Humanising the mouse genome piece by piece

Abstract

To better understand human health and disease, researchers create a wide variety of mouse models that carry human DNA. With recent advances in genome engineering, the targeted replacement of mouse genomic regions with orthologous human sequences has become increasingly viable, ranging from finely tuned humanisation of individual nucleotides and amino acids to the incorporation of many megabases of human DNA. Here, we examine emerging technologies for targeted genomic humanisation, we review the spectrum of existing genomically humanised mouse models and the insights such models have provided, and consider the lessons learned for designing such models in the future.

Fig. 1

Targeted genomic humanisation technologies. a HR in ES cells has been used to humanise loci up to ~200 kb (and beyond, using iterative targeting). A plasmid, or BAC, targeting vector carrying human sequence flanked by homology arms is transfected into ES cells by electroporation. Addition of Cas9:sgRNA, generating a targeted double strand break, increases HR efficiency. An antibiotic resistance selectable marker is included to enrich for ES cells harbouring the desired recombination. Selection cassettes are commonly flanked by frt sites for later excision by FLP recombinase, leaving a single frt genomic scar. b Recombinase-mediated cassette exchange (RMCE) can be used to humanise up to ~200 kb loci (can also be employed iteratively). In this example, a landing pad is first inserted at the target locus via HR (see part a), consisting of a selection cassette flanked by heterotypic lox sites. The same lox sites are inserted either side of the orthologous human locus within a BAC vector, which when electroporated into landing pad-harbouring ES cells will recombine in the presence of CRE recombinase. Cas9:sgRNA pairs can subsequently be utilised to delete the mouse locus. As an alternative to FLP/frt recombination, selection cassettes and other exogenous sequences can be flanked by PiggyBac inverted terminal repeats (ITR), which when inserted at an AATT recognition site, leave no genomic scar once excised with PiggyBac transposase. PiggyBAC transposition is less efficient than FLP/frt recombination, thus positive–negative selection cassettes (+/− s) such as HPRT (in HPRT−/− ES) or puroΔTK are used. c Introducing pathogenic mutations into humanised alleles can be achieved by HR in zygotes using a ssODN (~150 bp) donor template combined with a locus-specific Cas9:sgRNA (no selection required). A similar strategy can be used for small-scale humanisation projects (small genes or partial humanisation) using a long ssODN (<2 kb) as a donor template and a pair of Cas9:sgRNAs. d Knock-in of large inserts (up to 200 kb) in both mouse and rat zygotes has been achieved by combining Cas9:sgRNAs and short ssODN donors with hybrid homology at the break-points between donor and target site to facilitate HR

Fig. 2

How far to humanise. A summary of considerations when deciding on the extent of targeted genomic humanisation for a given gene of interest. Dark green boxes represent exons, lines between exons are introns, light green boxes are UTRs and blue regions represent humanisation. a Partial humanisation typically involves humanising specific residues and/or domains of interest. Fine-scale humanisation of specific amino-acid residues can be performed in isolation if they have known biochemical differences from mouse to human, or if only a small number of residues need to be altered to achieve a human protein sequence. Specific domains or exons can be humanised if, for example, they are known to be critical for human disease. Examples of translated protein products are given. b Full humanisation involves humanising the whole gene, including introns, to attain translation of the full human protein, potentially including human-specific splicing patterns, and for maximum translational potential. 5ʹ and 3ʹ-UTRs, promoters, and other regulatory sequences can be included, on a case-by-case basis, if understanding of gene regulation is the question at hand, if gene clusters are to be humanised or if pathogenic mutations fall within such flanking regions