PUBLICATION
Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM
- Authors
- Liu, Y., Liang, F., Dong, Z., Li, S., Ye, J., Qin, W.
- ID
- ZDB-PUB-210828-27
- Date
- 2021
- Source
- Cells 10(8): (Journal)
- Registered Authors
- Keywords
- CRISPR/Cas9, NNG PAM, ScCas9, gene editing, zebrafish
- MeSH Terms
-
- Animals
- CRISPR-Associated Protein 9/metabolism*
- CRISPR-Cas Systems
- Clustered Regularly Interspaced Short Palindromic Repeats/genetics*
- Gene Editing*
- Genome/genetics
- Mutation
- Nucleotide Motifs
- RNA, Guide, Kinetoplastida/genetics
- RNA, Guide, Kinetoplastida/metabolism
- Streptococcus/enzymology*
- Zebrafish/genetics*
- Zebrafish Proteins/genetics
- PubMed
- 34440868 Full text @ Cells
Citation
Liu, Y., Liang, F., Dong, Z., Li, S., Ye, J., Qin, W. (2021) Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM. Cells. 10(8):.
Abstract
The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Streptococcus pyogenes Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from S. canis (ScCas9), which has a more relaxed 5'-NNG-3' PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping