Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jul 9:18:639-651.
doi: 10.1016/j.omtm.2020.07.007. eCollection 2020 Sep 11.

AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity

Ngoc Tam Tran  1   2 , Cheryl Heiner  3 , Kristina Weber  3 , Michael Weiand  3 , Daniella Wilmot  4 , Jun Xie  1   2   5 , Dan Wang  1   2 , Alexander Brown  1   2 , Sangeetha Manokaran  1   5 , Qin Su  1   5 , Maria L Zapp  4 , Guangping Gao  1   2   5   6 , Phillip W L Tai  1   2
Affiliations

AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity

Ngoc Tam Tran et al. Mol Ther Methods Clin Dev. .

Abstract

The gene therapy field has been galvanized by two technologies that have revolutionized treating genetic diseases: vectors based on adeno-associated viruses (AAVs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas gene-editing tools. When combined into one platform, these safe and broadly tropic biotherapies can be engineered to target any region in the human genome to correct genetic flaws. Unfortunately, few investigations into the design compatibility of CRISPR components in AAV vectors exist. Using AAV-genome population sequencing (AAV-GPseq), we previously found that self-complementary AAV vector designs with strong DNA secondary structures can cause a high degree of truncation events, impacting production and vector efficacy. We hypothesized that the single-guide RNA (sgRNA) scaffold, which contains several loop regions, may also compromise vector integrity. We have therefore advanced the AAV-GPseq method to also interrogate single-strand AAV vectors to investigate whether vector genomes carrying Cas9-sgRNA cassettes can cause truncation events. We found that on their own, sgRNA sequences do not produce a high degree of truncation events. However, we demonstrate that vector genome designs that carry dual sgRNA expression cassettes in tail-to-tail configurations lead to truncations. In addition, we revealed that heterogeneity in inverted terminal repeat sequences in the form of regional deletions inherent to certain AAV vector plasmids can be interrogated.

Keywords: AAV-genome population sequencing; CRISPR-Cas; ITR; adeno-associated virus; gene therapy vectors; real-time sequencing; scAAV; sgRNA; single molecule; ssAAV.

PubMed Disclaimer

Figures

None
Graphical abstract
Figure 1
Figure 1
Schematic of SMRT Sequencing-Based AAV-GPseq Workflow Single-stranded or self-complementary (ssAAV and scAAV) genomes are purified from virions. The plus (+) and minus (−) strands of the ssAAV genomes undergo strand annealing in solution to form adapterable ends. ssAAVs are adaptered on both ends of the genome by ligation to SMRTbell adapters (green loops). scAAVs are adaptered only on one end. Libraries are subjected to SMRT sequencing to produce long reads that can be processed by strand-specific consensus reconstruction to separate plus- and minus-stranded genomes as independent reads. Since scAAVs are only adaptered on one end, a single pass encompasses both the forward and reverse strand of the full-length genome. Hence, strand-specific consensus does not impact representation.
Figure 2
Figure 2
SMRT Sequencing Summary of the ssAAV-SaCas9-sgRNA Vector (A) IGV display of reads mapping to the ssAAV-SaCas9-sgRNA cis plasmid reference. The top track displays the alignment summary in log10 scale. The bottom track displays individual reads mapping to the reference, squished down to visualize all reads. A linear diagram of the cis plasmid construct is displayed above the alignment tracks. Sequences matching the reference are displayed in gray, and those not matching the reference are colored as individual bases. Gaps are displayed as black dashes. In this squished view, they appear as speckles in the alignment. The display also shows soft-clipped bases to highlight truncated genomes that are in most of the cases in a self-complementary conformation. The blue arrow indicates a portion of reads with full-length genomes. The red arrow demarcates a population of truncated, self-complementary reads that are centered on the sgRNA cassette. The colored portion of the alignment summary track reflects sequence variation at the ITR regions that mark the distribution of flip- and flop-orientated ITRs. The ITR orientations within the reference are flop at both 5′ and 3′ ITRs. (B) Non-squished zoom-in displays of full-length genomes and truncated genomes from (A) (blue and red arrows). Each row is a continuous single SMRT read circular consensus. Mismatches at the 5′ and 3′ flanks indicate flip-orientated ITRs. Truncated genomes are self-complementary in structure, as revealed by their partial alignments, which spans half of the read. A color legend of the IGV-displayed matches, mismatches, gaps, and inserts is shown. (C) Lengths of all reads mapping to the vector reference were determined and their distributions are plotted. The relative abundances before normalization (blue trace) and after normalizing to the DNA (red trace) are shown. The relative read abundances of major peaks (brackets) are displayed as percentages of all mapped reads greater than 500 bp in length. Since the abundance of reads with lengths under 500 bp cannot be formally confirmed, they are discounted from the analysis.
Figure 3
Figure 3
Evaluation of Host-Cell DNAs Encapsidated within ssAAV-SaCas9-sgRNA Vectors (A) Venn diagrams of mapped read abundances related to the vector genome (white circles), the host-cell genome (gray circles), or a non-related DNA spike-in (red circle). Non-overlapping portions represent reads that map exclusively to either reference. Regions of overlap represent the counts of reads that co-mapped to both the host-cell genome and the vector genome. The percentages of co-mapped reads are displayed. (B) Histograms summarizing the number of unique regions throughout the host-cell genome (hg38) to which sequencing reads are mapped. Left graphs, without non-related DNA spike-in; right graphs, with spike-in. (C) Squished IGV display of chimeric reads mapped to the vector genome. Sequence regions that align to the vector reference are in gray, while those that do not are colored as their respective bases. A unifying feature of chimeric reads is that they are anchored at the 3′ ITR region. The lower left schematic illustrates the hypothesized self-complementary chimeric structures for reference.
Figure 4
Figure 4
Quantification of ITR Configuration and Heterogeneity with SMRT Sequencing (A) Stacked histogram of flip and flop configuration percentage among all full-length genomes. (B and C) Tabulation of read starts and ends to reveal frequency of partial ITRs. (B) Traces representing absolute counts of read alignment starts (red) and alignment ends (blue) for vector DNA. The vector genome from ITR to ITR is displayed above to indicate where start and stops are mapped. (C) Heatmap display (log10 scale) of read alignment starts and stops to indicate vector genome termini throughout the 145-nt ITR structure. All data reflect samples with or without treatment to heating and slow cooling (HT). Percentages of read termini among reads ending within the 145-nt ITR are displayed. (D–F) Quantification of ITR damage/repair of an 11-nt deletion within the C-domain found in certain AAV cis vectors in circulation. (D) mfold DNA structures of an intact ITR (left) and mutant ITR with 11-nt deletion within the C-domain. (E) IGV display of aligned SMRT reads zoomed into the ITR domain. All bases are displayed with their respective color scheme. Gaps are shown as dashed lines. Red arrows indicate reads containing the C-domain deletion. (F) Pie chart summarizing the absolute counts of ITRs carrying the C-domain deletion in the flip or flop orientations.
Figure 5
Figure 5
SMRT Sequencing of AAV Vectors Carrying Dual sgRNA Reveals Tail-to-Tail-Oriented Designs Yield Truncated Genomes (A and C) Squished IGV displays of SMRT sequencing reads of a single-strand vector carrying dual sgRNAs in a tail-to-tail orientation (ssAAV-2sgRNA-T2T, A) and a self-complementary vector carrying dual sgRNAs in a tail-to-head orientation (scAAV-2sgRNA-T2H, C) mapping to their respective references. Aligned sequences that match the reference are in gray, while mismatches are colored as individual bases. Gaps are displayed as dashes. Soft-clipped bases are shown to reveal complement strand misaligning to the reference. The squished view results in speckled appearance of gaps. Each alignment is accompanied by an alignment summary track and a diagram of the vector plasmid reference. (C) Box image displays the predicted structure of self-complementary (truncated) vector genomes. The scAAV-2sgRNA-T2H vector packages both scAAV genomes and unexpected ssAAV genomes spanning from the 5′ ITR to the 3′ ITR (reads marked by brackets). Arrows indicate the direction of the U6-driven sgRNAs (forward, magenta; reverse, cyan). (B and D) Traces displaying the relative abundances of mapped reads distributed by length for (A) and (C), respectively. Percentages of read abundances in major peaks (brackets) are shown.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wang D., Tai P.W.L., Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat. Rev. Drug Discov. 2019;18:358–378. - PMC - PubMed
    1. Broeders M., Herrero-Hernandez P., Ernst M.P.T., van der Ploeg A.T., Pijnappel W.W.M.P. Sharpening the molecular scissors: advances in gene-editing technology. iScience. 2020;23:100789. - PMC - PubMed
    1. Mou H., Kennedy Z., Anderson D.G., Yin H., Xue W. Precision cancer mouse models through genome editing with CRISPR-Cas9. Genome Med. 2015;7:53. - PMC - PubMed
    1. Maeder M.L., Stefanidakis M., Wilson C.J., Baral R., Barrera L.A., Bounoutas G.S., Bumcrot D., Chao H., Ciulla D.M., DaSilva J.A. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat. Med. 2019;25:229–233. - PubMed
    1. Ibraheim R., Song C.Q., Mir A., Amrani N., Xue W., Sontheimer E.J. All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo. Genome Biol. 2018;19:137. - PMC - PubMed

LinkOut - more resources