Long read sequencing reveals transgene concatemerization and vector sequences integration following AAV-driven electroporation of CRISPR RNP complexes in mouse zygotes

Abstract

Over the last decade CRISPR gene editing has been successfully used to streamline the generation of animal models for biomedical research purposes. However, one limitation to its use is the potential occurrence of on-target mutations that may be detrimental or otherwise unintended. These bystander mutations are often undetected using conventional genotyping methods. The use of Adeno-Associated Viruses (AAVs) to bring donor templates in zygotes is currently being deployed by transgenic cores around the world to generate knock-ins with large transgenes (i.e., 1-4 kb payloads). Thanks to a high level of efficiency and the relative ease to establish this technique, it recently became a method of choice for transgenic laboratories. However, a thorough analysis of the editing outcomes following this method is yet to be developed. To this end, we generated three different types of integration using AAVs in two different murine genes (i.e., Ace2 and Foxg1) and employed Oxford Nanopore Technologies long read sequencing to analyze the outcomes. Using a workflow that includes Cas9 enrichment and adaptive sampling, we showed that unintended on-target mutations, including duplication events and integration of viral sequences (sometimes reported using other workflows) can occur when using AAVs. This work highlights the importance of in-depth validation of the mutant lines generated and informs the uptake of this new method.

Keywords: CRISPR; adeno-associated-virus (AAV); concatemers; long read sequencing (LRS); mice; zygotes.

FIGURE 1

Generation of KI mouse lines by AAV-driven gene editing. The gene editing strategy for each knock-in (KI) line is illustrated. (a) The start codon of the murine Ace2 gene was targeted for Homology Directed Repair (HDR) with a donor template carrying the hACE2 coding DNA sequence upstream of a PolyA. (b) The stop codon of the mouse Foxg1 gene was targeted for HDR with an EGFP sequence at the c-terminus. (c) The start codon of the murine Foxg1 gene was targeted to insert a stop cassette and a triple FLAG tag. LHA = left homology arm, RHA = right homology arm, ITR = inverted terminal repeat sequence, hACE2 = human ACE2 coding sequence, SV40 pA = simian virus 40 polyadenylation signal, EGFP = Enhanced Green Fluorescence Protein sequence, EBFP2 = Enhanced Blue Fluorescent Protein sequence, 3xFLAG = triple FLAG tag, LSL = Lox-Stop-Lox. Purple arrows: location of the genotyping primers used in this study for the transgene-specific and the junction PCRs. Light blue arrows: endonucleases-induced double strand breaks.

FIGURE 2

PCR Genotyping outcome for the KI mouse lines. Illustrative cropped gel electrophoresis for the three KI mouse lines generated by CRISPR-READI. (a) Transgene-specific genotyping of the hACE2 founders (G0) showed that 4 out of 8 pups generated (50%) carried the hACE2 transgene. Targeted integration of the hACE2 transgene was confirmed by 5’ (b) and 3’ (c) junction PCRs. Founder #43215 may carry the AAV template as an episome or be randomly integrated. (d) Transgene-specific genotyping of the selected Foxg1-EGFP line (G1 generation). Targeted integration for the Foxg1-EGFP line was confirmed by 5’ (e) and 3’ (f) junction PCRs. (g) Transgene-specific genotyping of the selected Foxg1 conditional KI (Foxg1 cKI) line (G1 generation). Targeted integration for the Foxg1 cKI line was confirmed by 5’ (h) and 3’ (i) junction PCRs. L_1kb: Ladder (Bioline, HyperLadder 1 kb); L₁₀₀: Ladder (Bioline, HyperLadder 100 bp).

FIGURE 3

Cas9 enrichment strategy and resulting read depth for each KI. Illustration of the Cas9-based enrichment (nCATs method) of genomic DNA using four sgRNA and resulting read coverage. (a) Enrichment of the murine Ace2 targeted region using four sgRNA encompassing the insertion site of the hACE2 transgene. Example of reads (pink = 5’ to 3’ reads, blue = 3’ to 5’ reads, purple = mismatch) for each homozygous hACE2 mouse tested (#64005 and #65209). (b) Enrichment of the murine Foxg1 targeted region using four sgRNA encompassing the insertion site of the EGFP transgene. Example of reads for the heterozygous Foxg1-EGFP mouse tested (#80297). (c) Enrichment of the murine Foxg1 targeted region using four sgRNA encompassing the insertion site of the EBFP2-3xFLAG transgene. Example of reads for the two heterozygous Foxg1 cKI mouse tested (#88164 and #88312). G1-G4 = sgRNA1 to sgRNA4, WT = wildtype, KI = knock-in.

FIGURE 4

Visualization of the editing outcomes at the targeted loci following AAV-driven gene editing. Dot plots of the targeted loci show that 2 lines out of 5 (40%) carry an array of multicopies (i.e., concatemers) at the insertion site. x-axis = wildtype sequence of the targeted gene (left panels); theoretical KI sequences (right panels), y-axis = consensus sequences obtained by nanopore sequencing. A continuous line indicates full alignment, a broken line indicates a mismatch. (a) Dot plots analysis for the hACE2 homozygous mice reveals that mouse #65209 carries one copy whereas mouse #64005 carries three copies of the transgene. (b) Dot plots analysis for the Foxg1-EGFP heterozygous mouse reveals that mouse #80297 carries one copy of the transgene. Dot plots analysis for the Foxg1 cKI heterozygous mice reveals that mouse #88164 carries two copies (c) whereas mouse #88132 carries one copy of the transgene. (d) LHA = left homology arm, RHA = right homology arm, ITR = inverted terminal repeat sequence, hACE2 = human ACE2 coding sequence, SV40 pA = simian virus 40 polyadenylation signal, EGFP = Enhanced Green Fluorescence Protein sequence, 3xFLAG = triple FLAG tag, LSL = Lox-Stop-Lox.

FIGURE 5

Alignment of the consensus sequences with the respective AAV vectors. The alignment identifies partial insertion of AAV vector sequences in concatemer carriers. The alignment between the hACE2 concatemer carrier consensus sequence (mouse #64005) and the AAV vector sequences reveals that both the 5’ ITR (a) and the 3’ ITR (b) sequences align partially, illustrating the integration of these ITR sequences together with a small part of the vector containing the cloning sites (i.e., XhoI and AgeI). The alignment between the Foxg1 cKI concatemer carrier consensus sequence (mouse #88164) and the sequence of the AAV vector used to generate this KI reveals that the 3’ ITR sequence (c) aligns partially, illustrating the integration of this ITR sequence together with a small part of the vector containing the cloning site (i.e., SphI). Note that the alignment of mice carrying single copies and wildtype alleles did not show any integration of the AAV vector sequences. LHA = left homology arm, RHA = right homology arm, ITR = inverted terminal repeat sequence.

FIGURE 6

The presence of ITR sequences is only found in between copies of concatemer carriers. (a) Consensus sequence for the homozygous hACE2 mouse #64005 showing the location of the ITR sequences. (b) Schematic representation of the concatemer (3 copies) in mouse #64005 and associated 5’ junction PCR design. Note that the ITR sequences are only found at the junction of each copy, but not at the at the outermost extremities, where HDR occurred. As such, junction PCRs may not identify the presence of these ITRs, assuming that amplicons 2 and 3 may be too big to be generated.