Partial DNA-guided Cas9 enables genome editing with reduced off-target activity
Open accessAbstract
CRISPR-Cas9 is a versatile RNA-guided genome editing tool. Here we demonstrate that partial replacement of RNA nucleotides with DNA nucleotides in CRISPR RNA (crRNA) enables efficient gene editing in human cells. This strategy of partial DNA replacement retains on-target activity when used with both crRNA and sgRNA, as well as with multiple guide sequences. Partial DNA replacement also works for crRNA of Cpf1, another CRISPR system. We find that partial DNA replacement in the guide sequence significantly reduces off-target genome editing through focused analysis of off-target cleavage, measurement of mismatch tolerance and genome-wide profiling of off-target sites. Using the structure of the Cas9-sgRNA complex as a guide, the majority of the 3' end of crRNA can be replaced with DNA nucleotide, and the 5 - and 3'-DNA-replaced crRNA enables efficient genome editing. Cas9 guided by a DNA-RNA chimera may provide a generalized strategy to reduce both the cost and the off-target genome editing in human cells.
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Chemical modifications on sgRNAs can influence their off-target effect.
“Apart from sgRNA length adjustment, chemical modifications on sgRNAs can also influence their off-target effect.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Yin et al., 2018
Replacement of ribonucleotides by deoxyribonucleotides reduces off-target effects.
“The replacement of ribonucleotides by deoxyribonucleotides also reduced off-target effects”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Yin et al., 2018
AAV delivery in vivo lasts for years in terminally differentiated cell types.
“| Delivery methods improvement | Application in vivo [95–105] | AAV: Last for years in terminally differentiated cell types | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [99]
The Results section on therapeutic delivery reports Cas9 ribonucleoprotein electroporation in Jurkat T cells, not AAV delivery in vivo. The Cell culture and FACS methods describe lentiviral infection, but no passage found for AAV lasting years in terminally differentiated cells (searched: AAV, long-term expression, and terminally differentiated cells). (LLM, conf=0.76)
Experimental — accuracy not yet measuredLNP delivery in vivo eliminates off-target effects.
“| Delivery methods improvement | Application in vivo [95–105] | LNP: Can be quickly degraded in vivo | Eliminate off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [99]
The Discussion describes reduced off-target activity from partially DNA-replaced guides in mammalian cells and mentions improved delivery technologies only as a possible future combination. Neither the supplied passages nor searches for LNP, in vivo delivery, and elimination of off-target effects found a test of LNP delivery or evidence that it eliminates off-target effects. (LLM, conf=0.74)
Experimental — accuracy not yet measuredAAV delivery in vivo may cause accumulation of off-target mutations.
“| Delivery methods improvement | Application in vivo [95–105] | AAV: Last for years in terminally differentiated cell types | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [99]
The Results section on off-target editing reports indels in cultured cells, not accumulation of mutations after in vivo AAV delivery. Searches for AAV, prolonged expression, and in vivo off-target accumulation found no passage addressing that risk; page numbers were unavailable in our copy. (LLM, conf=0.74)
Experimental — accuracy not yet measured
LNP delivery in vivo can be quickly degraded.
“| Delivery methods improvement | Application in vivo [95–105] | LNP: Can be quickly degraded in vivo | Eliminate off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [99]
The Results section discusses electroporation of Cas9 ribonucleoproteins in Jurkat T cells, not LNP delivery in vivo. Searches for LNPs, lipid nanoparticles, and rapid in vivo degradation found no passage addressing the claim in our copy; page numbers were not provided. (LLM, conf=0.74)
Experimental — accuracy not yet measured
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“The replacement of ribonucleotides by deoxyribonucleotides also reduced off-target effects (Yin et al., 2018).”
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- Authors
- Yin, H., Song, C.-Q., Suresh, S., Kwan, S.-Y., Wu, Q., Walsh, S., Ding, J., Bogorad, R. L., Zhu, L. J., Wolfe, S. A., Koteliansky, V., Xue, W., Langer, R., & Anderson, D. G.
- Affiliations
- Koch Institute for Integrative Cancer Research At MIT (US)
- University of Massachusetts Chan Medical School (US)
- Skolkovo Institute of Science and Technology (RU)
- Harvard–MIT Division of Health Sciences and Technology (US)
- Corresponding author
- Xue, Wen
- Anderson, Daniel G
- ORCID
- Venue
- Nature Chemical Biology
- Published
- 2018-01-29
- Volume
- 14
- Issue
- 3
- Pages
- 311-316
- ISSN
- 1552-4450 (ISSN-L)
- 1552-4469
- PMC
- PMC5902734
- OpenAlex
- W2785793003
- Cited by
- 240
- References
- 48
- Citations by year
- 2026: 13 · 2025: 18 · 2024: 20 · 2023: 20 · 2022: 35 · 2021: 35 · 2020: 41 · 2019: 41 · 2018: 16 · 2017: 1
- FWCI
- 11.92
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Chemical Biology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- The Journal of the American Medical Association (JAMA) Network (American Medical Association) (submittedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
- Funders
- Massachusetts General Hospital
- Lung Cancer Research Foundation
- National Institutes of Health
- National Heart, Lung, and Blood Institute (P01HL131471, DP2HL137167)
- National Cancer Institute (U54CA151884, R00CA169512, P30CA014051)
- 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 › RNA Interference and Gene Delivery
- Keywords
- CRISPR/Cas9, off-target effects, genome editing specificity, CRISPR/Cas12a, mismatch tolerance, guide RNA design, human cells
- MeSH terms
- Gene Editing, RNA, Guide, CRISPR-Cas Systems, Alleles, Cell Separation, DNA, Flow Cytometry, Humans, Nucleotides, Oligonucleotides, RNA, Guide, Kinetoplastida, Jurkat Cells, Cell Line, Tumor, Green Fluorescent Proteins, HEK293 Cells, CRISPR-Cas Systems
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