Abstract

We have successfully used comparative genomics to identify putative regulatory elements within the human genome that contribute to the tissue specific expression of neuropeptides such as galanin and receptors such as CB1. However, a previous inability to rapidly delete these elements from the mouse genome has prevented optimal assessment of their function in-vivo. This has been solved using CAS9/CRISPR genome editing technology which uses a bacterial endonuclease called CAS9 that, in combination with specifically designed guide RNA (gRNA) molecules, cuts specific regions of the mouse genome. However, reports of “off target” effects, whereby the CAS9 endonuclease is able to cut sites other than those targeted, limits the appeal of this technology. We used cytoplasmic microinjection of gRNA and CAS9 mRNA into 1-cell mouse embryos to rapidly generate enhancer knockout mouse lines. The current study describes our analysis of the genomes of these enhancer knockout lines to detect possible off-target effects. Bioinformatic analysis was used to identify the most likely putative off-target sites and to design PCR primers that would amplify these sequences from genomic DNA of founder enhancer deletion mouse lines. Amplified DNA was then sequenced and blasted against the mouse genome sequence to detect off-target effects. Using this approach we were unable to detect any evidence of off-target effects in the genomes of three founder lines using any of the four gRNAs used in the analysis. This study suggests that the problem of off-target effects in transgenic mice have been exaggerated and that CAS9/CRISPR represents a highly effective and accurate method of deleting putative neuropeptide gene enhancer sequences from the mouse genome.

Highlights

  • Neuropeptides and their receptors play a crucial role in maintaining the homeostasis of the human nervous system and in maintaining health (Freimann et al, 2015; Davidson et al, 2011)

  • N90% survival was obtained after cytoplasmic injection of our guide RNA (gRNA)/ CAS9mRNA mixture and surviving two-cell embryos were transferred into host female mice

  • We analysed the effects of CAS9 on the four most likely off-target cut sites of four different gRNAs in three different founder mouse lines to test the hypothesis that, if offtarget cutting by CAS9/CRISPR was to be an issue in our analyses, that one or more of these predicted off-target sites would show evidence of deletions, insertions or base pair changes characteristic of targeted double strand cuts and subsequent non-homologous end joining repair mechanisms

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Summary

Introduction

Neuropeptides and their receptors play a crucial role in maintaining the homeostasis of the human nervous system and in maintaining health (Freimann et al, 2015; Davidson et al, 2011) Neuropeptides such as galanin play critical roles in inflammation (Pinter et al, 2013), mood, appetite and alcohol intake and their mis-expression has been associated with inflammatory pain, depression, alcohol abuse and obesity (Lang et al, 2015). We were able to identify a 1.5 kilobase (kb) region 42 kb upstream from the human GAL locus that we called GAL5.1 We cloned this sequence and made reporter constructs which were used to generate reporter mouse lines, using pronuclear microinjection, that expressed the β-galactosidase reporter gene in precisely the same cells that expressed GAL mRNA and galanin peptide (Davidson et al, 2011). The expense and time required to delete GAL5.1 from the mouse genome using existing embryonic stem cell targeting based technologies precluded this approach

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