Low Efficiency of Homology-Independent Targeted Integration for CRISPR/Cas9 Correction in the Vicinity of the SLC26A4 c.919-2A>G Variant
Abstract
1. Introduction
2. Results
2.1. The Surveyor Assay Results
2.2. Statistics of HITI Editing Efficiency
3. Discussion
4. Materials and Methods
4.1. HITI Plasmid System
4.2. Cell Culture and Transfection
4.3. Targeting Efficiency of sgRNAs and Selection
4.4. HITI
4.5. Amplicon-Based NGS and Data Processing of Reads
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
References
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No. | sgRNA and PAM Sequence | Out-of-Frame Scores |
---|---|---|
sgRNA1 | AAAGATGTTAAAAACTCCAT TGG | 53.9 |
sgRNA2 | ATTGCTACTGCCATTTCATA TGG | 69.8 |
sgRNA3 | TTAGAAAGTTCAGCATTATT TGG | 68.4 |
sgRNA4 | CATTATTTGGTTGACAAACA AGG | 70.5 |
Name | SLC-1 * | SLC-2 * | SLC-3 * |
---|---|---|---|
Raw reads | 17,435 | 18,381 | 22,062 |
Refined reads | 17,184 | 18,181 | 21,755 |
Refined reads with QueryWT | 16,466 (95.82%) | 16,561 (91.09%) | 20,688 (95.10%) |
Refined reads with QueryCI | 0 | 27 (0.15%) | 0 |
Refined reads with deletion | 14 (0.08%) | 193 (1.06%) | 19 (0.09%) |
Refined reads with insertion | 0 | 434 (2.39%) | 0 |
Refined reads with indel | 0 | 3 (0.02%) | 1 (<0.01%) |
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Ho, C.-H.; Tsai, C.-Y.; Chang, C.-C.; Hu, C.-J.; Huang, C.-Y.; Lu, Y.-C.; Lin, P.-H.; Lin, C.-H.; Lin, H.-I.; OuYang, C.-H.; et al. Low Efficiency of Homology-Independent Targeted Integration for CRISPR/Cas9 Correction in the Vicinity of the SLC26A4 c.919-2A>G Variant. Int. J. Mol. Sci. 2025, 26, 4980. https://doi.org/10.3390/ijms26114980
Ho C-H, Tsai C-Y, Chang C-C, Hu C-J, Huang C-Y, Lu Y-C, Lin P-H, Lin C-H, Lin H-I, OuYang C-H, et al. Low Efficiency of Homology-Independent Targeted Integration for CRISPR/Cas9 Correction in the Vicinity of the SLC26A4 c.919-2A>G Variant. International Journal of Molecular Sciences. 2025; 26(11):4980. https://doi.org/10.3390/ijms26114980
Chicago/Turabian StyleHo, Chang-Han, Cheng-Yu Tsai, Chi-Chieh Chang, Chin-Ju Hu, Cheng-Yen Huang, Ying-Chang Lu, Pei-Hsuan Lin, Chin-Hsien Lin, Han-I Lin, Chih-Hsin OuYang, and et al. 2025. "Low Efficiency of Homology-Independent Targeted Integration for CRISPR/Cas9 Correction in the Vicinity of the SLC26A4 c.919-2A>G Variant" International Journal of Molecular Sciences 26, no. 11: 4980. https://doi.org/10.3390/ijms26114980
APA StyleHo, C.-H., Tsai, C.-Y., Chang, C.-C., Hu, C.-J., Huang, C.-Y., Lu, Y.-C., Lin, P.-H., Lin, C.-H., Lin, H.-I., OuYang, C.-H., Hsu, C.-J., Liu, T.-C., Chen, Y.-T., Chan, Y.-H., Cheng, Y.-F., & Wu, C.-C. (2025). Low Efficiency of Homology-Independent Targeted Integration for CRISPR/Cas9 Correction in the Vicinity of the SLC26A4 c.919-2A>G Variant. International Journal of Molecular Sciences, 26(11), 4980. https://doi.org/10.3390/ijms26114980