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| Status |
Public on Nov 05, 2023 |
| Title |
Cas-SF01, an engineered Cas12i nuclease is an efficient genome editing tool in mammalian cells and plants |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
The type V-I CRISPR-Cas system is becoming increasingly attractive for its potential utility in gene editing. However, natural nucleases often exhibit low efficiency, limiting their application. Here, we utilized structure-guided rational design and combinatorial protein engineering to optimize an uncharacterized Cas12i nuclease, Cas12i3. Accordingly, we developed Cas-SF01, a Cas12i3 variant that exhibits significantly improved gene-editing activity in mammalian cells and plants. Cas-SF01 displays comparable or superior editing performance compared to SpCas9 or recently engineered Cas12 nucleases. Further analysis of PAM recognition showed that Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs. Additionally, we identified an amino acid substitution, D876R, that markedly reduced the off-target effect while maintaining high on-target activity, leading to the development of Cas-SF01HiFi (high-fidelity Cas-SF01). Finally, we demonstrated that Cas-SF01 has robust gene-editing activity in both the monocot plant rice and dicot plant pepper. Our results suggest that Cas-SF01 can serve as a robust gene-editing platform with high efficiency and specificity for future genome editing applications across different organisms.
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| Overall design |
to investigate the off-target effect of the Cas12i3 nuclease, we established the GUIDE-Seq experiment of two gene targetes in 293T cell.
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| Contributor(s) |
Duan Z, Liang Y |
| Citation(s) |
38379787 |
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| Submission date |
Jul 07, 2023 |
| Last update date |
Mar 07, 2024 |
| Contact name |
Weilong Yang |
| E-mail(s) |
yangweilong@whu.edu.cn
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| Organization name |
Southern University of Science and Technology
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| Street address |
No. 1088 Xueyuan Avenue, Nanshan District
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| City |
Shenzhen |
| State/province |
Guangdong |
| ZIP/Postal code |
518055 |
| Country |
China |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (8)
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| GSM7575047 |
293T,3days, rep1_4 |
| GSM7575048 |
293T,3days, rep2_1 |
| GSM7575049 |
293T,3days, rep2_2 |
| GSM7575050 |
293T,3days, rep2_3 |
| GSM7575051 |
293T,3days, rep2_4 |
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| Relations |
| BioProject |
PRJNA992370 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE236755_RAW.tar |
450.0 Kb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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