Abstract
The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating small insertion/deletion (indel) mutations, or by homology-directed repair (HDR). If ectopic donor templates are provided, the latter mechanism allows editing with single-nucleotide precision. The preference of mammalian cells to repair DSBs by NHEJ rather than HDR, however, limits the potential of CRISPR-Cas9 for applications where precise editing is needed. To enhance the efficiency of DSB repair by HDR from donor templates, we recently engineered a CRISPR-Cas9 system where the template DNA is bound to the Cas9 enzyme. In short, single-stranded oligonucleotides were labeled with O6-benzylguanine (BG), and covalently linked to a Cas9-SNAP-tag fusion protein to form a ribonucleoprotein-DNA (RNPD) complex consisting of the Cas9 nuclease, the sgRNA, and the repair template. Here, we provide a detailed protocol how to generate O6-benzylguanine (BG)-linked DNA repair templates, produce recombinant Cas9-SNAP-tag fusion proteins, in vitro transcribe single guide RNAs, and transfect RNPDs into various mammalian cells.
Highlights
[Background] The CRISPR-Cas[9] system efficiently induces site-directed double-stranded break (DSB), which are repaired by cell-autonomous mechanisms
Several attempts have been made to increase the efficiency of accurate DSB repair, including biochemical alteration the repair pathways (Chu et al, 2015; Maruyama et al, 2015; Yu et al, 2015; Song et al, 2016), limiting DSB-induction to the S/G2 phase of the cell cycle where homology-directed repair (HDR) is active (Lin et al, 2014; Gutschner et al, 2016; Howden et al, 2016), and optimizing length and symmetry of the repair template (Hendel et al, 2015; Rahdar et al, 2015; Yin et al, 2017; Richardson et al, 2016; Liang et al, 2017)
Recently demonstrated that temporal and spatial co-localization of the repair template to the Cas[9] complex enhances HDR rates (Carlson-Stevermer et al, 2017; Aird et al, 2018; Gu et al, 2018; Savić et al, 2018). These approaches do not involve any potentially harmful chemical treatment to alter endogenous cellular processes, and due to the shorter half-life of ribonucleoprotein complexes compared to DNA, they exhibit reduced risks of generating off-target mutations (Kim et al, 2014)
Summary
Phosphate buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023) 11. Fetal bovine serum (FBS) (Thermo Fisher Scientific, Life Technologies, catalog number: 10270106) for mESC culture 14. 2-β-mercaptoethanol (Thermo Fisher Scientific, Life Technologies, catalog number: 31350-010) 19. 3 M sodium acetate buffer solution (NaAc) (Sigma-Aldrich, catalog number: S7899-100ML) 27. NTP Set, 100 mM solution (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0481) 41. SYBRTM Gold nucleic acid gel stain, 10,000x Concentrate in DMSO (Thermo Fisher Scientific, InvitrogenTM, catalog number: S11494) 49. Tissue culture incubator set at 37 °C, 5% CO2 (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i, catalog number: 51026283) 9. Countess automated cell counter (Thermo Fisher Scientific, InvitrogenTM, catalog number: C10227) 15. Countess® cell counting chamber slides (Thermo Fisher Scientific, InvitrogenTM, catalog number: C10228) 16.
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