A most formidable arsenal: genetic technologies for building a better mouse

Abstract

The mouse is one of the most widely used model organisms for genetic study. The tools available to alter the mouse genome have developed over the preceding decades from forward screens to gene targeting in stem cells to the recent influx of CRISPR approaches. In this review, we first consider the history of mice in genetic study, the development of classic approaches to genome modification, and how such approaches have been used and improved in recent years. We then turn to the recent surge of nuclease-mediated techniques and how they are changing the field of mouse genetics. Finally, we survey common classes of alleles used in mice and discuss how they might be engineered using different methods.

Keywords: CRISPR; gene targeting; genetics; mice; transgenesis.

Figure 1.

Overview of CRISPR/Cas mechanism. (A) A typical CRISPR/Cas9 locus consists of a repeat-spacer array, transactivating CRISPR RNA (tracrRNA), and a Cas protein-coding region. (B) The repeat-spacer array encodes the CRISPR RNAs (crRNA), which form a heteroduplex with the tracrRNA and guide the Cas9 endonuclease to its target. The crRNA:tracrRNA duplex can be replaced with a single guide RNA (gRNA). (C) Binding of the gRNA is dictated by the protospacer sequence and the protospacer adjacent motif (PAM) site located downstream. The specific PAM site is unique for the various orthologs of Cas9. (R) A/G;(W) A/T; (Y) C/T (D) Cas9 creates DSBs via its RuvC and HNH nuclease domains. Cas9 nickases can be made via a single amino acid substitution in either of the nuclease domains. (E) Cas12a, another single nuclease Cas system, uses a single crRNA, an increased protospacer region, an upstream PAM site, and a single RuvC domain that induces a 5-nt 5′ overhang.

Figure 2.

CRISPR/Cas base editors and prime editor. (A) Base editor systems can induce nucleotide changes without the need for DSBs in the target DNA. Cas9n (D10A) is used to nick the unedited strand to facilitate incorporation of the deaminated nucleotide. (B) Base editors use a deaminase domain to convert C to T (or A to G). (C) The conversions can occur within the ssDNA deaminase activity window upstream of the PAM site. (D) The width of the activity window is dictated by the base editor system. (E) The prime editor system uses a Mo-MLV RT domain to reverse-transcribe small insertions and deletions into the target DNA. A specialized pegRNA contains a protospacer, scaffold sequence, an RT template, and a primer binding sequence. Cas9n (H840A) nicks the targeted strand of DNA to allow the RT domain to reverse transcribe the template into the DNA strand. (F) The PE3 system uses an additional gRNA to induce a nick in the unedited strand at a nearby locus to facilitate incorporation of the edited DNA.

Figure 3.

Typical workflow. The typical workflow required for the creation of a mouse model varies between gene targeting or CRISPR. For gene targeting, mESC clones should be screened via PCR and Southern blot to verify correct integration prior to injection into blastocysts. Chimeras are chosen based on coat color contribution and bred. Their pups are then screened via PCR (and coat color if crossing to a different strain) for germline transmission of the intended mutation. For CRISPR, gRNA cutting efficiency should be verified via PCR and Surveyor (or T7E1) assays. After introducing the CRISPR reagents to embryos, founder pups should be screened for both the correct mutation and any off-target effects. CRISPR has the potential to significantly reduce the time needed to create a mouse model.

Figure 4.

Designing alleles via gene targeting or CRISPR/Cas. Various genetic alterations can be constructed using either gene targeting or CRISPR. Gene targeting constructs typically consist of sequence containing the intended edit and a positive selection marker flanked by homology arms to facilitate integration. A negative selection marker is located outside of the homology arms to select against random insertion. For CRISPR, repair constructs are mainly used for conditional, tag, and reporter alleles and do not require the use of selection markers. Base or prime editing systems can be used for smaller point mutations. The conditional reporter is depicted here at the commonly targeted ROSA26 locus using a splice acceptor (SA) to drive the reporter off the endogenous promoter. Alternatively, an exogenous promoter such as CAG can be used. Orange triangles represent FRT sites, red triangles are loxP sites, red stars are point mutations, single white triangles are nicks, and double white triangles are DSBs.