Homology arms of targeting vectors for gene insertions and CRISPR/Cas9 technology: size does not matter; quality control of targeted clones does
-
Silvia Petrezselyova
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology has brought rapid progress in mammalian genome editing (adding, disrupting or changing the sequence of specific sites) by increasing the frequency of targeted events. However, gene knock-in of DNA cassettes by homologous recombination still remains difficult due to the construction of targeting vectors possessing large homology arms (from 2 up to 5 kb). Here, we demonstrate that in mouse embryonic stem cells the combination of CRISPR/Cas9 technology and targeting vectors with short homology arms (~ 0.3 kb) provides sufficient specificity for insertion of fluorescent reporter cassettes into endogenous genes with similar efficiency as those with large conventional vectors. Importantly, we emphasize the necessity of thorough quality control of recombinant clones by combination of the PCR method, Southern hybridization assay and sequencing to exclude undesired mutations. In conclusion, our approach facilitates programmed integration of exogenous DNA sequences at a target locus and thus could serve as a basis for more sophisticated genome engineering approaches, such as generation of reporters and conditional knock-out alleles.
References
1. Capecchi, M.R. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat. Rev. Genet. 6 (2005) 507-512.Search in Google Scholar
2. Gaj, T., Gersbach, C.A. and Barbas C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31 (2013) 397-405.Search in Google Scholar
3. Hasty, P., Rivera-Perez, J. and Bradley A. The length of homology required for gene targeting in embryonic stem cells. Mol. Cell Biol. 11 (1991) 5586-5591.Search in Google Scholar
4. Deng, C. and Capecchi, M.R. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol. Cell Biol. 12 (1992) 3365-3371.Search in Google Scholar
5. Shulman, M.J., Nissen, L. and Collins C. Homologous recombination in hybridoma cells: dependence on time and fragment length. Mol. Cell Biol. 10 (1990) 4466-4472.Search in Google Scholar
6. Ran, F.A., Hsu, P.D., Lin, C.Y., Gootenberg, J.S., Konermann, S., Trevino, A.E., Scott, D.A., Inoue, A., Matoba, S., Zhang, Y. and Zhang, F. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154 (2013) 1380-1309.Search in Google Scholar
7. Copeland, N.G., Jenkins, N.A. and Court, D.L. Recombineering: a powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2 (2001) 769-779.Search in Google Scholar
8. Engert, S., Liao, W.P., Burtscher, I. and Lickert, H. Sox17-2A-iCre: a knock-in mouse line expressing Cre recombinase in endoderm and vascular endothelial cells. Genesis 47 (2009) 603-610.Search in Google Scholar
9. Hsu, P.D., Scott, D.A., Weinstein, J.A., Ran, F.A., Konermann, S., Agarwala, V., Li, Y., Fine, E.J., Wu, X., Shalem, O., Cradick, T.J., Marraffini, L.A., Bao, G. and Zhang, F. DNA targeting specificity of RNAguided Cas9 nucleases. Nat. Biotechnol. 31 (2013) 827-832.Search in Google Scholar
10. Uetzmann, L., Burtscher, I. and Lickert, H. A mouse line expressing Foxa2- driven Cre recombinase in node, notochord, floorplate, and endoderm. Genesis 10 (2008) 515-522.10.1002/dvg.20410Search in Google Scholar PubMed
11. Hitz, C., Wurst, W. and Kuhn, R. Conditional brain-specific knockdown of MAPK using Cre/loxP regulated RNA interference. Nucleic Acids Res. 35 (2007) e90.10.1093/nar/gkm475Search in Google Scholar PubMed PubMed Central
12. Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E. and Tsien, R.Y. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22 (2004) 1567-1572.Search in Google Scholar
13. Li, K., Wang, G., Andersen, T., Zhou, P. and Pu, W.T. Optimization of genome engineering approaches with the CRISPR/Cas9 system. PLoS One 9 (2014) e105779.10.1371/journal.pone.0105779Search in Google Scholar PubMed PubMed Central
14. Hisano, Y., Sakuma, T., Nakade, S., Ohga, R., Ota, S., Okamoto, H., Yamamoto, T. and Kawahara, A. Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish. Sci. Rep. 5 (2015) 8841.Search in Google Scholar
15. Paix, A., Wang, Y., Smith, H.E., Lee, C.Y., Calidas, D., Lu, T., Smith, J., Schmidt, H., Krause, M.W. and Seydoux, G. Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics 198 (2014) 1347-1356. Search in Google Scholar
© University of Wroclaw, Poland
Articles in the same Issue
- How taste works: cells, receptors and gustatory perception
- Non-cooperative immobilization of residual water bound in lyophilized photosynthetic lamellae
- Coexistence of rare variant HbD Punjab [α2β2121(Glu→Gln)] and alpha 3.7 kb deletion in a young boy of Hindu family in West Bengal, India
- Somatic stem cell aging and malignant transformation – impact on therapeutic application
- 1H NMR-based metabolic profiling for evaluating poppy seed rancidity and brewing
- Homology arms of targeting vectors for gene insertions and CRISPR/Cas9 technology: size does not matter; quality control of targeted clones does
- Death domain associated protein (Daxx), a multi-functional protein
- In silico screening of alleged miRNAs associated with cell competition: an emerging cellular event in cancer
- MGL induces nuclear translocation of EndoG and AIF in caspase-independent T cell death
- Cerivastatin represses atherogenic gene expression through the induction of KLF2 via isoprenoid metabolic pathways
- Proteasomes raise the microtubule dynamics in influenza A (H1N1) virus-infected LLC-MK2 cells
- Purinergic signaling and the functioning of the nervous system cells
- The effect of cultureware surfaces on functional and structural components of differentiated 3T3-L1 preadipocytes
- Ramipril inhibits high glucose-stimulated up-regulation of adhesion molecules via the ERK1/2 MAPK signaling pathway in human umbilical vein endothelial cells
- The effect of nicotine on the expressions of the α7 nicotinic receptor gene and Bax and Bcl-2 proteins in the mammary gland epithelial-7 breast cancer cell line and its relationship to drug resistance
- The cloning, expression and purification of recombinant human neuritin from Escherichia coli and the partial analysis of its neurobiological activity
Articles in the same Issue
- How taste works: cells, receptors and gustatory perception
- Non-cooperative immobilization of residual water bound in lyophilized photosynthetic lamellae
- Coexistence of rare variant HbD Punjab [α2β2121(Glu→Gln)] and alpha 3.7 kb deletion in a young boy of Hindu family in West Bengal, India
- Somatic stem cell aging and malignant transformation – impact on therapeutic application
- 1H NMR-based metabolic profiling for evaluating poppy seed rancidity and brewing
- Homology arms of targeting vectors for gene insertions and CRISPR/Cas9 technology: size does not matter; quality control of targeted clones does
- Death domain associated protein (Daxx), a multi-functional protein
- In silico screening of alleged miRNAs associated with cell competition: an emerging cellular event in cancer
- MGL induces nuclear translocation of EndoG and AIF in caspase-independent T cell death
- Cerivastatin represses atherogenic gene expression through the induction of KLF2 via isoprenoid metabolic pathways
- Proteasomes raise the microtubule dynamics in influenza A (H1N1) virus-infected LLC-MK2 cells
- Purinergic signaling and the functioning of the nervous system cells
- The effect of cultureware surfaces on functional and structural components of differentiated 3T3-L1 preadipocytes
- Ramipril inhibits high glucose-stimulated up-regulation of adhesion molecules via the ERK1/2 MAPK signaling pathway in human umbilical vein endothelial cells
- The effect of nicotine on the expressions of the α7 nicotinic receptor gene and Bax and Bcl-2 proteins in the mammary gland epithelial-7 breast cancer cell line and its relationship to drug resistance
- The cloning, expression and purification of recombinant human neuritin from Escherichia coli and the partial analysis of its neurobiological activity
