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.

Selective genome editing such as gene knock-in has recently been achieved by administration of chemical enhancer or inhibitor of particular DNA double-strand break (DSB) repair pathways, as well as overexpression of pathway-specific genes. In this study, we attempt to enhance the efficiency further to secure robust gene knock-ins, by using the local accumulation of DSB repair molecules (LoAD) system. We identify CtIP as a strong enhancer of microhomology-mediated end-joining (MMEJ) repair by genetic screening, and show the knock-in-enhancing effect of CtIP LoADing. Next-generation sequencing reveals that CtIP LoADing highly increases the frequency of MMEJ-mediated integration. Selection-free, simultaneous triple gene knock-ins are also achieved with the CtIP-LoADing strategy. Moreover, by replacing the LoADing molecules and targeting strategies, this system can be applied for other specific genome engineering purposes, such as introducing longer deletions for gene disruption, independently introducing multiple mutations without chromosomal deletion, and efficiently incorporating a single-stranded oligodeoxynucleotide donor.

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Efficiency and accuracy are paramount in genome editing. While CRISPR-Cas nucleases are efficient at editing target genes, their accuracy is limited because following DNA cleavage by Cas proteins, error-prone repair mechanisms introduce random mutations. Improving the accuracy of CRISPR-Cas by reducing random repairs using DNA- or RNA-based templates can compromise efficiency. To simultaneously improve both editing efficiency and accuracy, we created a frameshifting genome-editing technology by fusing Cas9 with DNA polymerases. These Frame Editors (FEs) introduce precise and controlled frameshifts into target loci via specific DNA repairs near Cas9-induced cleavage loci. We demonstrate two types of FEs: the insertion-inducing frame editor (iFE) and the deletion-inducing frame editor (dFE). For iFE, DNA polymerase beta (POLB) is fused with Cas9, which increases the frequency of 1-bp insertions. For dFE, T4 DNA polymerase (T4pol) is fused with Cas9, which increases the frequency of 1-bp deletions. Both types of FEs reduce the number of random mutations at target loci compared with Cas9. We show that off-target editing can be reduced by substituting Cas9 with high-fidelity variants, such as HiFi Cas9 or LZ3 Cas9. Thus, FEs can introduce frameshifts into target loci with much improved mutation profiles compared with Cas9 alone and without the requirement for template sequences, offering a new strategy for repairing pathogenic frameshifts.

Base Editor, a technique that utilizes Cas9 nickase fused with deaminase to introduce single base substitutions, has significantly facilitated the creation of valuable genome variants in medical and agricultural fields. However, a phenomenon known as RNA off-target effects is recognized with Base Editor, resulting in unintended substitutions in the transcriptome. It has been reported that such substitutions often occur in specific base motifs (ACW), but whether these motif mutations are dominant has not been investigated. In this study, we constructed a pipeline for analyzing RNA off-target effects, called the Pipeline for CRISPR-induced Transcriptome-wide Unintended RNA Editing (PiCTURE), and analyzed RNA-seq data previously reported. We found minor RNA off-target effects associated with the reported base motifs, and most were indistinguishable in motif analysis.Consequently, we trained a Large Language Model (LLM) specialized for DNA base sequences on RNA off-target sequences and developed a classifier for assessing the risk of RNA off-target effects based on the sequences. When the model's estimations were applied to the RNA off-target data for BE4-rAPOBEC1 and BE4-RrA3F, satisfactory determination results were obtained. This study is the first to demonstrate the efficacy of machine learning approaches in determining RNA off-target effects caused by Base Editor and presents a predictive model for the safer use of Base Editor.