CRISPR

Not applicable

This “safe harbor landing pad” allele on the A/J background provides a tool for precise, site-specific insertion of single-copy sequence into the Gt(ROSA)26Sor intron 1 in a manner that is in the desired orientation and free from vector sequence. In practice, a plasmid containing a single cognate 38 bp attB-GT site and the donor sequence is first converted into a vector-less DNA minicircle donor. The DNA minicircle is delivered along with the Bxb1 integrase (mRNA or protein) into the pronucleus of fertilized oocytes containing the 48 bp attP-GT landing pad attachment site. The result is a unidirectional, single-copy integration of the sequence into the Gt(ROSA)26Sor locus. The inserted sequence will be flanked by the recombined attachments sites, now called Bxb1 attL and Bxb1 attR, but will be: (1) free from residual vector sequence from the plasmid backbone, (2) present as a single-copy (no concatemers), and (3) inserted precisely into a known and verifiable locus (Gt(ROSA)26Sor). [Low et al., 2022] The donating investigator indicates that 5% of integrations result in aberrant integration events. In addition, a recombinase mediated knock-in (RMKI) strategy is successful with inserts up to approximately 15 kb.

The RosaBxb-GT allele was generated in the laboratory of Dr. Michael V. Wiles (The Jackson Laboratory) using CRISPR/Cas9 endonuclease-mediated genome editing/oligo-mediated homology directed repair. An 18-mer TRU-guide RNA was selected to target near the XbaI site of intron 1 in the Gt(ROSA)26Sor locus on Chromosome 6. A 200 nt ssDNA donor (with 76 nt homology arms flanking the 48 bp Bxb1 attP-GT attachment site), the TRU-guide and Cas9 protein were introduced into A/J zygotes by electroporation. Progeny were screened by PCR amplification and DNA sequencing to identify correctly targeted pups. Founder mice were then back-crossed at least once to A/J mice for germline transmission and allele verification before intercrossing to generate homozygotes.

A/J

The MMRRC Centers have developed a genetic QC pipeline using MiniMUGA array genotyping to provide additional information on strain backgrounds for MMRRC congenic and inbred strains. For more information on when data may be available, or to request genotyping for a strain of interest, please contact csmmrrc@jax.org. Older strains may not have this information.

Aamir Zuberi, Ph.D., The Jackson Laboratory.
Michael Wiles, The Jackson Laboratory.