Establishment of expanded and streamlined pipeline of PITCh knock-in - a web-based design tool for MMEJ-mediated gene knock-in, PITCh designer, and the variations of PITCh, PITCh-TG and PITCh-KIKO

Abstract

The emerging genome editing technology has enabled the creation of gene knock-in cells easily, efficiently, and rapidly, which has dramatically accelerated research in the field of mammalian functional genomics, including in humans. We recently developed a microhomology-mediated end-joining-based gene knock-in method, termed the PITCh system, and presented various examples of its application. Since the PITCh system only requires very short microhomologies (up to 40 bp) and single-guide RNA target sites on the donor vector, the targeting construct can be rapidly prepared compared with the conventional targeting vector for homologous recombination-based knock-in. Here, we established a streamlined pipeline to design and perform PITCh knock-in to further expand the availability of this method by creating web-based design software, PITCh designer ( http://www.mls.sci.hiroshima-u.ac.jp/smg/PITChdesigner/index.html ), as well as presenting an experimental example of versatile gene cassette knock-in. PITCh designer can automatically design not only the appropriate microhomologies but also the primers to construct locus-specific donor vectors for PITCh knock-in. By using our newly established pipeline, a reporter cell line for monitoring endogenous gene expression, and transgenesis (TG) or knock-in/knockout (KIKO) cell line can be produced systematically. Using these new variations of PITCh, an exogenous promoter-driven gene cassette expressing fluorescent protein gene and drug resistance gene can be integrated into a safe harbor or a specific gene locus to create transgenic reporter cells (PITCh-TG) or knockout cells with reporter knock-in (PITCh-KIKO), respectively.

Keywords: CRISPR–Cas9; gene knock-in; genome engineering; microhomology-mediated end-joining (MMEJ); web tool.

Figure 1.

Schematic illustrations of the PITCh knock-in for C-terminal tagging (A) and transgenesis or knock-in/knockout (B). In the C-terminal tagging, a promoterless EGFP-2A-Puro^R cDNA is integrated into the genome to monitor endogenous gene expression and protein localization. In the transgenesis or knock-in/knockout, a constitutive promoter-driven CMV-EGFP-2A-Puro^R-polyA cassette is integrated to establish a cell line with the stable expression of exogenous gene (PITCh-TG) or to disrupt gene function by knocking-in the exogenous gene cassette (PITCh-KIKO), respectively. The CMV-EGFP-2A-Puro^R-polyA cassette should be integrated with the opposite orientation in the endogenous gene to avoid promoter interference. Puro^R, puromycin resistance gene.

Figure 2.

Detailed design of PITCh-TG in human AAVS1 locus. Black lines indicate the target sequences of sgRNAs. Black boxes indicate PAM sequences. Black triangles indicate DSB sites. MH, microhomology. Puro^R, puromycin resistance gene.

Figure 3.

Experimental example of PITCh-TG. (A) Schematic illustration of knock-in allele. The genomic context was visualized using SnapGene Viewer software (Chicago, IL, USA) (http://www.snapgene.com/) with various annotations including primers used for genotyping and probes for Southern blotting. Red boxes indicate left and right microhomology regions. The cut region indicates a DNA fragment generated by restriction enzymes used in Southern blotting. (B) Gel images and summary of genotyping. The clone IDs are shown at the top of each panel. Red letters indicate the clones which the knock-in was successful. A, B, and C in the upper panels indicate the PCR products of non-knock-in allele, 5′ junction, and 3′ junction, respectively. The lower panel represents the results of the out-out PCR, simultaneously amplifying both the knock-in and the non-knock-in alleles. M, ladder marker. (C) Southern blotting. The results of inner and outer probes are shown. WT, wild type; KI, knock-in.

Figure 4.

Screenshots of PITCh designer, representing the sequence submission (A), selection of target bases (B), and the example of the design results (C).