sgRNA design and plasmid construction
Cas9/sgRNA expression vectors and donor plasmids used in this study are
listed in the Supplementary Table S2. Target site-specific sgRNA
sequences were designed by the CRISPOR bioinformatics tool (version
4.95) (Concordet and Haeussler, 2018). Duplex of single stranded oligos
were cloned into Cas9_T2A_mCherry and sgRNA expression vectors
(Addgene #64324) (Chu et al., 2015). Donor plasmids were constructed
via the uracil-specific excision reagent (USER) cloning method (Lee et
al., 2015b) with uracil-containing primers listed in Supplementary Table
S3. The primers used for DCDs contain sgRNA/PAM sequences for the
backbone fragment. The PCR products were purified from 1% agarose TAE
gel using NucleoSpin® Gel and PCR Cleanup kit (Macherey-Nagel, Duren,
Germany). Four PCR amplicons including homology arms (5’HA and 3’HA),
backbone (CCD or DCD), and promoter (short variant of CMV or human EF-1α
promoter or Chinese hamster EF-1α promoter) were assembled with USER
enzyme (New England Biolabs, Ipswich, MA); 3’ homology arm lengths were
optimized for the truncation of EGFP (~0.8kb) and
TagRFP657 (~0.65kb) expression in donor plasmid. All
templates for the PCR reactions are listed in Supplementary Table S4.
The reaction mixtures were then transformed into E.coli One Shot®
Mach1TM competent cells (Life technologies, Thermo
Scientific, Rockford, IL) according to standard procedures. Plasmids
were harvested from a single colony on LB agar plates containing
ampicillin. All constructs were purified using NucleoBond Xtra Midi EF
(Macherey-Nagel) according to the manufacturer’s instructions, and
validated by sanger sequencing by primers listed in Supplementary Table
S3.