Increasing the specificity of CRISPR systems with engineered RNA secondary structures
Open accessAbstract
CRISPR (clustered regularly interspaced short palindromic repeat) systems have been broadly adopted for basic science, biotechnology, and gene and cell therapy. In some cases, these bacterial nucleases have demonstrated off-target activity. This creates a potential hazard for therapeutic applications and could confound results in biological research. Therefore, improving the precision of these nucleases is of broad interest. Here we show that engineering a hairpin secondary structure onto the spacer region of single guide RNAs (hp-sgRNAs) can increase specificity by several orders of magnitude when combined with various CRISPR effectors. We first demonstrate that designed hp-sgRNAs can tune the activity of a transactivator based on Cas9 from Streptococcus pyogenes (SpCas9). We then show that hp-sgRNAs increase the specificity of gene editing using five different Cas9 or Cas12a variants. Our results demonstrate that RNA secondary structure is a fundamental parameter that can tune the activity of diverse CRISPR systems.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Modifications at the 5′ end of the gRNA, such as addition of secondary structures or unpaired nucleotides, lower off-target activity.
“Modifications at the 5′end of the gRNA, such as the addition of secondary structures (Kocak et al., 2019) or unpaired nucleotides (Kulcsár et al., 2020), have also been found to lower off-target activity.”
From Emerging applications of gene editing technologies for the development of climate-resilient crops (2025) · cites this paper as Kocak et al., 2019
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
The citation lists haven’t been fetched yet
References 69 · Cited by 351
Research works printed but not linked: 1
- [66]Kuscu C,, Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nature biotechnology, 2014. 24837660
- Authors
- Kocak, D. D., Josephs, E. A., Bhandarkar, V., Adkar, S. S., Kwon, J. B., & Gersbach, C. A.
- Affiliations
- University of North Carolina at Greensboro (US)
- Duke University (US)
- Corresponding author
- Gersbach, Charles A.
- Venue
- Nature Biotechnology
- Published
- 2019-04-15
- Volume
- 37
- Issue
- 6
- Pages
- 657-666
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC6626619
- OpenAlex
- W2939661038
- Cited by
- 351
- References
- 69
- Citations by year
- 2026: 28 · 2025: 37 · 2024: 47 · 2023: 35 · 2022: 56 · 2021: 55 · 2020: 58 · 2019: 33
- FWCI
- 16.79
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- NC Digital Online Collection of Knowledge and Scholarship (The University of North Carolina at Greensboro) (submittedVersion)
- Funders
- National Science Foundation (DP2OD008586, R01DA036865, 1709527, 1830957)
- Foundation for the National Institutes of Health
- Sarepta Therapeutics
- Paul G. Allen Frontiers Group
- National Institutes of Health (EFMA-1830957, 1dp2od008586-01)
- National Institute on Drug Abuse (R01DA036865)
- National Cancer Institute (P30CA014236)
- National Institute of General Medical Sciences (T32GM007171, P50GM098792, T32GM145449)
- National Institute of Arthritis and Musculoskeletal and Skin Diseases (P30AR066527, R01AR069085)
- Division of Materials Research
- Eunice Kennedy Shriver National Institute of Child Health and Human Development (T32HD040372)
- 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, single guide RNA, hairpin secondary structure, off-target effects, gene editing specificity, CRISPR/Cas12a, SpCas9, Streptococcus pyogenes, transcriptional activator, RNA engineering, gene therapy safety
- MeSH terms
- Gene Editing, RNA, Guide, CRISPR-Cas Systems, Biotechnology, Nucleic Acid Conformation, RNA, Streptococcus pyogenes, RNA, Guide, Kinetoplastida, CRISPR-Cas Systems
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
