Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

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.

In the past few years, several types of artificial transcriptional activator, based on CRISPR-Cas9, have been developed and refined. Of these, in synergistic activation mediator and SunTag systems, the effector proteins, expressed in trans , can be recruited to the target sites via the MS2 RNA-binding system and GCN4-scFv antibody system, respectively. Here, we report a strong transcriptional activation system achieved by fusing GCN4 repeat to MS2 coat protein to accumulate numbers of activators, fused to scFv antibodies. By targeting the CDH1 gene, we show that our novel system, named “TREE,” results in a greater effect of activating exogenous reporter and endogenous gene. Moreover, by targeting another gene, RANKL , we consistently show the superiority of the TREE system with fewer single-guide RNAs compared to conventional systems. Our TREE system is a promising tool for transcriptional activation and can potentially contribute to other dCas9-mediated technologies such as epigenome editing and chromosome visualization.

Highly efficient gene knock-out and knock-in have been achieved by harnessing CRISPR-Cas9 and its advanced technologies such as Prime Editor. In addition, various bioinformatics resources have become available to quantify and qualify the efficiency and accuracy of CRISPR edits, which significantly increased the user-friendliness of the general next-generation sequencing (NGS) analysis in the context of genome editing. However, there is no specialized and integrated software for investigating the preference in the genomic context involved in the efficiency and accuracy of genome editing using CRISPR-Cas9 and beyond. Here, we address this issue by establishing a novel analysis platform of NGS data for profiling the outcome of template-free knock- out and short homology-based editing, named MaChIAto (Microhomology-associatedChromosomalIntegration/editingAnalysistools) (https://github.com/KazukiNakamae/MaChIAto). MaChIAto accommodates the classification and profiling of the NGS reads to uncover the tendency of the corresponding method of genome editing. In the profiling function, MaChIAto can summarize the mutation patterns along with the editing efficiency, and > 70 kinds of feature analysis, e.g., correlation analysis with thermodynamics and secondary structure parameters, are available. Additionally, the classifying function of MaChIAto is based on, but much stricter than, that of the existing tool, which is achieved by implementing a novel method of parameter adaptation utilizing Bayesian optimization. To demonstrate the functionality of MaChIAto, we analyzed the NGS data of knock- out, short homology-based knock-in, and Prime Editing. We confirmed that some features of (epi-)genomic context affected the efficiency and accuracy. These results show that MaChIAto is a helpful tool for understanding the best design for CRISPR edits. More importantly, it is the first tool for discovering features in the short homology-based knock-in outcomes. MaChIAto would help researchers profile editing data and generate prediction models for CRISPR edits, further contributing to revealing a “black box” process to produce a variety of CRISPR and Prime Editing outcomes.

Highly efficient gene knock-out and knock-in have been achieved by harnessing CRISPR-Cas9 and its advanced technologies such asPrime Editor. In addition, various bioinformatics resources have become available to quantify and qualify the efficiency and accuracy of CRISPR edits, which significantly increased the user-friendliness of the general next-generation sequencing (NGS) analysis in the context of genome editing. However, there is no specialized and integrated software for investigating the preference in the genomic context involved in the efficiency and accuracy of genome editing using CRISPR-Cas9 and beyond. Here, we address this issue by establishing a novel analysis platform of NGS data for profiling the outcome of template-free knock-out and short homology-based editing, named MaChIAto (Microhomology-associated Chromosomal Integration/editing Analysis tools)