The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template
Abstract
:1. Introduction
2. Materials and Methods
2.1. Preparing Cell-Free Extract
2.2. Reaction Conditions
2.3. Transformation, Selection, DNA Isolation, PCR, and Analysis
2.4. Statistical Analysis–Fisher’s Exact Test p < 0.05
3. Results
3.1. Target Site in Genetic Readout System
3.1.1. Precise HDR is Influenced by the Length of the Homology Arms of the Donor DNA Template in a Cas12a-Catalyzed Reaction
3.1.2. Precise HDR is Influenced by the Length of the Homology Arms of the Donor DNA Template in a Cas9-Catalyzed Reaction
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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* Cas12a vs. Cas9-20HDR-NS | ||||
HDR | Indel | Total | ||
Cas12a 1364 20HDR-NS | 42 | 22 | 64 | |
Cas9 1364 20HDR-NS | 20 | 41 | 61 | |
Marginal column totals | 62 | 63 | 125 | |
p-value < 0.05 | 0.0003 * | |||
* Cas12a vs. Cas9 50HDR-NS | ||||
HDR | Indel | Total | ||
Cas12a 1364 50HDR-NS | 34 | 28 | 62 | |
Cas9 1364 50HDR-NS | 3 | 58 | 61 | |
Marginal column totals | 37 | 86 | 123 | |
p-value < 0.05 | 0.0001 * | |||
* Cas12a vs. Cas9-75HDR-NS | ||||
HDR | Indel | Total | ||
Cas12a 1364 75HDR-NS | 26 | 30 | 56 | |
Cas9 1364 75HDR-NS | 2 | 61 | 63 | |
Marginal column totals | 28 | 91 | 119 | |
p-value < 0.05 | 0.00001 * |
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Hewes, A.M.; Sansbury, B.M.; Kmiec, E.B. The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template. Genes 2020, 11, 1160. https://doi.org/10.3390/genes11101160
Hewes AM, Sansbury BM, Kmiec EB. The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template. Genes. 2020; 11(10):1160. https://doi.org/10.3390/genes11101160
Chicago/Turabian StyleHewes, Amanda M., Brett M. Sansbury, and Eric B. Kmiec. 2020. "The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template" Genes 11, no. 10: 1160. https://doi.org/10.3390/genes11101160
APA StyleHewes, A. M., Sansbury, B. M., & Kmiec, E. B. (2020). The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template. Genes, 11(10), 1160. https://doi.org/10.3390/genes11101160