5 alpha chemically competent e coli high efficiency

Donor plasmids were created to introduce SRY into the ZFX locus on the X-chromosome 10 kb downstream of the ZFX gene. The donor plasmids were commercially synthesized (GeneWiz) to contain the endogenous Bos taurus SRY promoter and coding sequence (Accession U15569)^{41 (link)}. 1 kb homology arms were commercially synthesized (GeneWiz) containing regions flanking the cut site and inserted into the donor plasmids using Gibson Assembly Master Mix (NEB), with (hmejSRYp) and without (hrSRYp) the endogenous CRISPR target site flanking the homology arms (Fig. 2). Plasmids were clonally amplified using 5-alpha Chemically Competent E. coli (High Efficiency) (NEB) and extracted using the EndoFree Plasmid Maxi Kit (Qiagen).

The guide-RNA (gRNA) targeting the H11 safe harbor locus on bovine chromosome 17 was designed as previously described [15 (link)] (TAGCCATAAGACTACCTAT) and commercially synthesized (Synthego, Redwood City, CA, USA). The donor plasmid construct was designed as previously described [9 ], containing the endogenous bovine sex-determining region Y, (SRY) promoter and coding sequence [14 (link)], the green fluorescent protein (GFP) coding sequence and SV40 promoter, and 1 kb homology arms flanked on either side by the CRISPR target site (Fig. 1a). Each piece was commercially synthesized (GeneWiz, LLC, South Plainfield, NJ, USA) and inserted into a pUC19 plasmid using Gibson Assembly Master Mix (New England Biolabs, Inc., Ipswich, MA). Plasmids were clonally amplified using 5-alpha Chemically Competent E. coli (High Efficiency) (New England Biolabs, Inc., Ipswich, MA) and extracted using the EndoFree Plasmid Maxi Kit (Qiagen, Inc., Valencia, CA).