High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets
By B. Kleinstiver, V. Pattanayak, Michelle S. Prew, S. Tsai, Nhu T. Nguyen, Zongli Zheng, J. Keith Joung. Nature, 2016
1,880 citations recorded by the source corpus.
CRISPR–Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.
Subfields: Molecular Biology
View Paper: https://www.semanticscholar.org/paper/3c8b586afbd22f28d46d52e6dc5c6f9ebf48a0a6
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In Molecular Biology
Citations in the corpus
- Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition (2016, background): “Researchers from the Joung laboratory improved the on-target specificity of the Cas9 nuclease with another independently discovered CRISPR variant SpCas9-HF1 (37) that can be used for more precise genomic edits in mammalian cells.”
- Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects (2020, background): “created SpCas9-HF1 (high-fidelity variant 1) by introducing alanine substitutions at four residues in SpCas9 to disrupt nonspecific contacts with the phosphorylated framework of the target DNA strand, which interacts with gRNA.(295) These engineered variants of SpCas9 have been engineered by reducin”
- CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future (2020, background): “A major caveat is pre-existing immunity in humans against 2 of these orthologs—SpCas9 and SaCas9, leaving CjCas9 as the only current option for this cohort of patients.”
- Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018, methodology): “This method is applicable for genome editing of any model organism with minimal off-target effects [27, 28].”
- Review of empirical and emerging breeding methods and tools for yam ( Dioscorea spp.) improvement: Status and prospects (2019, background): “Some approaches such as DNA-RNA chimeric guides, Cpf1, a single RNA endonuclease that employs a T-rich PAM on the 5′ side of the guide, and specific point mutations have been developed to mitigate this limitation (Kleinstiver et al., 2016; Zetsche et al., 2016).”
- CRISPR technology: A decade of genome editing is only the beginning (2023, methodology): “To reduce the offtarget effects of CRISPR-Cas nucleases resulting from unintended binding and cleavage, researchers have utilized a combination of rational design and selection to develop highfidelity Cas variants [such as SpCas9-HF1 (101), evoCas9 (102), HiFiCas9 (103)] and the Cas9_ R63A/Q768A var”