprogrammed Modular Integrases Enables Precise Genomic Integration of Large DNA Sequences

doi: 10.1101/2024.05.09.593242

Figure Lengend Snippet: a. Sequence of the pseudo-sites recognized by the TRAC and AAVS1 Bxb1 variants shown in panel b. b. Bxb1 peptide sequences of evolved Bxb1 variants for each of the two TRAC and AAVS1 half-sites generated by the strategy shown in . c. Results from our standard PCR-based NGS assay demonstrating targeted integration at the depicted TRAC site. See for additional details. d. Results from a digital PCR-based assay demonstrating targeted integration at the depicted AAVS1 site. See for additional details.

Article Snippet: All constructs were sequence confirmed using Sanger sequencing services provided by Elim Biopharm Inc., and whole plasmid sequences were verified using either the Nextera XT DNA library prep kit (Illumina, Catalog #FC-131-1096) or whole plasmid sequencing services (Plasmidsaurus Inc.; Elim Biopharma Inc.).

Techniques: Sequencing, Generated, Digital PCR

Journal: bioRxiv

Article Title: Systematic Development of Reprogrammed Modular Integrases Enables Precise Genomic Integration of Large DNA Sequences

doi: 10.1101/2024.05.09.593242

Figure Lengend Snippet: a. Schematic of a system for testing evolved Bxb1 variants against thousands of artificial targets in a plasmid target library as described in . b. Sequence examples for the target library and the donor, including assay design information for PCR-based detection of recombination events. c. Sequence example for all plasmid target library members of the AAVS1 site highlighted in . The plasmid target library includes up to six distinct members for each endogenous target site. Four members are designed to support the identification of Bxb1 variants targeting the RD and ZD motifs of both endogenous half-sites. Two additional members are designed to confirm activity of stacked Bxb1 variants against the corresponding half-sites. Bxb1 variants are screened individually against the plasmid target library.

Article Snippet: All constructs were sequence confirmed using Sanger sequencing services provided by Elim Biopharm Inc., and whole plasmid sequences were verified using either the Nextera XT DNA library prep kit (Illumina, Catalog #FC-131-1096) or whole plasmid sequencing services (Plasmidsaurus Inc.; Elim Biopharma Inc.).

Techniques: Plasmid Preparation, Sequencing, Activity Assay

Journal: bioRxiv

Article Title: Systematic Development of Reprogrammed Modular Integrases Enables Precise Genomic Integration of Large DNA Sequences

doi: 10.1101/2024.05.09.593242

Figure Lengend Snippet: a. Utilization of a single-stranded AAV (ssAAV) donor for Bxb1-mediated targeted integration. We tested ssAAV as a donor against an attB landing pad in K562 cells and noticed measurable integration levels that can be increased through co-delivery of an oligonucleotide that is complementary to the attP sequence, therefore making the ssAAV donor partially double-stranded. b. Utilization of a self-complementary AAV (scAAV) donor for Bxb1-mediated targeted integration.

Article Snippet: All constructs were sequence confirmed using Sanger sequencing services provided by Elim Biopharm Inc., and whole plasmid sequences were verified using either the Nextera XT DNA library prep kit (Illumina, Catalog #FC-131-1096) or whole plasmid sequencing services (Plasmidsaurus Inc.; Elim Biopharma Inc.).

Techniques: Sequencing

Journal: Nature Biotechnology

Article Title: Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity

doi: 10.1038/s41587-022-01533-6

Figure Lengend Snippet: a , Summary of TadA-8e variants evolved and characterized in this work. The variants are representative of conserved mutations after nine passages of PANCE or after 159 hours of PACE. For a full list of mutations, see Supplementary Figs. and . b , Method for assessing base editing of target plasmids in E. coli . Cells are co-transformed with a target plasmid (blue) and a base editor plasmid (purple). Base editor expression is induced with arabinose. After 16 hours, cells are harvested, and the target plasmid is analyzed by high-throughput sequencing. c , Base editing in E. coli of a protospacer matching the selection circuit target site. C•G-to-T•A edits are shown in blue. A•T-to-G•C edits are shown in magenta. Dots represent individual biological replicates, and bars represent mean ± s.d. from four independent biological replicates. d , Locations of evolved mutations in the cryo-EM structure of ABE8e (PDB: 6VPC ) .

Article Snippet: Plasmid DNA was amplified using the Illustra Templiphi 100 Amplification Kit (GE Healthcare Life Sciences) before Sanger sequencing (Quintara Biosciences).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Next-Generation Sequencing, Selection, Cryo-EM Sample Prep

Journal: Nature Biotechnology

Article Title: Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity

doi: 10.1038/s41587-022-01533-6

Figure Lengend Snippet: a , Base editing activity window for ABE8e with 2×UGI, TadCBEa and TadCBEa V106W across nine different target genomic sites in HEK293T. Dots represent average editing across all sites containing the specified base at the indicated position within the protospacer. Individual data points used for this analysis are in Fig. and Supplementary Figs. and . b , Method for measuring Cas-independent off-target DNA editing with the orthogonal R-loop assay. c , Average Cas-independent off-target editing across all cytosines within six orthogonal R-loops (SaR1–SaR6) generated by dead S. aureus Cas9. d , Off-target RNA editing. RNA was harvested from HEK293T cells 48 hours after transfection with the indicated base editor. After cDNA synthesis, CTNNB1 , IP90 and RSL1D1 were amplified and analyzed by high-throughput sequencing. For c and d , dots represent individual biological replicates, and bars represent mean ± s.d. of three ( c ) or four ( d ) independent biological replicates.

Article Snippet: Plasmid DNA was amplified using the Illustra Templiphi 100 Amplification Kit (GE Healthcare Life Sciences) before Sanger sequencing (Quintara Biosciences).

Techniques: Activity Assay, Generated, Transfection, cDNA Synthesis, Amplification, Next-Generation Sequencing

Journal: Nature Biotechnology

Article Title: Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity

doi: 10.1038/s41587-022-01533-6

Figure Lengend Snippet: a , Overall efficiency and selectivity of base editors analyzed through editing of the library. Data show the average fraction of edited sequencing reads across all library members between protospacer positions −9 to 20, where positions 21–23 are the PAM. b , BE4max, TadCBEa–e, TadCBEd V106W and TadDE editing profiles across 10,638 genomically integrated target sites. The editing window is defined as the protospacer positions for which average editing efficiency is ≥30% of the average peak editing efficiency. Window plots for all variants tested in the library experiment can be found in Supplementary Fig. . c , Sequence motifs of TadCBEd and TadCBEd V106W for cytosine and adenine base editing outcomes determined by performing regression on editing efficiencies. Opacity of sequence motifs is proportional to the test R on a held-out set of sequences. Complete sequence motif plots for all variants are in Supplementary Fig. .

Article Snippet: Plasmid DNA was amplified using the Illustra Templiphi 100 Amplification Kit (GE Healthcare Life Sciences) before Sanger sequencing (Quintara Biosciences).

Techniques: Sequencing

Journal: Nature Biotechnology

Article Title: Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity

doi: 10.1038/s41587-022-01533-6

Figure Lengend Snippet: mRNA encoding the indicated base editor or GFP as a negative control was electroporated into human T cells ( n = 4 donors) along with two synthetic guide RNAs targeting CXCR4 ( a ) or CCR5 ( b ) at the specified protospacers. Target cytosines are blue, target adenines are magenta, and PAM sequences are underlined. After 3 days, genomic DNA was harvested from T cell lysates and analyzed by high-throughput sequencing. The gray boxes indicate the desired location of stop codon installation in CXCR4 and CCR5 . The targeted cytidine to yield T AG ( CXCR4 ) and T AA ( CCR5 ) stop codons upon cytosine base editing is underlined. c , mRNA encoding the indicated base editor or GFP as a negative control was electroporated into HSPCs along with a synthetic guide RNA targeting the BCL11A enhancer. After 3 days, genomic DNA was harvested from cell lysates and analyzed by high-throughput sequencing. C•G-to-T•A base editing is shown in shades of blue, and A•T-to G•C-base editing is shown in shades of magenta. Dots represent individual biological replicates, and bars represent mean ± s.d. from n = 4 donors ( a and b ) or n = 3 donors ( c ).

Article Snippet: Plasmid DNA was amplified using the Illustra Templiphi 100 Amplification Kit (GE Healthcare Life Sciences) before Sanger sequencing (Quintara Biosciences).

Techniques: Negative Control, Next-Generation Sequencing