Speciation and adaptation research meets genome editing

Abstract

Understanding the genetic basis of reproductive isolation and adaptive traits in natural populations is one of the fundamental goals in evolutionary biology. Genome editing technologies based on CRISPR-Cas systems and site-specific recombinases have enabled us to modify a targeted genomic region as desired and thus to conduct functional analyses of target loci, genes and mutations even in non-conventional model organisms. Here, we review the technical properties of genome editing techniques by classifying them into the following applications: targeted gene knock-out for investigating causative gene functions, targeted gene knock-in of marker genes for visualizing expression patterns and protein functions, precise gene replacement for identifying causative alleles and mutations, and targeted chromosomal rearrangement for investigating the functional roles of chromosomal structural variations. We describe examples of their application to demonstrate functional analysis of naturally occurring genetic variations and discuss how these technologies can be applied to speciation and adaptation research. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.

Keywords: CRISPR-Cas; chromosomal rearrangement; genome editing; knock-in; knock-out; site-specific recombination.

Figure 1.

Targeted gene knock-out and gene knock-in using targetable nucleases. (a) A double strand break (DSB) at a target site is repaired by non-homologous end joining (NHEJ), which can lead to small insertions and deletions around the target sites. (b) Targeted integration of an exogenous DNA fragment can be achieved by several different approaches. A double-stranded DNA (dsDNA) donor with long (approx. 1 kb) or short (approx. 25 bp) homology arms is integrated around the DSB site through homologous recombination (HR) or microhomology-mediated end joining (MMEJ), respectively. A single-stranded DNA (ssDNA) donor is integrated via a DSB repair pathway known as single-stranded template repair (SSTR). A dsDNA donor without any homology arms can be integrated through NHEJ between the targeted genome DNA molecule and the exogenous donor DNA molecule. (c–e) Genome editing of the csf1 gene in an Indonesian medaka fish (Oryzias woworae) is shown as a representative example of genome-edited organisms [40]. (c) Targeted knock-out (KO) of the csf1 gene by deleting 11 bp of the coding sequence. A homozygous KO mutant (csf1^Δ11/Δ11) male (upper) lacked red pigmented cells that are present in the pectoral and tail fins of wild-type (WT; csf1^+/+) males (lower). (d,e) Images of a knock-in fish harbouring a green fluorescent protein (GFP) with a minimal promoter and a poly(A) signal inserted at the csf1 gene. GFP fluorescence in a male (upper) and a female fish (lower) are shown to visualize sexual dimorphism in the spatial expression patterns of the csf1 gene. (d) Fluorescent images of the anterior half of adult fish. A stronger GFP signal is found in the male than in the female. The GFP signal in the lens of the eye is a non-specific signal derived from basal activity of a minimal promoter. (e) Magnified views of the pectoral fins in bright-field (left) and GFP fluorescent observations (right). GFP is expressed in a wider area in the male fin than in the female fin. Scale bars indicate 200 µm. (Online version in colour.)

Figure 2.

Schematic illustrations of chromosomal engineering using targetable nucleases and site-specific recombinases. (a) Double strand breaks (DSBs) by targetable nucleases facilitate targeted chromosomal rearrangements. Simultaneous induction of two DSBs on a single chromosome can induce a large deletion or an inversion between the two cleavage sites. Simultaneous induction of two DSBs on two different chromosomes can induce translocation between non-homologous chromosomes or recombination between homologous chromosomes. (b) Targeted replacement of a genomic region by recombinase-mediated cassette exchange (RMCE). Open and closed triangles indicate two different recognition sites of site-specific recombinases (SSRs), which will recombine with an identical recognition site but not with other sites (e.g. loxP/lox511 for Cre-lox system and FRT/FRT3 for FLP-FRT system). This allows for double-reciprocal recombination between a target genomic region and an exogenous DNA. (c) Targeted chromosomal engineering using SSRs. Open and closed triangles indicate two different SSR targets for unidirectional recombination (e.g. lox66/lox71 for Cre-lox system and attP/attB for PhiC31 integrase system). Recombination between these two recognition sites on a single chromosome in opposite directions results in an inversion of the target region, whereas recombination between target sites on two separate chromosomes can induce translocation between the two chromosomes. (Online version in colour.)