HIGHLY ACCURATE HIFI READS FOR
GENE EDITING IN PLANTS + ANIMALSWith highly accurate HiFi reads, you can discover, design, and validate genome engineering approaches with confidence, ease,and sensitivity. Gene editing starts with having the confidence of WGS information and the tools to validate editing outcomes.

Sequence with confidence
Agriscientists know that to combat climate change and feed an ever-growing population, a variety of different programmablemolecular techniques are needed to increase our food supply and shorten the development time to bring new crops to market.
Gene editing and HiFi sequencing provide fast, reliable
solutions that can:• Accelerate the breeding process• Modify target genes to improve traits• Improve plant resistances to biotic and abiotic stresses

Characterize
CRISPR-Cas9
editing outcomes
Evaluate onand off-target
effects

Use WGS ortargeted-based
approaches• Increase plant architecture to better resist climate change

The advantages of HiFi reads for gene editing approaches• Generate high-quality reference genomes to design editing • Confirm edits even in high heterozygous or GC-rich regions
experiments• Evaluate off-target effects to confirm no unintended effects• Validate constructs with high accuracy

Reference genome assembly(whole genome sequencing)
RNA transcript validation(Iso-Seq® analysis)

Construct validation (targeted sequencing)
Off-target validation (variant calling)Gene editing workflow at a glance

Sample prep

Library prep

SMRT sequencing

Data analysis

Flexible DNA input
requirements down to 5 ng

Adjustable multiplexing
options to maximize
throughput

Run a single SMRT® Cell
8M at a time or several
consecutively for set-it-andforget-it sequencing

Use SMRT® Link or opensource tools to analyze yourHiFi data

Sequence beyond your target

Comprehensive variant detection

Understanding the extent of CRISPR-Cas9 editing requires
long read lengths and high accuracy to capture both on- andoff- target effects to fully evaluate editing outcomes on atargeted and genome-wide scale.1–4

Detect all variant types to ensure both base-level
modifications and structural rearrangements are captured.
HiFi long read lengths span complete genes or regions ofinterest with Sanger-quality, base-level resolution.
% errors relative to HiFi reads

3,7009008007006005004003002001000Single nucleotide variants

HiFi reads

IndelsIllumina

Structural variants

Nanopore

Variant calling performance against Genome in a Bottle benchmarks for
PacBio® HiFi reads (35-fold, Sequel® II system, 2.0 chemistry); Illumina (35-fold,NovaSeq); Oxford Nanopore (60-fold, PromethION R9.4.1).

Analysis of the PigA locus edited with the CRISPR-Cas9 method using selectedgRNAs. SMRT® sequencing of a 5.5 kb amplicon around gRNA cut sites (verticallines) enabled detection of large-scale deletions and structural changes missedby other methods.
KEY REFERENCES

Learn more about plant + animal gene editing
pacb.com/ag-blog-gene-editing
Research use only. Not for use in diagnostic procedures. © 2022 Pacific Biosciences of California,Inc. (“PacBio”). All rights reserved. Information in this document is subject to change without notice.
PacBio assumes no responsibility for any errors or omissions in this document. Certain notices,terms, conditions and/or use restrictions may pertain to your use of PacBio products and/or thirdparty products. Refer to the applicable PacBio terms and conditions of sale and to the applicable
license terms at pacb.com/license. Pacific Biosciences, the PacBio logo, PacBio, Circulomics,Omniome, SMRT, SMRTbell, Iso-Seq, Sequel, Nanobind, and SBB are trademarks of PacBio. All othertrademarks are the sole property of their respective owners.

© 2022 PacBio. All rights reserved. 102-326-513 REV01 20JUN2022

1. Tran, N. T. et al. (2020) AAV-genome population sequencing of vectors packagingCRISPR components reveals design-influenced heterogeneity. Molecular Therapy.
Methods & Clinical Development, 18, 639-651
2. Larrea, A. and Tai, P.W.L. (2019) Highly accurate SMRT sequencing for gene editing
applications. PacBio Webinar.
3. Kosicki, M. et al. (2018) Repair of Double-strand breaks induced by CRISPR-Cas9
leads to large deletions and complex rearrangements. Nature Biotechnology, 36,765-711.
4. Höijer, l. et al. (2020) Amplification-free long read sequencing reveals unforeseen
CRISPR-Cas9 off-target activity. Genome Biology. 21, 290.