PUBLICATION
Key sequence features of CRISPR RNA for dual-guide CRISPR-Cas9 ribonucleoprotein complexes assembled with wild-type or HiFi Cas9
- Authors
- Okada, K., Aoki, K., Tabei, T., Sugio, K., Imai, K., Bonkohara, Y., Kamachi, Y.
- ID
- ZDB-PUB-220216-33
- Date
- 2022
- Source
- Nucleic acids research 50(5): 2854-2871 (Journal)
- Registered Authors
- Kamachi, Yusuke
- Keywords
- none
- MeSH Terms
-
- CRISPR-Associated Protein 9*
- RNA, Guide, Kinetoplastida*/genetics
- CRISPR-Cas Systems*/genetics
- Gene Editing*
- Zebrafish/genetics
- Animals
- RNA/genetics
- Ribonucleoproteins/genetics
- PubMed
- 35166844 Full text @ Nucleic Acids Res.
Citation
Okada, K., Aoki, K., Tabei, T., Sugio, K., Imai, K., Bonkohara, Y., Kamachi, Y. (2022) Key sequence features of CRISPR RNA for dual-guide CRISPR-Cas9 ribonucleoprotein complexes assembled with wild-type or HiFi Cas9. Nucleic acids research. 50(5):2854-2871.
Abstract
Specific sequence features of the protospacer and protospacer-adjacent motif (PAM) are critical for efficient cleavage by CRISPR-Cas9, but current knowledge is largely derived from single-guide RNA (sgRNA) systems assessed in cultured cells. In this study, we sought to determine gRNA sequence features of a more native CRISPR-Cas9 ribonucleoprotein (RNP) complex with dual-guide RNAs (dgRNAs) composed of crRNA and tracrRNA, which has been used increasingly in recent CRISPR-Cas9 applications, particularly in zebrafish. Using both wild-type and HiFi SpCas9, we determined on-target cleavage efficiencies of 51 crRNAs in zebrafish embryos by assessing indel occurrence. Statistical analysis of these data identified novel position-specific mononucleotide features relevant to cleavage efficiencies throughout the protospacer sequence that may be unique to CRISPR-Cas9 RNPs pre-assembled with perfectly matched gRNAs. Overall features for wild-type Cas9 resembled those for HiFi Cas9, but specific differences were also observed. Mutational analysis of mononucleotide features confirmed their relevance to cleavage efficiencies. Moreover, the mononucleotide feature-based score, CRISPR-kp, correlated well with efficiencies of gRNAs reported in previous zebrafish RNP injection experiments, as well as independently tested crRNAs only in RNP format, but not with Cas9 mRNA co-injection. These findings will facilitate design of gRNA/crRNAs in genome editing applications, especially when using pre-assembled RNPs.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping