Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis
Abstract
:1. Introduction
2. Results
2.1. Design and Pre-Selection of Efficient MdPDS Specific crRNAs for LbCas12a
2.2. MdPDS Mutagenesis by T-DNA Transformation of CRISPR/LbCas12a Construct
2.3. High-Throughput Genotyping Indels Using Fluorescent PCR Combined with Capillary Gel Electrophoresis
2.4. Verification of GE Events by Amplicon Deep Sequencing
2.5. Characterization of GE Events in Apple, Introduced by CRISPR/LbCas12a
2.6. Tracing GE Events in Chimeric Regenerates
3. Discussion
4. Materials and Methods
4.1. Oligonucleotides and Kits
4.2. Guide RNA Design and Determination of DNA Cleavage Efficiency
4.3. Transformation of the CRISPR/LbCas12a Construct
4.4. Detection of Transgenic Elements by PCR
4.5. Genotyping of Indels by Fluorescent PCR Capillary Gel Electrophoresis
4.6. Amplicon Deep Sequencing of MdPDS Target Sites
4.7. Cloning and Sanger Sequencing
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Material | Locus A | Locus D | |||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Deep Sequencing | Fragment Length Analysis | Deep Sequencing | Fragment Length Analysis | ||||||||||||||||
Regenerate | Shoot | Reads analyzed | Type | Diff. from wt | [%] Reads | Allele size | Diff. to wt | Peak Height | Sum Peak Heights | [%] Peak Height | Reads analyzed | Type | Diff. from wt | [%] Reads | Allele size | Diff. from wt | Peak Height | Sum Peak Heights | [%] Peak Height |
Gala | 163,807 | wt | 0 | 100.0 | 430 | 0 | 13,617 | 13,617 | 100.0 | 151,045 | wt | 0 | 100.0 | 377 | 0 | 21,545 | 21,545 | 100.0 | |
18 219 | a | 197,114 | Del | −84 | 62.9 | 346 | −84 | 11,069 | 18,988 | 58.3 | 167,120 | Del | −12 | 51.0 | 365 | −12 | 13,582 | 26,958 | 50.4 |
wt | 0 | 35.1 | 430 | 0 | 7480 | 39.4 | Del | −2 | 49.0 | 375 | −2 | 13,376 | 49.6 | ||||||
Del | −4 | 2.1 | 426 | −4 | 439 | 2.3 | |||||||||||||
18 234 | 1 | 215,296 | Del | −1 | 38.6 | 429 | −1 | 6854 | 17,093 | 40.1 | 136,560 | wt | 0 | 43.8 | 377 | 0 | 9734 | 23,160 | 42.0 |
Del | −4 | 33.4 | 426 | −4 | 5267 | 30.8 | Del | −8 | 27.8 | 369 | −8 | 6586 | 28.4 | ||||||
wt | 0 | 25.6 | 430 | 0 | 4552 | 26.6 | Del | −10 | 16.2 | 367 | −10 | 3769 | 16.3 | ||||||
Del | −8 | 2.4 | 422 | −8 | 420 | 2.5 | Del | −13 | 7.2 | 364 | −13 | 1657 | 7.2 | ||||||
Del | −17 | 1.9 | 360 | −17 | 499 | 2.2 | |||||||||||||
Del | −16 | 1.7 | 361 | −16 | 406 | 1.8 | |||||||||||||
Del | −9 | 1.4 | n.d. | ||||||||||||||||
358 | −19 | 509 | 2.2 | ||||||||||||||||
18 238 | a | 202,366 | Del a | −4 | 80.4 | 426 | −4 | 5580 | 6604 | 84.5 | 125,784 | wt | 0 | 79.9 | 377 | 0 | 7954 | 9010 | 88.3 |
wt | 0 | 14.8 | 430 | 0 | 1024 | 15.5 | Del | −16 | 11.6 | 361 | −16 | 1056 | 11.7 | ||||||
Del | −6 | 2.8 | n.d. | Del g | −6 | 3.6 | n.d. | ||||||||||||
Del b | −5 | 1.9 | n.d. | Del | −9 | 2.7 | n.d. | ||||||||||||
Del h | −13 | 2.2 | n.d. | ||||||||||||||||
b | 183,884 | Del a | −4 | 78.2 | 426 | −4 | 11,714 | 15,395 | 76.1 | 143,519 | Del i | −18 | 23.1 | 359 | −18 | 4320 | 19,405 | 22.3 | |
wt | 0 | 19.1 | 430 | 0 | 3149 | 20.5 | Del k | −12 | 22.4 | 365 | −12 | 4453 | 22.9 | ||||||
Del | −10 | 2.7 | 420 | −10 | 532 | 3.5 | wt | 0 | 19.5 | 377 | 0 | 3917 | 20.2 | ||||||
Del | −14 | 16.8 | 363 | −14 | 3507 | 18.1 | |||||||||||||
Del | −20 | 16.2 | 357 | −20 | 3208 | 16.5 | |||||||||||||
Del h | −13 | 2.0 | n.d. | ||||||||||||||||
c | 172,597 | Del b | −5 | 40.4 | 425 | −5 | 1545 | 3904 | 39.6 | 155,911 | Del k | −12 | 39.8 | 365 | −12 | 3956 | 5,335 | 74.2 | |
Del a | −4 | 37.2 | 426 | −4 | 1300 | 33.3 | Del | −12 | 38.9 | ||||||||||
wt | 0 | 22.4 | 430 | 0 | 1059 | 27.1 | wt | 0 | 19.5 | 377 | 0 | 910 | 17.1 | ||||||
Del | −8 | 1.8 | 369 | −8 | 150 | 2.8 | |||||||||||||
370 | −7 | 173 | 3.2 | ||||||||||||||||
371 | −6 | 146 | 2.7 | ||||||||||||||||
d | 192,845 | Del a | −4 | 63.2 | 426 | −4 | 6797 | 10,654 | 63.8 | 126,005 | Del | −14 | 44.8 | 363 | −14 | 5807 | 13,844 | 41.9 | |
wt | 0 | 36.8 | 430 | 0 | 3857 | 36.2 | Del | −4 | 30.6 | 373 | −4 | 4289 | 31.0 | ||||||
Del i | −18 | 13.2 | 359 | −18 | 1857 | 13.4 | |||||||||||||
wt | 0 | 7.7 | 377 | 0 | 1010 | 7.3 | |||||||||||||
Del | −19 | 2.0 | 358 | −19 | 500 | 3.6 | |||||||||||||
Del g | −6 | 1.7 | 371 | −6 | 381 | 2.8 | |||||||||||||
18 268 | 201,374 | wt | 0 | 93.6 | 430 | 0 | 15,210 | 16,634 | 91.4 | 207,734 | wt | 0 | 92.2 | 377 | 0 | 23,847 | 26,738 | 89.2 | |
Del | −10 | 4.2 | 420 | −10 | 919 | 5.5 | Del | −38 | 2.2 | 339 | −38 | 743 | 2.8 | ||||||
Del | −14 | 2.2 | 416 | −14 | 505 | 3.0 | Del | −15 | 2.0 | 362 | −15 | 754 | 2.8 | ||||||
Del | −5 | 1.8 | 372 | −5 | 737 | 2.8 | |||||||||||||
Del | −11 | 1.8 | 366 | −11 | 657 | 2.5 | |||||||||||||
18 298 | c | 201,668 | Del c | −12 | 43.6 | 418 | −12 | 7547 | 14,362 | 52.5 | 182,157 | Del m | −20 | 50.4 | 357 | −20 | 10,278 | 20,324 | 50.6 |
wt | 0 | 37.2 | 430 | 0 | 6815 | 47.5 | Del o | −9 | 49.6 | 368 | −9 | 10,046 | 49.4 | ||||||
Art e | −165 | 17.0 | n.d. | ||||||||||||||||
Art f | −172 | 2.3 | n.d. | ||||||||||||||||
d | 204,297 | Del c | −12 | 52.0 | 418 | −12 | 9693 | 18,744 | 51.7 | 182,047 | Del m | −20 | 51.6 | 357 | −20 | 12,387 | 24,866 | 49.8 | |
Del d | −4 | 48.0 | 426 | −4 | 9051 | 48.3 | Del o | −9 | 48.4 | 368 | −9 | 12,479 | 50.2 | ||||||
e | 210,743 | Del c | −12 | 50.8 | 418 | −12 | 4822 | 9280 | 52.0 | 188,583 | Del m | −20 | 52.7 | 357 | −20 | 7393 | 14,335 | 51.6 | |
wt | wt | 28.5 | 430 | 0 | 3066 | 33.0 | Del o | −9 | 47.3 | 368 | −9 | 6942 | 48.4 | ||||||
Del | −7 | 10.3 | 423 | −7 | 1392 | 15.0 | |||||||||||||
Art e | −165 | 8.2 | n.d. | ||||||||||||||||
Art f | −172 | 2.3 | n.d. | ||||||||||||||||
i | 221,032 | Del c | −12 | 49.7 | 418 | −12 | 9484 | 19,358 | 49.0 | 197,422 | Del m | −20 | 48.9 | 357 | −20 | 13,184 | 27,323 | 48.3 | |
wt | 0 | 45.1 | 430 | 0 | 9175 | 47.4 | Del o | −9 | 46.2 | 368 | −9 | 12,774 | 46.8 | ||||||
Del d | −4 | 3.1 | 426 | −4 | 699 | 3.6 | wt | 0 | 4.9 | 377 | 0 | 1365 | 5.0 | ||||||
Art e | −165 | 2.0 | n.d. |
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Schröpfer, S.; Flachowsky, H. Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis. Int. J. Mol. Sci. 2021, 22, 12611. https://doi.org/10.3390/ijms222212611
Schröpfer S, Flachowsky H. Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis. International Journal of Molecular Sciences. 2021; 22(22):12611. https://doi.org/10.3390/ijms222212611
Chicago/Turabian StyleSchröpfer, Susan, and Henryk Flachowsky. 2021. "Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis" International Journal of Molecular Sciences 22, no. 22: 12611. https://doi.org/10.3390/ijms222212611