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In order to aid generation and analysis of CRISPR deletions in F1 cells, we have collected all SNPs between the 129 and Cast genomes based on the Sanger Institute Mouse Genomes Project and located them to their coordinates on the mm9 reference genome. Note that SNPs common to both genomes are not marked.


In our paper identifying the Sox2 Control Region, we studied the effect of distal regulatory elements on Sox2 transcription using genome editing. Although reporter gene constructs can provide useful guidance on which elements are active, the best way to examine regulatory element activity is in an endogenous context. One method of doing this is to delete the regulatory elements in the cell using CRISPR/Cas9-mediated deletion.

We used F1 cells of M. musculus129/M. castaneus to study the elements responsible for Sox2 regulation. Given that disruption of one allele of an enhancer could result in changes to transcription in cis, but not in trans, protein levels of the target gene will not be eliminated by monoallelic disruption. Consequently there is less chance of phenotypic changes due to disruption of a regulatory cascade, and we can monitor transcriptional changes in the altered allele without confounding effects.

CRISPR/Cas9-mediated deletion can occur on either allele. To distinguish between alleles in our F1 cells, we have collected all the SNPs between 129 and Cast from the Sanger Institute Mouse Genomes Project and located them to their coordinates on the mm9 reference genome.

These SNPs can be displayed at the UCSC Mouse Genome Browser by following this link. The first base in the 129CastTrack is the 129 SNP, and the second is the Cast SNP. For SNPs at which confidence in the SNP is low, the base is displayed in lower case.

Usage of our SNP track

We used CRISPR to target the Sox2 Control Region (SCR) for deletion (Zhou et al, 2014). Guide RNAs targeted the regions designated gRNA104 and gRNA112 in A below. The 129 or Cast alleles were specifically amplified by PCR using the primer pr3’112R1 and allele specific versions of the primer pr5’104F1. As shown for isolate #15 in B, these allele specific primer pairs are used to distinguish the deleted 129 allele from the undeleted Cast allele.

targeting

We designed allele specific primers using our SNP database to monitor transcription levels of both alleles of Sox2 in our deleted lines.

Quantitative RT-qPCR revealed that in clones lacking the 129 enhancer (ΔSCR129), transcription of the 129 allele of Sox2 was greatly reduced, whereas in clones lacking the Cast enhancer (ΔSCRCast), transcription of the Cast allele of Sox2 was virtually eliminated.

targeting