How to create state-of-the-art genetic model systems: strategies for optimal CRISPR-mediated genome editing

Abstract

Model systems with defined genetic modifications are powerful tools for basic research and translational disease modelling. Fortunately, generating state-of-the-art genetic model systems is becoming more accessible to non-geneticists due to advances in genome editing technologies. As a consequence, solely relying on (transient) overexpression of (mutant) effector proteins is no longer recommended since scientific standards increasingly demand genetic modification of endogenous loci. In this review, we provide up-to-date guidelines with respect to homology-directed repair (HDR)-mediated editing of mammalian model systems, aimed at assisting researchers in designing an efficient genome editing strategy.

Figure 1.

Exploiting HDR for insertion or replacement of DNA. Left panel: DNA Insertion strategy. A nuclease induced DSB near the stop codon triggers HDR mediated insertion of sequence in-between the homology regions of the donor template. Right panel: DNA replacement strategy. In this example, replacing an exon for a mutant variant will be accommodated via its excision by dual targeting of the nuclease to both ends of the exon (using two gRNA), followed by HDR between the DNA break extremities and the homology regions of the donor template. HDR via the 5’ and 3’ homology regions is indicated in blue and red respectively.

Figure 2.

Key features of SpCas9 and LbCpf1. Schematic representation of SpCas9 (A) and LbCpf1 (B). Ribonucleoprotein heteroduplexed with target DNA. DNA is indicated with grey lines, unless specified otherwise (PAM and protospacer). Red lines are RNAs. Light grey shape at the back represents protein structure. DNA strand cleavage is indicated using red arrow heads.

Figure 3.

Nuclease target site choice. Schematic representation of a dsDNA with 5 candidate gRNA (coloured arrows). To ensure optimal HDR of a donor template at the hypothetical integration site, the possible gRNA are ranked with respect to their cleavage dynamics (on and off-target scores), as well as in relation to their location and orientation towards the intended integration site.

Figure 4.

Internal homology increases the risk of undesired recombination events. (A) Positional divergence between DNA cleavage (DSB, indicated with red arrow heads) and the intended integration site (green arrow heads) creates the possibility for an alternative mode of recombination wherein an internal homology region participates. In the green panel, the internal homology region is indicated (dashed grey lines), but HDR is mediated via the intended homology arms at the extremities of the donor template (indicated in blue and red). Alternatively, the internal homology region (red) participates with the upstream homology arm (blue) in HDR, thereby failing to integrate the insert from the donor template (red panel). Since the size of internal homology is proportional to the probability of undesired recombination events, there are two widely used preventive strategies that minimize the extent of internal homology. (B) Internal recombination can be prevented by introducing (silent) mutations (red stars) in the internal homology region of the donor template (recoding). (C) Alternatively, in case of extensive internal homology, the region can be excised from the genome using dual targeting of the nuclease to introduce two DSBs that flank the internal homology sequence.

Figure 5.

Intronic integration and removal of a selection cassette. Schematic representation of HDR via a large DNA donor template that includes an autonomous selection cassette (in the opposite transcriptional orientation) to provide neomycin resistance. The selection cassette is integrated in the intron downstream of the integration site. Ideally the positioning of the cassette is in close proximity to the intended modification (mutant exon) to minimize internal homology. However, it is essential that sequences and relative locations of important regulatory elements for correct splicing remain intact (such as the 5’ splice donor site and the 3’ branch point). After clonal selection, the cassette is ideally removed from the genome in order to exclude undesired influence of the selection cassette on the expression levels of the endogenous gene. Multiple strategies exist for removal, for instance via Cre/Flp mediated recombination of LoxP or Frt sites that flank the cassette (red triangles). The minimal left-over sequence, in this case a single recombination site of ∼30nt, is often referred to as a scar sequence. Scar-free removal strategies are available (see text).

Figure 6.

PCR-based identification of correctly modified alleles. PCR-based assays can be employed to expedite screening of clones for correctly modified alleles (A). For insertion strategies, zygosity can be determined by discrimination between PCR amplicon sizes spanning the integration site. Subsequently, sequence integrity can be confirmed by sequencing of the amplicons. (B) Discrimination based on size is not always possible when replacing DNA sequences with modified versions. Sequence specific primers can be used to generate amplicons that are specific for WT or modified alleles.

Figure 7.

Parameters for donor template design. (A) Schematic representation of ssODN template integration stimulated by a DSB (red arrow heads). Due to 5’ DNA end resection by DNA repair pathway associated proteins, an ssODN can be designed to either hybridize in the sense or antisense orientation. Complete hybridization from the 3’ end of the ssODN towards the DNA strand extremity (DSB site) is advised. In this example, ssODN hybridization with the antisense genomic resected overhang (red) would lead to mismatches, whereas hybridization with the sense overhang (blue) does not. Furthermore, homology should be distributed in an asymmetric fashion in favour of the 5’ homology arm. Phosphorothioate bonds prevent ssODN degradation. (B) Schematic representation of HDR using long ssDNA templates. (C) Schematic representation of HDR using PCR amplified dsDNA donor. Homology arms up to 80nt can be added to conventional PCR primers as 3’ overhangs. (D) Schematic representation of HDR using a donor vector. Vectors can be assembled as complex donor templates from multiple sources. Flanking of the template with nuclease target sites identical to the genomic target site allows excision and linearization of the template concurrent with genomic cleavage.

Figure 8.

Designing genome editing strategies: 3 real examples. (A) Schematic workflow of the practical steps and the sequence information for the process of generating ΔF508 CFTR mutant cell lines. Top: The dsDNA sequence of the CFTR gene is presented around the intended modification site. In between the corresponding amino acid sequence is depicted. As a donor template, an asymmetric antisense ssODN is advised (see text). (B) Schematic workflow of the practical steps and the sequence information for the process of generating an mNeongreen knock-in at the C-terminus of hACTB in cell lines. Top: The dsDNA sequence of the hACTB gene is presented around its endogenous stop codon. The corresponding amino acid sequence is depicted in between the dsDNA. An ssDNA donor template is depicted below the schematic representation of the hACBT locus. Rationale for strategy design is described in the text. (C) Schematic workflow of the practical steps and the sequence information for the process of generating a CreERT2 knock-in in the hKRT20 locus via a P2A fusion at its C-terminus in human colon organoids. Top: A stretch of dsDNA sequence of the 3’UTR of the hKRT20 gene is presented. Below the locus is a schematic representation of the donor plasmid. Rationale for strategy design is described in the text. Yellow arrow indicates gRNA. Cleavage sites (DSB) are indicated with red arrow heads. PAM sequences are underscored.