One 35mm-dish of mES cells of approximately 80% confluence were transfected using Amaxa Nucleofector machine (Lonza, USA), program A-30, 0.2cm cuvette and Nucleofector Kit (VPH-1001). For testing promoter activity in 129×B6 F1 ES cells, hybrid CBA (CBh) promoter from pX330 and cytomegalovirus (CMV) early enhancer/chicken ß actin (CAG) promoter from pCAGGS were compared to drive expression of the enhanced green fluorescent protein (EGFP). For optimization, different amount of supercoiled pCBh-EGFP-N1 plasmid were tested for nucleofection. For Dip2a?65kb DONOR free electroporation, 1.5µg of each supercoiled pX330-sgRNA were used and 300 cells per 100mm-dish were seeded and maintained for 72 hours for clone picking. For electroporation with Dip2a?65kb DONOR, 1µg of each supercoiled pX330-sgRNA and 2µg of supercoiled DONOR DNA were used. 24hs later, cells were selected with 200µg/ml G418 for 5 days. Single isolated colonies were picked and grown in duplicates on 96 well plates for screening by PCR. For Dip2a lacZ knockin Nucleofection, 3µg supercoiled DONOR plasmid and 3µg supercoiled pX330-sgRNA plasmid per 35mm-dish were used.
ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. However, the most mutations reported up to date are small insertions and deletions. In this report, CRISPR/Cas9 system was used to make large DNA fragment deletions and insertions, including entire Dip2a gene deletion, about 65kb in size, and ß-galactosidase (lacZ) reporter gene insertion of larger than 5kb in mouse. About 11.8% (11/93) are positive for 65kb deletion from transfected and diluted ES clones. High targeting efficiencies in ES cells were also achieved with G418 selection, 46.2% (12/26) and 73.1% (19/26) for left and right arms respectively. Targeted large fragment deletion efficiency is about 21.4% of live pups or 6.0% of injected embryos. Targeted insertion of lacZ reporter with NEO cassette showed 27.1% (13/48) of targeting rate by ES cell transfection and 11.1% (2/18) by direct zygote injection. The procedures have bypassed in vitro transcription by directly co-injection of zygotes or co-transfection of embryonic stem cells with circular plasmid DNA. The methods are technically easy, time saving, and cost effective in generating mouse models and will certainly facilitate gene function studies. Lonza Summary: In this study, the authors show that CRISPR-Cas9 is a simple system and can be used to manipulate large genome fragments with high efficiency. The result indicates that a deletion of a 65kb DNA fragment or insertion of a 5.3kb DNA fragment by homologous recombination are highly efficient both by ES cell transfection and by direct injection of zygotes. Furthermore, CRISPR/Cas9 can mediate efficient homologous recombination (HR) of large DNA fragment in ES cells, much more efficient than traditional gene targeting.