Shota Nakade

Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9

Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR in some cell type and organisms represent technical hurdles for the application of HR-mediated knock-in technology. Here, we introduce an alternative strategy for gene knock-in using transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) mediated by microhomology-mediated end-joining, termed the PITCh (Precise Integration into Target Chromosome) system. TALEN-mediated PITCh, termed TAL-PITCh, enables efficient integration of exogenous donor DNA in human cells and animals, including silkworms and frogs. We further demonstrate that CRISPR/Cas9-mediated PITCh, termed CRIS-PITCh, can be applied in human cells without carrying the plasmid backbone sequence. Thus, our PITCh-ing strategies will be useful for a variety of applications, not only in cultured cells, but also in various organisms, including invertebrates and vertebrates.

Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish

The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10–40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes (tyrosinase and krtt1c19e) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish.

MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems

Programmable nucleases enable engineering of the genome by utilizing endogenous DNA double-strand break (DSB) repair pathways. Although homologous recombination (HR)-mediated gene knock-in is well established, it cannot necessarily be applied in every cell type and organism because of variable HR frequencies. We recently reported an alternative method of gene knock-in, named the PITCh (Precise Integration into Target Chromosome) system, assisted by microhomology-mediated end-joining (MMEJ). MMEJ harnesses independent machinery from HR, and it requires an extremely short homologous sequence (5–25 bp) for DSB repair, resulting in precise gene knock-in with a more easily constructed donor vector. Here we describe a streamlined protocol for PITCh knock-in, including the design and construction of the PITCh vectors, and their delivery to either human cell lines by transfection or to frog embryos by microinjection. The construction of the PITCh vectors requires only a few days, and the entire process takes ∼1.5 months to establish knocked-in cells or ∼1 week from injection to early genotyping in frog embryos.

Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

BackgroundAlthough CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining.ResultsHere, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes.ConclusionsOur results provide a technical platform for high-throughput knock-in.

Cas9, Cpf1 and C2c1/2/3―What's next?

ABSTRACT Since the rapid emergence of clustered regulatory interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system, developed as a genome engineering tool in 2012–2013, most researchers in the life science field have had a fixated interest in this fascinating technology. CRISPR-Cas9 is an RNA-guided DNA endonuclease system, which consists of Cas9 nuclease defining a few targeting base via protospacer adjacent motif complexed with easily customizable single guide RNA targeting around 20-bp genomic sequence. Although Streptococcus pyogenes Cas9 (SpCas9), one of the Cas9 proteins that applications in genome engineering were first demonstrated, still has wide usage because of its high nuclease activity and broad targeting range, there are several limitations such as large molecular weight and potential off-target effect. In this commentary, we describe various improvements and alternatives of CRISPR-Cas systems, including engineered Cas9 variants, Cas9 homologs, and novel Cas proteins other than Cas9. These variations enable flexible genome engineering with high efficiency and specificity, orthogonal genetic control at multiple gene loci, gene knockdown, or fluorescence imaging of transcripts mediated by RNA targeting, and beyond.

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.

Homeolog-specific targeted mutagenesis in Xenopus laevis using TALENs

Transcription activator-like effector nucleases (TALENs) have previously been used for targeted genome editing in various organisms including Xenopus laevis. However, because of genomic polyploidization, X. laevis usually possess homeologous genes (homeologs) with quite similar sequences that make the analysis of gene function difficult. In the present study, we show methodological examples of targeted gene modification of X. laevis homeologs. The X. laevis cytoglobin gene (cygb) consists of two homeologs (xlcygba and xlcygbb), and molecular phylogenetic analysis suggested that they have potentially different functions. Thus, there is a need to establish a method of homeolog-specific gene disruption to clarify gene functions in detail. Here, we show successful examples of homeolog-specific and simultaneous gene disruption for xlcygba and xlcygbb. We found that selective digestion can be performed with at least three mismatches in TALEN target sites in both homeologs. This report paves the way for the functional analyses of X. laevis homeologs, even those containing nearly identical sequences.

Biased genome editing using the local accumulation of DSB repair molecules system

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.Genome editing using CRISPR can be enhanced by manipulating DNA double-strand break repair pathways. Here the authors demonstrate LoAD, local accumulation of repair molecules, which shifts repair to microhomology-mediated end-joining.

Cancer induction and suppression with transcriptional control and epigenome editing technologies

Cancer epigenetics is one of the most important research subjects in dissecting cancer mechanisms and therapeutic targets because the emergence and malignant transformation of various cancers are caused by unnatural expression of cancer-related genes attributed to their epigenetic errors. The original concept of cancer epigenetics basically stands on the analysis of the epigenetic status in naturally occurring cancer cells; however, the rapidly emerging technology called epigenome editing would change this situation drastically. Epigenome editing, the most promising derivative technology of genome editing, can modify the epigenetic states at the pre-defined genomic locus using the programmable effectors, consisting of various epigenetic factors combined with site-specific DNA-binding domains. This technology can be utilized in a reversible manner; i.e., cancer modeling can be achieved by introducing aberrant epigenetic marks in normal cells, and cancer suppression can be achieved by correcting the epigenetic errors in cancer cells. In this review, we summarize the basics of epigenome editing and cancer epigenetics, followed by the current examples of cancer induction and suppression with the transcriptional control and epigenome editing technologies.

Unexpected heterogeneity derived from Cas9 ribonucleoprotein-introduced clonal cells at the HPRT1 locus

Single‐cell cloning is an essential technique for establishing genome‐edited cell clones mediated by programmable nucleases such as CRISPR‐Cas9. However, residual genome‐editing activity after single‐cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR‐Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single‐cell cloning of Cas9 RNP‐transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single‐cell cloning at 24, 48, 72 hr and 1 week post‐transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high‐resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long‐term incubation before single‐cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single‐cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures.