Modeling human disease in rodents by CRISPR/Cas9 genome editing

Abstract

Modeling human disease has proven to be a challenge for the scientific community. For years, generating an animal model was complicated and restricted to very few species. With the rise of CRISPR/Cas9, it is now possible to generate more or less any animal model. In this review, we will show how this technology is and will change our way to obtain relevant disease animal models and how it should impact human health.

Fig. 1

Humanization of whole genomic fragment. a Schematic representation of the BAC knock-in by 2-hit 2-oligo. The endogenous gene is knocked out by the insertion of a >100-kb BAC (Knock-In), which contains the human locus. Two sgRNAs targeting the human BAC and the genome to modify are co-injected with 2 ssODNs which have an homology for both the BAC and the genomic DNA (Yoshimi et al. 2016). b Schematic representation of the 3-hit 2-oligo method to replace a rat cluster genes (>58 kb), with the orthologous human gene (6.2 kb). Three gRNAs for upstream and downstream of the rat cluster and for the human gene containing plasmid cut the targeting sites, and two ssODNs ligate to each cut end (Yoshimi et al. 2016). c Schematic representation of a humanization in mouse ES cells. Two sgRNAs leading to double-strand break 5′ and 3′ of the region to humanize highly increased the number of recombined clones. Well-characterized ES cells clones can then be processed through germline

Fig. 2

CRISpr-MEdiated REarrangement mechanisms (CRISMERE). a–c Standard chromosomic recombination when Cas9 edits the genome in G1. After mitosis, two alleles distinct from the initial WT allele will be obtained. a Intra-chromosomal recombination between two DSBs on a single chromosome. b Trans-allelic recombination between two DSBs each on one of the two chromosome. c Trans-allelic recombination between three (or four DSBs) on the two chromosomes ending with head to head, tail to tail duplication. d–f Standard chromosomic recombination when Cas9 edits the genome in G2. d Schematic of the event that should take place in the eggs where Cas9 edits the genome in cis configuration in G2 leading to monosomic and trisomic daughter cells after mitosis. Trans-allelic recombination between two DSBs on the two chromatids in G2. e Trans-allelic recombination between three DSBs when two breaks are located on only one chromatid and the third break is located on another chromatid. f Trans-allelic recombination between four DSBs when two breaks are located on one chromatid and the two other are located on another chromatid. As much as nine possible alleles can be obtained