Uploaded May 2026 | Updated September 2026, 1 hour ago
CRISPR-Cas9 gene editing can create outcomes that are hard to characterize with short reads or PCR-based targeted sequencing. In this webinar, Nadia Sellami, PhD, hosts the session; Elizabeth Tseng introduces how PacBio HiFi sequencing and PureTarget amplification-free target enrichment can help you assess on-target and off-target edits, larger structural variants, mosaicism, and methylation signal from native DNA.
Then Adam Ameur, PhD, shares how SciLifeLab National Genomics Infrastructure and Uppsala University used a custom PureTarget panel to revisit CRISPR-Cas9 edited zebrafish samples. The study generated 1168x average target-region coverage, QV39 read quality, a 3 bp off-target deletion, a 1053 bp on-target deletion, and up to 18 different alleles in one F0 zebrafish.
*Key takeaways*
- PureTarget sequences unamplified DNA, retaining methylation information while avoiding PCR bias.
- A custom 7-target panel covered 4 on-target genes and 3 off-target sites across 32 zebrafish samples.
- PureTarget data showed more even allele balance than LR-PCR in heterozygous F1 zebrafish.
- High-coverage HiFi reads helped resolve inherited off-target editing, larger deletions, and mosaicism.
- The Q&A covers target size, detection frequency, base-editing use cases, coverage targets, and where whole-genome sequencing may still be needed.
*Featured speakers*
- Nadia Sellami, PhD, Global Lead, Biopharma, PacBio
- Elizabeth Tseng, Associate Director, Integrated Marketing, PacBio
- Adam Ameur, PhD, Associate Professor and Senior Bioinformatician, SciLifeLab National Genomics Infrastructure, Uppsala University, Sweden
*Chapters*
00:00 Welcome and speaker introductions
01:58 HiFi sequencing for gene editing research
03:50 Long reads, high accuracy, and methylation signal
05:11 Amplicon-based sequencing and what short reads miss
07:03 PureTarget versus PCR-based targeted sequencing
07:42 How PureTarget works
09:08 Adam Ameur: PureTarget for CRISPR-Cas9 zebrafish
10:26 Long-read sequencing at SciLifeLab/NGI
11:43 PureTarget custom panel setup
12:37 Off-target editing, structural variation, and mosaicism
14:00 Zebrafish sample design and LR-PCR baseline
15:18 Custom panel: 7 on- and off-target regions
16:00 PureTarget performance and allele balance
17:02 Why LR-PCR can be biased
18:17 PureTarget read quality around the Cas9 site
19:18 Off-target edit in the F1 generation
20:03 1053 bp on-target deletion in sh2b3
20:59 Mosaicism in F0 zebrafish
21:56 SIQ analysis of editing alleles
22:47 Germ-cell mosaicism in F1 siblings
24:15 How germ-cell mosaicism may arise
24:58 Summary: off-target edits, SVs, and mosaicism
25:26 5mC methylation as an added layer
26:38 PureTarget summary and future targets
27:48 Acknowledgements
28:15 Q&A: molecule size between CRISPR sites
29:13 Q&A: other PureTarget applications
30:02 Q&A: detection frequency
30:53 Q&A: when amplification-based methods still fit
31:39 Q&A: inherited mosaicism
32:27 Q&A: translocations and deletion frequency
33:44 Q&A: base-editing off-target identification
34:27 Q&A: targets, multiplexing, and SMRT Cell use
36:07 Q&A: input DNA and coverage
37:42 Q&A: length bias for structural variants
39:23 Q&A: accounting for unknown off-target edits
40:12 Q&A: yield variation
40:49 Q&A: method development versus verification goals
42:15 Q&A: off-target read yield
43:02 Q&A: SIQ software availability
43:34 Future directions and closing
*Resources*
- Preprint record: Accurate characterization of CRISPR-Cas9 genome editing outcomes and mosaicism with near-perfect long reads: sciety.org/articles/activity/10.1101/2025.09.08.674810
- PureTarget custom panels technical note: programs.pacb.com/l/1652/2025-10-03/458hnf
- PureTarget custom panels PDF: pacb.com/wp-content/uploads/Technical-note-A-practical-guide-to-amplification-free-custom-PureTarget-panels.pdf
- SIQ full text: pmc.ncbi.nlm.nih.gov/articles/PMC9453813
Which gene editing outcome is hardest for your team to characterize: off-target edits, larger SVs, mosaicism, or methylation changes?
*Comment below.*
Subscribe for more PacBio webinars on HiFi sequencing, targeted sequencing, and gene editing research.
For Research Use Only. Not for use in diagnostic procedures.
#HiFiSequencing #GeneEditing #PureTarget
CRISPR-Cas9 gene editing can create outcomes that are hard to characterize with short reads or PCR-based targeted sequencing. In this webinar, Nadia Sellami, PhD, hosts the session; Elizabeth Tseng introduces how PacBio HiFi sequencing and PureTarget amplification-free target enrichment can help you assess on-target and off-target edits, larger structural variants, mosaicism, and methylation signal from native DNA.
Then Adam Ameur, PhD, shares how SciLifeLab National Genomics Infrastructure and Uppsala University used a custom PureTarget panel to revisit CRISPR-Cas9 edited zebrafish samples. The study generated 1168x average target-region coverage, QV39 read quality, a 3 bp off-target deletion, a 1053 bp on-target deletion, and up to 18 different alleles in one F0 zebrafish.
*Key takeaways*
- PureTarget sequences unamplified DNA, retaining methylation information while avoiding PCR bias.
- A custom 7-target panel covered 4 on-target genes and 3 off-target sites across 32 zebrafish samples.
- PureTarget data showed more even allele balance than LR-PCR in heterozygous F1 zebrafish.
- High-coverage HiFi reads helped resolve inherited off-target editing, larger deletions, and mosaicism.
- The Q&A covers target size, detection frequency, base-editing use cases, coverage targets, and where whole-genome sequencing may still be needed.
*Featured speakers*
- Nadia Sellami, PhD, Global Lead, Biopharma, PacBio
- Elizabeth Tseng, Associate Director, Integrated Marketing, PacBio
- Adam Ameur, PhD, Associate Professor and Senior Bioinformatician, SciLifeLab National Genomics Infrastructure, Uppsala University, Sweden
*Chapters*
00:00 Welcome and speaker introductions
01:58 HiFi sequencing for gene editing research
03:50 Long reads, high accuracy, and methylation signal
05:11 Amplicon-based sequencing and what short reads miss
07:03 PureTarget versus PCR-based targeted sequencing
07:42 How PureTarget works
09:08 Adam Ameur: PureTarget for CRISPR-Cas9 zebrafish
10:26 Long-read sequencing at SciLifeLab/NGI
11:43 PureTarget custom panel setup
12:37 Off-target editing, structural variation, and mosaicism
14:00 Zebrafish sample design and LR-PCR baseline
15:18 Custom panel: 7 on- and off-target regions
16:00 PureTarget performance and allele balance
17:02 Why LR-PCR can be biased
18:17 PureTarget read quality around the Cas9 site
19:18 Off-target edit in the F1 generation
20:03 1053 bp on-target deletion in sh2b3
20:59 Mosaicism in F0 zebrafish
21:56 SIQ analysis of editing alleles
22:47 Germ-cell mosaicism in F1 siblings
24:15 How germ-cell mosaicism may arise
24:58 Summary: off-target edits, SVs, and mosaicism
25:26 5mC methylation as an added layer
26:38 PureTarget summary and future targets
27:48 Acknowledgements
28:15 Q&A: molecule size between CRISPR sites
29:13 Q&A: other PureTarget applications
30:02 Q&A: detection frequency
30:53 Q&A: when amplification-based methods still fit
31:39 Q&A: inherited mosaicism
32:27 Q&A: translocations and deletion frequency
33:44 Q&A: base-editing off-target identification
34:27 Q&A: targets, multiplexing, and SMRT Cell use
36:07 Q&A: input DNA and coverage
37:42 Q&A: length bias for structural variants
39:23 Q&A: accounting for unknown off-target edits
40:12 Q&A: yield variation
40:49 Q&A: method development versus verification goals
42:15 Q&A: off-target read yield
43:02 Q&A: SIQ software availability
43:34 Future directions and closing
*Resources*
- Preprint record: Accurate characterization of CRISPR-Cas9 genome editing outcomes and mosaicism with near-perfect long reads: sciety.org/articles/activity/10.1101/2025.09.08.674810
- PureTarget custom panels technical note: programs.pacb.com/l/1652/2025-10-03/458hnf
- PureTarget custom panels PDF: pacb.com/wp-content/uploads/Technical-note-A-practical-guide-to-amplification-free-custom-PureTarget-panels.pdf
- SIQ full text: pmc.ncbi.nlm.nih.gov/articles/PMC9453813
Which gene editing outcome is hardest for your team to characterize: off-target edits, larger SVs, mosaicism, or methylation changes?
*Comment below.*
Subscribe for more PacBio webinars on HiFi sequencing, targeted sequencing, and gene editing research.
For Research Use Only. Not for use in diagnostic procedures.
#HiFiSequencing #GeneEditing #PureTarget
