Application brief

HIGHLY ACCURATE HIFI READSFOR GENE EDITING RESEARCH
With highly accurate long reads (HiFi reads), powered by Single Molecule, Real-Time (SMRT®) sequencing
technology, you can more comprehensively validate gene editing techniques such as CRISPR-Cas9 approaches.

Sequence with confidence• Detect and accurately measure the efficiency of bothon- and off-target effects
Use amplificationor amplificationfree approaches• Assess insertional mutagenesis in greater detail• Help assess the potential safety of resulting constructs

CharacterizeCRISPR-Cas9
editing outcomes

The advantages of HiFi reads for gene editing research• Long read lengths up to 20 kb to span complete genes
or regions of interest• More comprehensively assess variants from base-level
modifications to structural rearrangements• High accuracy of 99.9% (Q30) to provide
Sanger-quality, base-level resolution• Uniform coverage to detect variants in repetitive andextreme GC-regions in an unbiased manner

Comprehensive variant detection
HiFi reads

105k

Other CCS reads

Number of reads

120k

90k
75k
60k
45k
30k
15k

3,700% errors relative to HiFi reads

High accuracy

900800700
60050040030020010000Q10Q20Q30Q40Single nucleotide variants≥Q50

HiFi reads

Predicted accuracy (Phred scale)
Data from a 15 kb size-selected human library using the SMRTbell® express
template prep kit 2.0 on a Sequel® IIe system (2.0 chemistry, Sequel IIe system
software v10, 30-hour movie).

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).

WorkflowSample prep

Library prep

HiFi sequencing

Data analysis

Use a range of starting
materials that fit your project,including DNA amplicons ortarget capture-based methods

Implement standardized andautomatable workflows that
support a range of target types
and up to 96-target multiplexing

Generate 1–3 million HiFi reads
up to 20 kb in length on theSequel IIe or Revio™ systems

Use SMRT® Link, a webbased end-to-end workflow
to demultiplex, analyze, andvisualize sequencing data

How can HiFi sequencing power your gene editing research?
Understand the effects of haplotype and SNVs ongene editing through allele-specific resolution

Understanding the extent of CRISPR-Cas9 editing requires
long read lengths and high accuracy to capture bothon- and off-target effects. Accurately identifying genome
modifications like large-scale deletions, insertions andstructural changes is necessary to fully understand editing
outcomes.1,2

Genetic variation such as SNVs may introduce allele-specificCas9 cleavage. HiFi sequencing can discriminate and resolve
editing efficiency with an allele-specific approach if SNVs
cause differential gRNA binding.

Frequency

Sequence beyond your target to fully understandthe potential outcomes of gene editing approaches

HiFi reads reveal allele-specific SNVs within a gRNA binding site (A) and demonstratedifferential editing efficiency between reference and alternative alleles (B).5

Avoid PCR biases and limitations by using
amplification-free approaches

SMRT sequencing of amplicons around CRISPR-Cas9 cleavage sites revealed large
deletions and insertions around both on-target and off-target cleavage sites (markedwith 0 bp).1

Many approaches to detect on- and off-target sites rely on
PCR amplification, which can introduce biases and can failto capture cleavage sites in regions of the genome that arerepetitive or GC-rich. Single-Molecule Real-Time Off-targetsequencing (SMRT-OTS) takes advantage of PacBio longread technology to overcome those limitations.

Assess insertional mutagenesis in greater detail
Gene insertion with homology-directed repair (HDR) at aCRISPR target locus can lead to small and large indels. HiFi
sequencing can characterize these mutations as well as
concatenations and other complex changes as a result ofHDR-based gene insertion.3

Detect ultra-rare off-target mutations
HiFi sequencing can efficiently detect ultra-rare substitution
mutations in whole genomes with a sensitivity of ~1×10 ‐8mutations per base pair and can efficiently detect on- andoff-target base editing in E. coli.4

Conceptual workflow of the SMRT-OTS method for Cas9 cleavage detection.5

Learn more about targeted sequencing with PacBio for gene editing validation: pacb.com/target
KEY REFERENCES
1.
2.
3.

Höijer I, Emmanouilidou A, Östlund R, et al (2022) CRISPR-Cas9 induces large structuralvariants at on-target and off-target sites in vivo that segregate across generations. NatCommun 13:627. https://doi.org/10.1038/s41467-022-28244-5
Kosicki M, Tomberg K, Bradley A (2018) Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements.
Nat Biotechnol 36:765–771. https://doi.org/10.1038/nbt.4192
Tei C, Hata S, Mabuchi A, et al (2023) Comparable analysis of multiple DNA doublestrand break repair pathways in CRISPR-mediated endogenous tagging. https://doi.
org/10.1101/2023.06.28.546861

4.
5.

Miranda JA, Fenner K, McKinzie PB, et al (2023) Unbiased whole genome detection of ultrarare
off-target mutations in genome-edited cell populations by HiFi sequencing. Environ Mol
Mutagen. https://doi.org/10.1002/em.22566
Höijer I, Johansson J, Gudmundsson S, et al (2020) Amplification-free long-read sequencing
reveals unforeseen CRISPR-Cas9 off-target activity. Genome Biol 21:290.
https://doi.org/10.1186/s13059-020-02206-w

Research use only. Not for use in diagnostic procedures. © 2023 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 third-party products. Refer to the applicable PacBio terms and conditions of sale and to theapplicable license terms at pacb.com/license. Pacific Biosciences, the PacBio logo, PacBio, Circulomics, Omniome, SMRT, SMRTbell, Iso-Seq, Sequel, Nanobind,SBB, Revio, Onso, Apton, and Kinnex are trademarks of PacBio.

102-326-595 REV01 04DEC2023