Abstract
Although CRISPR/Cas9 genome editing has provided numerous opportunities to interrogate the functional significance of any given genomic site, there is a paucity of data on the extent of molecular scars inflicted on the mouse genome. Here we interrogate the molecular consequences of CRISPR/Cas9-mediated deletions at 17 sites in four loci of the mouse genome. We sequence targeted sites in 632 founder mice and analyse 54 established lines. While the median deletion size using single sgRNAs is 9 bp, we also obtain large deletions of up to 600 bp. Furthermore, we show unreported asymmetric deletions and large insertions of middle repetitive sequences. Simultaneous targeting of distant loci results in the removal of the intervening sequences. Reliable deletion of juxtaposed sites is only achieved through two-step targeting. Our findings also demonstrate that an extended analysis of F1 genotypes is required to obtain conclusive information on the exact molecular consequences of targeting events.
Highlights
clustered regularly interspaced short palindromic repeat (CRISPR)/Cas[9] genome editing has provided numerous opportunities to interrogate the functional significance of any given genomic site, there is a paucity of data on the extent of molecular scars inflicted on the mouse genome
We investigated the molecular consequences on targeting sites with single single guide RNA (sgRNA) and identified prevalent asymmetric deletions, preferred sites at which deletion occurs and large deletions
We targeted one enhancer bound by STAT5 and NFIB, a transcription factor (TF) involved in epithelial cell differentiation[28,29,30] in the Csn (F) locus (Supplementary Fig. 1)
Summary
By directly injecting the Cas[9] machinery into mouse zygotes, we targeted five enhancers bound by the cytokine-sensing TF STAT5 in three genomic loci, Stat5a (A)[20], Socs[2] (B)[26] and Wap (C)[19] (Fig. 1). We targeted one enhancer bound by STAT5 and NFIB, a TF involved in epithelial cell differentiation[28,29,30] in the Csn (F) locus (Supplementary Fig. 1). These genomic sites were targeted individually or in combination. We targeted individual TF-binding sites with only one corresponding sgRNA (Type 1), targeting individual TF-binding sites with more than one sgRNA (Type 2) and more than one TF-
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