Hindriksen Sanne, Bramer Arne J, Truong My Anh, Vromans Martijn J M, Post Jasmin B, Verlaan-Klink Ingrid, Snippert Hugo J, Lens Susanne M A, Hadders Michael A
Center for Molecular Medicine, Section Molecular Cancer Research, University Medical Center Utrecht, Universiteitsweg 100, CG, Utrecht, The Netherlands.
PLoS One. 2017 Jun 22;12(6):e0179514. doi: 10.1371/journal.pone.0179514. eCollection 2017.
The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B.
CRISPR/Cas9系统是一种用于基因组编辑的高效工具。强大的基因组编辑的关键在于CRISPR/Cas9机制的有效递送。病毒递送系统是转导外源基因的有效载体，但常用的病毒载体携带的遗传信息容量有限。然而，杆状病毒能够携带大片段外源DNA。在此，我们研究了杆状病毒载体作为基于CRISPR/Cas9的基因组编辑工具的递送载体的用途。我们展示了用Cas9和一个sgRNA序列转导一组细胞系，这导致染色体乘客复合体（CPC）的所有四个靶向亚基的有效敲除。我们进一步表明，将同源定向修复模板引入同一CRISPR/Cas9杆状病毒中有助于引入特定的点突变和内源性基因标签。用荧光报告基因YFP标记CPC募集因子Haspin，使我们能够研究其天然定位以及向黏连蛋白亚基Pds5B的募集。
