Rosetta2 de3

Streptococcus pyogenes Cas9 (pMJ915, Addgene #69090) with two nuclear localization signal peptides and an HA tag at the C-terminus was expressed in Rosetta2 DE3 (UC Berkeley Marcolab) cells. Cell pellets were sonicated, clarified, Ni2+ -affinity purified (HisTraps, GE life sciences), TEV cleaved, cation-exhanged (HiTrap SP HP, GE life sciences), size excluded (Sephacryl S-200, GE life sciences) and eluted at 40 in 20 mM HEPES KOH pH 7.5, 5% glycerol, 150 mM KCl, 1 mM dithiothreitol (DTT)^{55 (link)}. Alternatively, Streptococcus pyogenes Cas9-NLS was obtained from the QB3 MacroLab at UC Berkeley.
sgRNAs were synthesized by Synthego as modified gRNAs with 2′-O-methyl analogs and 3′ phosphorothioate internucleotide linkages at the first three 5′ and 3′ terminal RNA residues using protospacer sequences described in (Supplementary Data 3).
crRNAs/tracrRNAs were chemically synthesized (Edit-R, Dharmacon Horizon) using protospacer sequences described in (Supplementary Data 3).
ssDonors were obtained by ordering unmodified Ultramer oligonucleotides (Integrated DNA Technologies).
dsDonor was obtained by purifying plasmid DNA from bacterial cultures containing the indicated plasmid (Qiagen) or by SPRI purification of long double-stranded PCR amplicons.

Escherichia coli strain Rosetta 2 DE3 carrying plasmid pMJ806 was obtained from Addgene. The Cas9 protein was prepared following the protocol as described (Anders and Jinek, 2014). For in vitro transcription of guide RNA, pT7‐sgRNA derived gRNA‐specific construct was linearized at a unique restriction enzyme site NcoI, followed by dephosphorylation with Shrimp Alkaline Phosphatase (rSAP) (New England Biolabs, Ipswich, MA), and then purified with DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA). sgRNA was synthesized by using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs) following the manufacturer's instructions and purified with RNA Clean and Concentrator (Zymo Research, Irvine, CA). For each DNA/ribonucleoprotein reaction, 1 μg of sgRNA and 1 μg of Cas9 protein were mixed in Cas9 reaction buffer and incubated at room temperature for 15 min to form a ribonucleoprotein (RNP) complex. The resulting RNP was used for 2–3 reactions each with about 1 μg of PCR product in a total volume of 10 μL at 37 °C for 3 h. After inactivated at 65 °C for 15 min, the reactions were analysed with electrophoresis in 2% agarose gel.