Optimizing sgRNA length to improve target specificity and efficiency for the GGTA1 gene using the CRISPR/Cas9 gene editing system


Autoři: Anders W. Matson aff001;  Nora Hosny aff001;  Zachary A. Swanson aff001;  Bernhard J. Hering aff001;  Christopher Burlak aff001
Působiště autorů: Schulze Diabetes Institute, Department of Surgery, University of Minnesota School of Medicine, Minneapolis, MN, United States of America aff001;  Department of Medical Biochemistry and Molecular Biology, Suez Canal University, Faculty of Medicine, Ismailia, Egypt aff002
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0226107

Souhrn

The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood of off-target mutation while maintaining a high efficiency rate of desired genetic mutations for the GGTA1 gene. Extension of sgRNA length, responsible for recognition of the target DNA sequence for Cas9 cleavage, resulted in improved specificity for the GGTA1 gene and less off-target DNA cleavage. Three PAM sites were selected within exon 1 of the porcine GGTA1 gene and ten sgRNA of variable lengths were designed across these three sites. The sgRNA was tested against synthetic double stranded DNA templates replicating both the native GGTA1 DNA template and the two most likely off-target binding sites in the porcine genome. Cleavage ability for native and off-target DNA was determined by in vitro cleavage assays. Resulting cleavage products were analyzed to determine the cleavage efficiency of the Cas9/sgRNA complex. Extension of sgRNA length did not have a statistical impact on the specificity of the Cas9/sgRNA complex for PAM1 and PAM2 sites. At the PAM3 site, however, an observed increase in specificity for native versus off-target templates was seen with increased sgRNA length. In addition, distance between PAM site and the start codon had a significant impact on cleavage efficiency and target specificity, regardless of sgRNA length. Although the in vitro assays showed off-target cleavage, Sanger sequencing revealed that no off-target mutations were found in GGTA1 knockout cell lines or piglet. These results demonstrate an optimized method for improvement of the CRIPSR/Cas9 gene editing system by reducing the likelihood of damaging off-target mutations in GGTA1 knocked out cells destined for xenotransplant donor production.

Klíčová slova:

DNA – DNA cleavage – Genetic engineering – Mutation – Polymerase chain reaction – Sequence alignment – Sequence motif analysis – Swine


Zdroje

1. Naeimi Kararoudi M, Hejazi SS, Elmas E, Hellström M, Naeimi Kararoudi M, Padma AM, et al. Clustered regularly interspaced short palindromic repeats/Cas9 gene editing technique in xenotransplantation. Front Immunol [Internet]. 2018;9(1711):1–7. Available from: https://www.frontiersin.org/article/10.3389/fimmu.2018.01711/full

2. Sato M, Miyoshi K, Nagao Y, Nishi Y, Ohtsuka M, Nakamura S, et al. The combinational use of CRISPR/Cas9-based gene editing and targeted toxin technology enables efficient biallelic knockout of the α-1,3- galactosyltransferase gene in porcine embryonic fibroblasts. Xenotransplantation [Internet]. 2014;21(3):291–300. Available from: doi: 10.1111/xen.12089 24919525

3. Burlak C, Paris LL, Lutz AJ, Sidner RA, Estrada J, Li P, et al. Reduced binding of human antibodies to cells from GGTA1/CMAH knockout pigs. Am J Transpl [Internet]. 2014;14(8):1895–900. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4366649/

4. Estrada J, Martens G, Li P, Adams A, Newell K, Ford M, et al. Evaluation of human and nonhuman primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes. Xenotransplantation [Internet]. 2015;22(3):194–202. Available from: doi: 10.1111/xen.12161 25728481

5. Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed genome editing in human cells. Elife [Internet]. 2013;2:1–9. Available from: https://doi.org/10.7554/eLife.00471

6. Jiang F, Doudna JA. The structural biology of CRISPR-Cas systems. Curr Opin Struct Biol [Internet]. 2015;30:100–11. Available from: doi: 10.1016/j.sbi.2015.02.002 25723899

7. Jiang F, Doudna JA. CRISPR–Cas9 structures and mechanisms. Annu Rev Biophys [Internet]. 2017;46:505–29. Available from: doi: 10.1146/annurev-biophys-062215-010822 28375731

8. Gaj T. ZFN, TALEN and CRISPR/Cas based methods for genome engineering. Trends Biotechnol [Internet]. 2014;31(7):397–405. Available from: https://www.ncbi.nlm.nih.gov/pubmed/23664777

9. Peng R, Lin G, Li J. Potential pitfalls of CRISPR/Cas9-mediated genome editing. FEBS J [Internet]. 2016;283(7):1218–31. Available from: doi: 10.1111/febs.13586 26535798

10. Fu Y, Foden J a, a C, Maeder ML, Reyon D, Joung K, et al. High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol [Internet]. 2013;31(9):822–6. Available from: doi: 10.1038/nbt.2623 23792628

11. Yang H, Wu Z. Genome Editing of Pigs for Agriculture and Biomedicine. Front Genet [Internet]. 2018;9(September):360. Available from: https://www.frontiersin.org/article/10.3389/fgene.2018.00360/full

12. Sander JD, Zaback P, Joung JK, Voytas DF. Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic Acids Res [Internet]. 2007;35:599–605. Available from: https://www.ncbi.nlm.nih.gov/pubmed/20435679

13. Sander JD, Maeder ML, Reyon D, Voytas DF, Joung JK, Dobbs D. ZiFiT (zinc finger targeter): an updated zinc finger engineering tool. Nucleic Acids Res [Internet]. 2010;38:462–8. Available from: https://www.ncbi.nlm.nih.gov/pubmed/20435679

14. Takara. Guide-it TM sgRNA in vitro transcription and screening systems user manual [Internet]. 2017. Available from: takarabio.com

15. Bae S, Park J, Kim J-S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 2014;30(10):1473–5. doi: 10.1093/bioinformatics/btu048 24463181

16. York IA, Brehm MA, Zendzian S, Towne CF, Rock KL. Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims MHC class I-presented peptides in vivo and plays an important role in immunodominance. Proc Natl Acad Sci. 2006;103(24):9202–7. doi: 10.1073/pnas.0603095103 16754858

17. Han Q, Cai T, Tagle DA, Robinson H, Li J. Substrate specificity and structure of human aminoapidate aminotransferase/kynurenine aminotransferase II. Biosci Rep [Internet]. 2008;28(4):205–15. Available from: https://www.ncbi.nlm.nih.gov/pubmed/18620547 doi: 10.1042/BSR20080085 18620547

18. Barić I, Fumic K, Glenn B, Schulze A, Finkelstein JD, James SJ, et al. S-adenosylhomocysteine hydrolase deficiency in a human: A genetic disorder of methionine metabolism. Proc Natl Acad Sci [Internet]. 2004;101(12):4234–9. Available from: https://www.pnas.org/content/101/12/4234 doi: 10.1073/pnas.0400658101 15024124

19. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK, Unit P, et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol [Internet]. 2014;32(3):279–84. Available from: https://www.ncbi.nlm.nih.gov/pubmed/24463574 doi: 10.1038/nbt.2808 24463574

20. Ran FA, Hsu PD, Lin C, Gootenberg JS, Trevino A, Scott DA, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell [Internet]. 2014;154(6):1380–9. Available from: https://www.cell.com/abstract/S0092-8674(13)01015-5

21. Zhang JP, Li XL, Neises A, Chen W, Hu LP, Ji GZ, et al. Different effects of sgRNA length on CRISPR-mediated gene knockout efficiency. Sci Rep [Internet]. 2016;6:1–10. Available from: doi: 10.1038/s41598-016-0001-8

22. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol [Internet]. 2013;31(9):827–32. Available from: doi: 10.1038/nbt.2647 23873081

23. Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res [Internet]. 2014;24:132–41. Available from: doi: 10.1101/gr.162339.113 24253446

24. Dang Y, Jia G, Choi J, Ma H, Anaya E, Ye C, et al. Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency. Genome Biol [Internet]. 2015;16(280):1–10. Available from: http://dx.doi.org/10.1186/s13059-015-0846-3


Článek vyšel v časopise

PLOS One


2019 Číslo 12