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Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM
Yunxing Liu1, Fang Liang2, Zijiong Dong2
1State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen 518055, China.
Cells
|August 27, 2021
Summary
The Cas9 from Streptococcus canis (ScCas9) enzyme enables gene editing in zebrafish by targeting a relaxed NNG PAM sequence. Optimizing its delivery and guide RNA improved ScCas9 activity, expanding zebrafish genome editing capabilities.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR/Cas9 gene editing is crucial for zebrafish research.
- Streptococcus pyogenes Cas9 (SpCas9) is limited by its NGG PAM, restricting genome editing targets in zebrafish.
- Cas9 from Streptococcus canis (ScCas9) offers a relaxed 5'-NNG-3' PAM, showing potential for broader applications.
Purpose of the Study:
- To characterize and optimize the gene editing activity of ScCas9 in zebrafish.
- To evaluate ScCas9 as a novel nuclease for zebrafish genome editing.
- To expand the editable range within the zebrafish genome using a relaxed PAM system.
Main Methods:
- Delivered ScCas9 as a ribonucleoprotein complex in zebrafish.
- Utilized synthetic modified crRNA:tracrRNA duplexes for guide RNA delivery.
- Assessed ScCas9-induced mutations at various zebrafish loci.
Main Results:
- ScCas9 successfully induced mutations in zebrafish when delivered as a ribonucleoprotein complex.
- The use of synthetic modified crRNA:tracrRNA duplexes significantly enhanced ScCas9 activity at several loci.
- This study represents the first evaluation of ScCas9 activity in an animal model.
Conclusions:
- Optimized ScCas9 is an effective new nuclease for zebrafish genome editing, targeting relaxed NNG PAMs.
- The improved ScCas9 system expands the targeting scope for zebrafish gene editing.
- This work provides a valuable tool for advancing genetic studies in zebrafish.
