Highly Parallel Profiling of Cas9 Variant Specificity
Open accessAbstract
Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.
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Although some Cas9 variants have comparable editing activity and specificity with SpCas9 at a few target sites, high-throughput analysis is still lacking.
“Although some Cas9 variants have comparable editing activity and specificity with SpCas9 at a few target sites, high-throughput analysis is still lacking.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [23]
The Introduction says earlier off-target methods were relatively low-throughput, and the Results say a systematic comparison of SpCas9 specificity variants had not previously been reported. The Results also report comparable on-target activity for some variants, but not comparable specificity: each tested variant improved specificity relative to SpCas9. Moreover, this paper itself performs a high-throughput comparison across 59 guides, so it does not support the claim that such analysis is still lacking. (LLM, conf=0.84)
Experimental — accuracy not yet measuredIn several previous reports, LZ3 Cas9, Sniper Cas9, and HiFi Cas9 had comparable editing activity to SpCas9-WT in human cells or other animal embryos.
“It worth mentioning that in several previous reports, LZ3 Cas9, Sniper Cas9, and HiFi Cas9 had comparable editing activity to the SpCas9-WT in human cells or other animal embryos.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [23]
The Results report on-target activity comparable to WT SpCas9 for LZ3 Cas9, HiFi Cas9, and Sniper-Cas9. But the paper describes LZ3 as a newly generated variant, so it does not support attributing the finding for all three variants to several previous reports. The cited passages do not establish the animal-embryo setting. (LLM, conf=0.88)
Experimental — accuracy not yet measuredLZ3 Cas9 did not display a stable 1 bp site-directed insertion pattern.
“Although LZ3 Cas9 did not display a stable 1 bp site-directed insertion pattern, as shown in previous studies according to the DSB repair results”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [23]
The Results report that LZ3 Cas9 had a markedly different +1 insertion-frequency profile from WT SpCas9 across 58 guides, with frequencies influenced by target sequence. The Discussion likewise describes a differential +1 insertion profile, but neither passage establishes that LZ3 lacked a stable 1 bp insertion pattern. (LLM, conf=0.80)
Experimental — accuracy not yet measured
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- Authors
- Schmid-Burgk, J. L., Gao, L., Li, D., Gardner, Z., Strecker, J., Lash, B., & Zhang, F.
- Affiliations
- Broad Institute (US)
- Corresponding author
- Zhang, Feng
- Venue
- Molecular Cell
- Publisher
- Elsevier BV
- Published
- 2020-05
- Volume
- 78
- Issue
- 4
- Pages
- 794-800.e8
- ISSN
- 1097-2765 (ISSN-L)
- PMC
- PMC7370240
- OpenAlex
- W3011375666
- Cited by
- 197
- References
- 29
- Citations by year
- 2026: 11 · 2025: 25 · 2024: 42 · 2023: 34 · 2022: 38 · 2021: 34 · 2020: 13
- FWCI
- 9.65
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Molecular Cell (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-nd)
- Funders
- National Institutes of Health
- G. Harold and Leila Y. Mathers Foundation
- Howard Hughes Medical Institute
- Edward Mallinckrodt, Jr. Foundation
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Keywords
- Cas9 variants, off-target cleavage, DNA double-strand breaks, genome editing specificity, programmable nucleases
- MeSH terms
- Gene Editing, CRISPR-Associated Protein 9, RNA, Guide, CRISPR-Cas Systems, Humans, Genetic Variation, RNA, Guide, Kinetoplastida, K562 Cells, DNA Breaks, Double-Stranded, HEK293 Cells, CRISPR-Cas Systems
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