Citation

  • Authors: Wang, Y., Liu, K. I., Sutrisnoh, N. B., Srinivasan, H., Zhang, J., Li, J., Zhang, F., Lalith, C. R. J., Xing, H., Shanmugam, R., Foo, J. N., Yeo, H. T., Ooi, K. H., Bleckwehl, T., Par, Y. Y. R., Lee, S. M., Ismail, N. N. B., Sanwari, N. A. B., Lee, S. T. V., Lew, J., Tan, M. H.
  • Year: 2018
  • Journal: Genome Biol 19 62
  • Applications: in vitro / DNA, oligonucleotide and DNA cotransfection / jetPRIME
  • Cell type: HEK-293T
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293T, 293T

Method

For NHEJ-mediated editing experiments, HEK-293T cells were transfected with 500 ng CRISPR plasmids in 12-well plates. For HDR-mediated editing experiments with ssODNs, HEK-293FT cells were co-transfected with 300 ng CRISPR plasmids and 300 ng ssODNs. For HDR-mediated editing experiments with donor plasmids, HEK293FT cells were co-transfected with 300 ng CRISPR plasmids and 300 ng donor templates. For CRISPR applications.

Abstract

BACKGROUND: While CRISPR-Cas systems hold tremendous potential for engineering the human genome, it is unclear how well each system performs against one another in both non-homologous end joining (NHEJ)-mediated and homology-directed repair (HDR)-mediated genome editing. RESULTS: We systematically compare five different CRISPR-Cas systems in human cells by targeting 90 sites in genes with varying expression levels. For a fair comparison, we select sites that are either perfectly matched or have overlapping seed regions for Cas9 and Cpf1. Besides observing a trade-off between cleavage efficiency and target specificity for these natural endonucleases, we find that the editing activities of the smaller Cas9 enzymes from Staphylococcus aureus (SaCas9) and Neisseria meningitidis (NmCas9) are less affected by gene expression than the other larger Cas proteins. Notably, the Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) are able to perform precise gene targeting efficiently across multiple genomic loci using single-stranded oligodeoxynucleotide (ssODN) donor templates with homology arms as short as 17 nucleotides. Strikingly, the two Cpf1 nucleases exhibit a preference for ssODNs of the non-target strand sequence, while the popular Cas9 enzyme from Streptococcus pyogenes (SpCas9) exhibits a preference for ssODNs of the target strand sequence instead. Additionally, we find that the HDR efficiencies of Cpf1 and SpCas9 can be further improved by using asymmetric donors with longer arms 5' of the desired DNA changes. CONCLUSIONS: Our work delineates design parameters for each CRISPR-Cas system and will serve as a useful reference for future genome engineering studies.

Pubmed