A HIT-trapping strategy for rapid generation of reversible and conditional alleles using a universal donor

Targeted mutagenesis in model organisms is key for gene functional annotation and biomedical research. Despite technological advances in gene editing by the CRISPR-Cas9 systems, rapid and efficient introduction of site-directed mutations remains a challenge in large animal models. Here, we developed a robust and flexible insertional mutagenesis strategy, homology-independent targeted trapping (HIT-trapping), which is generic and can efficiently target-trap an endogenous gene of interest independent of homology arm and embryonic stem cells. Further optimization and equipping the HIT-trap donor with a site-specific DNA inversion mechanism enabled one-step generation of reversible and conditional alleles in a single experiment. As a proof of concept, we successfully created mutant alleles for 21 disease-related genes in primary porcine fibroblasts with an average knock-in frequency of 53.2%, a great improvement over previous approaches. The versatile HIT-trapping strategy presented here is expected to simplify the targeted generation of mutant alleles and facilitate large-scale mutagenesis in large mammals such as pigs.

Following the completion of animal genome sequencing projects, rapid and efficient mutagenesis strategies are needed for analyzing gene function and for creating human disease models. Gene trapping is a high-throughput mutagenesis strategy whereby random vector insertion can be achieved across the mouse genome. A typical gene-trap vector contains a promoter-less reporter/selection gene flanked by an upstream splice acceptor (SA) and a downstream poly(A) signal. Upon insertion into an intron of a gene, the vector both inactivates the trapped gene and enables the gene-specific expression of a reporter gene (Gossler et al. 1989; Stanford et al. 2001). To date, gene-trapping approaches have been successfully applied toward large-scale mutagenesis in mouse embryonic stem cells (mESCs) and generation of gene knockout mice (Skarnes et al. 2004). The main drawback of random gene trapping is that gene-trap alleles are not specifically engineered to target genes of interest in advance. Therefore, methods to streamline the introduction of predesigned, site-specific modifications into the genome by homologous recombination would represent a significant technological advance. Previously, a hybrid approach combining gene targeting and gene trapping (targeted trapping) enabled mutation of expressed genes in mESCs with high efficiency, using a gene-trap construct flanked by homologous sequences of the target locus (Friedel et al. 2005). Also, homologous recombination is commonly used for creating conditional alleles, which is essential to avoid embryonic lethality and to study the stage- and tissue-specific functions of genes (Branda and Dymecki 2004). However, both standard gene trapping and targeted trapping are only suitable for genes expressed in embryonic stem (ES) cells. Furthermore, construction of targeting donor vectors with homology arms is labor intensive and costly, and the low efficiency of homologous recombination is also a rate-limiting step for gene targeting in mammalian genomes.Recently, by taking advantage of precise genomic double-strand breaks (DSBs) created by the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system (Ran et al. 2013; Doudna and Charpentier 2014; Hsu et al. 2014), homology-directed repair (HDR) efficiency was substantially enhanced (Porteus and Carroll 2005), and even donors with short homology arms (Orlando et al. 2010) or single-stranded DNA oligonucleotides (Chen et al. 2011; Quadros et al. 2017) were found to be compatible with site-specific integration. However, each targeting donor for HDR still needs to be customized with gene-specific homology sequences. Because of the lack of ES cells for certain animals such as pigs, sheep, and cattle, the genome must be edited either in a zygote embryo or in a somatic cell for somatic cell nuclear transfer (SCNT) (Reddy et al. 2020). It is still not feasible to achieve large-scale insertional mutagenesis including conditional knockouts in these important species with random gene trapping or HDR-based methods. Also, the problem of genetic mosaicism in embryo editing remains unresolved (Mehravar et al. 2019), prompting a need for technological advances to accelerate genetic modification in somatic cells.Alternatively, the generally more efficient nonhomologous end joining (NHEJ) pathway has been exploited for site-specific insertion of exogenous DNA by simultaneous cleavage of both donor plasmid and genome using programmable nucleases (Cristea et al. 2013; Maresca et al. 2013; Brown et al. 2016; Suzuki et al. 2016; Sawatsubashi et al. 2018). In contrast to HDR-based strategies, NHEJ-mediated insertions do not require gene-specific homology arms, enabling diverse sites to be targeted with a universal donor vector. Therefore, we speculated that a gene-trap cassette could be inserted into a specific locus easily through this mechanism in any cell type.Here, by combining NHEJ-mediated knock-in and gene trapping, we developed a strategy for targeted mutagenesis, especially in somatic cells with low HDR activity, referred to as HIT-trapping. By using a universal donor, this strategy allows us to (1) create null alleles, (2) produce a fluorescent reporter signal that could potentially allow cells with null alleles to be identified very quickly, and (3) produce reversible and conditional alleles that would be very helpful to have in most animal models but are often cumbersome to create.