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Article ; Online: Highly Multiplexed Analysis of CRISPR Genome Editing Outcomes in Mammalian Cells.

Ishiguro, Soh / Yachie, Nozomu

Methods in molecular biology (Clifton, N.J.)

2021 Volume 2312, Page(s) 193–223

Abstract: CRISPR-Cas-based genome editing has enabled efficient genetic engineering of a range of organisms and sparked revolutions in many fields of biology. After Streptococcus pyogenes Cas9 was first demonstrated for mammalian genome editing, many CRISPR- ... ...

Abstract	CRISPR-Cas-based genome editing has enabled efficient genetic engineering of a range of organisms and sparked revolutions in many fields of biology. After Streptococcus pyogenes Cas9 was first demonstrated for mammalian genome editing, many CRISPR-associated (Cas) protein variants have been isolated from different species and adopted for genome editing. Furthermore, various effector domains have been fused to these Cas proteins to expand their genome-editing abilities. Although the number of genome-editing tools has been rapidly increasing, the throughput of cell-based characterization of new genome-editing tools remains limited. Here we describe a highly multiplexed genome editing and sequencing library preparation protocol that allows high-resolution analysis of mutation outcomes and frequencies induced by hundreds to thousands of different genome-editing reagents in mammalian cells. We have successful experiences of developing several key genome-editing tools using this protocol. The protocol also is designed to be compatible with robotic liquid handling systems for further scalability.
MeSH term(s)	CRISPR-Associated Protein 9/genetics ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Gene Expression Regulation ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Multiplex Polymerase Chain Reaction ; Transfection
Chemical Substances	CRISPR-Associated Protein 9 (EC 3.1.-)
Language	English
Publishing date	2021-07-06
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-1441-9_12
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Article ; Online: DNA-GPS: A theoretical framework for optics-free spatial genomics and synthesis of current methods.

Greenstreet, Laura / Afanassiev, Anton / Kijima, Yusuke / Heitz, Matthieu / Ishiguro, Soh / King, Samuel / Yachie, Nozomu / Schiebinger, Geoffrey

Cell systems

2023 Volume 14, Issue 10, Page(s) 844–859.e4

Abstract: While single-cell sequencing technologies provide unprecedented insights into genomic profiles at the cellular level, they lose the spatial context of cells. Over the past decade, diverse spatial transcriptomics and multi-omics technologies have been ... ...

Abstract	While single-cell sequencing technologies provide unprecedented insights into genomic profiles at the cellular level, they lose the spatial context of cells. Over the past decade, diverse spatial transcriptomics and multi-omics technologies have been developed to analyze molecular profiles of tissues. In this article, we categorize current spatial genomics technologies into three classes: optical imaging, positional indexing, and mathematical cartography. We discuss trade-offs in resolution and scale, identify limitations, and highlight synergies between existing single-cell and spatial genomics methods. Further, we propose DNA-GPS (global positioning system), a theoretical framework for large-scale optics-free spatial genomics that combines ideas from mathematical cartography and positional indexing. DNA-GPS has the potential to achieve scalable spatial genomics for multiple measurement modalities, and by eliminating the need for optical measurement, it has the potential to position cells in three-dimensions (3D).
MeSH term(s)	Genomics/methods ; Gene Expression Profiling ; DNA/genetics
Chemical Substances	DNA (9007-49-2)
Language	English
Publishing date	2023-09-25
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2854138-8
ISSN	2405-4720 ; 2405-4712
ISSN (online)	2405-4720
ISSN	2405-4712
DOI	10.1016/j.cels.2023.08.005
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Article ; Online: Pooled CRISPR screening of high-content cellular phenotypes using ghost cytometry.

Tsubouchi, Asako / An, Yuri / Kawamura, Yoko / Yanagihashi, Yuichi / Nakayama, Hirofumi / Murata, Yuri / Teranishi, Kazuki / Ishiguro, Soh / Aburatani, Hiroyuki / Yachie, Nozomu / Ota, Sadao

Cell reports methods

2024 Volume 4, Issue 3, Page(s) 100737

Abstract: Recent advancements in image-based pooled CRISPR screening have facilitated the mapping of diverse genotype-phenotype associations within mammalian cells. However, the rapid enrichment of cells based on morphological information continues to pose a ... ...

Abstract	Recent advancements in image-based pooled CRISPR screening have facilitated the mapping of diverse genotype-phenotype associations within mammalian cells. However, the rapid enrichment of cells based on morphological information continues to pose a challenge, constraining the capacity for large-scale gene perturbation screening across diverse high-content cellular phenotypes. In this study, we demonstrate the applicability of multimodal ghost cytometry-based cell sorting, including both fluorescent and label-free high-content phenotypes, for rapid pooled CRISPR screening within vast cell populations. Using the high-content cell sorter operating in fluorescence mode, we successfully executed kinase-specific CRISPR screening targeting genes influencing the nuclear translocation of RelA. Furthermore, using the multiparametric, label-free mode, we performed large-scale screening to identify genes involved in macrophage polarization. Notably, the label-free platform can enrich target phenotypes without requiring invasive staining, preserving untouched cells for downstream assays and expanding the potential for screening cellular phenotypes even when suitable markers are absent.
MeSH term(s)	Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; Flow Cytometry ; Phenotype ; Cell Separation ; Genetic Testing ; Mammals
Language	English
Publishing date	2024-03-15
Publishing country	United States
Document type	Journal Article
ISSN	2667-2375
ISSN (online)	2667-2375
DOI	10.1016/j.crmeth.2024.100737
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Article ; Online: DNA event recorders send past information of cells to the time of observation.

Ishiguro, Soh / Mori, Hideto / Yachie, Nozomu

Current opinion in chemical biology

2019 Volume 52, Page(s) 54–62

Abstract: While current omics and single cell technologies have enabled measurements of high-resolution molecular snapshots of cells at a large scale, these technologies all require destruction of samples and prevent us from analyzing dynamic changes in molecular ... ...

Abstract	While current omics and single cell technologies have enabled measurements of high-resolution molecular snapshots of cells at a large scale, these technologies all require destruction of samples and prevent us from analyzing dynamic changes in molecular profiles, phenotypes, and behaviors of individual cells in a complex system. One possible direction to overcome this issue is the development of a cell-embedded 'event recorder' system, whereby molecular and phenotypic information of a cell(s) can be obtained at the time of observation with their past event information stored in 'heritable polymers' of the same cell. This concept has been demonstrated by many synthetic cellular circuits that monitor and transmit a certain set of environmental and intracellular signals into DNA, and have now been further accelerated by recent CRISPR-related technologies. Notably, the discovery of the RT-Cas1-Cas2 system, which acquires sequences of cellular transcripts into a specific host genomic region, has enabled recording of a broader range of molecular profile histories in the DNA tapes of cells, to understand the dynamics of complex biological processes that cannot be addressed by current technologies.
MeSH term(s)	Animals ; CRISPR-Cas Systems ; Cell Communication ; Cells ; DNA/genetics ; Humans ; Recombination, Genetic ; Transcriptome
Chemical Substances	DNA (9007-49-2)
Language	English
Publishing date	2019-06-11
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	1439176-4
ISSN	1879-0402 ; 1367-5931
ISSN (online)	1879-0402
ISSN	1367-5931
DOI	10.1016/j.cbpa.2019.05.009
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Article ; Online: Engineered Campylobacter jejuni Cas9 variant with enhanced activity and broader targeting range.

Nakagawa, Ryoya / Ishiguro, Soh / Okazaki, Sae / Mori, Hideto / Tanaka, Mamoru / Aburatani, Hiroyuki / Yachie, Nozomu / Nishimasu, Hiroshi / Nureki, Osamu

Communications biology

2022 Volume 5, Issue 1, Page(s) 211

Abstract: The RNA-guided DNA endonuclease Cas9 is a versatile genome-editing tool. However, the molecular weight of the commonly used Streptococcus pyogenes Cas9 is relatively large. Consequently, its gene cannot be efficiently packaged into an adeno-associated ... ...

Abstract	The RNA-guided DNA endonuclease Cas9 is a versatile genome-editing tool. However, the molecular weight of the commonly used Streptococcus pyogenes Cas9 is relatively large. Consequently, its gene cannot be efficiently packaged into an adeno-associated virus vector, thereby limiting its applications for therapeutic genome editing. Here, we biochemically characterized the compact Cas9 from Campylobacter jejuni (CjCas9) and found that CjCas9 has a previously unrecognized preference for the N
MeSH term(s)	CRISPR-Cas Systems ; Campylobacter jejuni/genetics ; Gene Editing ; Humans
Language	English
Publishing date	2022-03-08
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2399-3642
ISSN (online)	2399-3642
DOI	10.1038/s42003-022-03149-7
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Article ; Online: Deep distributed computing to reconstruct extremely large lineage trees.

Konno, Naoki / Kijima, Yusuke / Watano, Keito / Ishiguro, Soh / Ono, Keiichiro / Tanaka, Mamoru / Mori, Hideto / Masuyama, Nanami / Pratt, Dexter / Ideker, Trey / Iwasaki, Wataru / Yachie, Nozomu

Nature biotechnology

2022 Volume 40, Issue 4, Page(s) 566–575

Abstract: Phylogeny estimation (the reconstruction of evolutionary trees) has recently been applied to CRISPR-based cell lineage tracing, allowing the developmental history of an individual tissue or organism to be inferred from a large number of mutated sequences ...

Abstract	Phylogeny estimation (the reconstruction of evolutionary trees) has recently been applied to CRISPR-based cell lineage tracing, allowing the developmental history of an individual tissue or organism to be inferred from a large number of mutated sequences in somatic cells. However, current computational methods are not able to construct phylogenetic trees from extremely large numbers of input sequences. Here, we present a deep distributed computing framework to comprehensively trace accurate large lineages (FRACTAL) that substantially enhances the scalability of current lineage estimation software tools. FRACTAL first reconstructs only an upstream lineage of the input sequences and recursively iterates the same produce for its downstream lineages using independent computing nodes. We demonstrate the utility of FRACTAL by reconstructing lineages from >235 million simulated sequences and from >16 million cells from a simulated experiment with a CRISPR system that accumulates mutations during cell proliferation. We also successfully applied FRACTAL to evolutionary tree reconstructions and to an experiment using error-prone PCR (EP-PCR) for large-scale sequence diversification.
MeSH term(s)	Algorithms ; Cell Lineage/genetics ; Mutation ; Phylogeny ; Software
Language	English
Publishing date	2022-01-06
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1311932-1
ISSN	1546-1696 ; 1087-0156
ISSN (online)	1546-1696
ISSN	1087-0156
DOI	10.1038/s41587-021-01111-2
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Zs.A 2167: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: Fast and global detection of periodic sequence repeats in large genomic resources.

Mori, Hideto / Evans-Yamamoto, Daniel / Ishiguro, Soh / Tomita, Masaru / Yachie, Nozomu

Nucleic acids research

2018 Volume 47, Issue 2, Page(s) e8

Abstract: Periodically repeating DNA and protein elements are involved in various important biological events including genomic evolution, gene regulation, protein complex formation, and immunity. Notably, the currently used genome editing tools such as ZFNs, ... ...

Abstract	Periodically repeating DNA and protein elements are involved in various important biological events including genomic evolution, gene regulation, protein complex formation, and immunity. Notably, the currently used genome editing tools such as ZFNs, TALENs, and CRISPRs are also all associated with periodically repeating biomolecules of natural organisms. Despite the biological importance of periodically repeating sequences and the expectation that new genome editing modules could be discovered from such periodical repeats, no software that globally detects such structured elements in large genomic resources in a high-throughput and unsupervised manner has been developed. We developed new software, SPADE (Search for Patterned DNA Elements), that exhaustively explores periodic DNA and protein repeats from large-scale genomic datasets based on k-mer periodicity evaluation. With a simple constraint, sequence periodicity, SPADE captured reported genome-editing-associated sequences and other protein families involving repeating domains such as tetratricopeptide, ankyrin and WD40 repeats with better performance than the other software designed for limited sets of repetitive biomolecular sequences, suggesting the high potential of this software to contribute to the discovery of new biological events and new genome editing modules.
MeSH term(s)	Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/chemistry ; Genomics/methods ; Humans ; Repetitive Sequences, Amino Acid ; Repetitive Sequences, Nucleic Acid ; Software ; Transcription Activator-Like Effectors/chemistry ; Zinc Finger Nucleases/chemistry
Chemical Substances	Transcription Activator-Like Effectors ; DNA (9007-49-2) ; Zinc Finger Nucleases (EC 3.1.-)
Language	English
Publishing date	2018-12-01
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gky890
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Zs.A 1067: Show issues

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Article ; Online: CRISPR/Cas9-mediated base-editing enables a chain reaction through sequential repair of sgRNA scaffold mutations.

Fukushima, Tsuyoshi / Tanaka, Yosuke / Adachi, Keito / Masuyama, Nanami / Tsuchiya, Akiho / Asada, Shuhei / Ishiguro, Soh / Mori, Hideto / Seki, Motoaki / Yachie, Nozomu / Goyama, Susumu / Kitamura, Toshio

Scientific reports

2021 Volume 11, Issue 1, Page(s) 23889

Abstract: Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two ... ...

Abstract	Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting in cellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.
MeSH term(s)	CRISPR-Cas Systems ; Cytosine Deaminase/genetics ; Cytosine Deaminase/metabolism ; DNA Repair ; Gene Editing ; HEK293 Cells ; Humans ; Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; TATA Box/genetics ; Thymidine/genetics
Chemical Substances	RNA, Guide, CRISPR-Cas Systems ; Cytosine Deaminase (EC 3.5.4.1) ; Thymidine (VC2W18DGKR)
Language	English
Publishing date	2021-12-13
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-021-02986-6
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Article ; Online: An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis.

Hino, Tomohiro / Omura, Satoshi N / Nakagawa, Ryoya / Togashi, Tomoki / Takeda, Satoru N / Hiramoto, Takafumi / Tasaka, Satoshi / Hirano, Hisato / Tokuyama, Takeshi / Uosaki, Hideki / Ishiguro, Soh / Kagieva, Madina / Yamano, Hiroyuki / Ozaki, Yuki / Motooka, Daisuke / Mori, Hideto / Kirita, Yuhei / Kise, Yoshiaki / Itoh, Yuzuru /

Matoba, Satoaki / Aburatani, Hiroyuki / Yachie, Nozomu / Karvelis, Tautvydas / Siksnys, Virginijus / Ohmori, Tsukasa / Hoshino, Atsushi / Nureki, Osamu

Cell

2023 Volume 186, Issue 22, Page(s) 4920–4935.e23

Abstract: SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus ... ...

Abstract	SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.
MeSH term(s)	Animals ; Humans ; Mice ; CRISPR-Cas Systems ; Cryoelectron Microscopy ; Gene Editing ; Mutation ; Genetic Therapy
Language	English
Publishing date	2023-09-29
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	187009-9
ISSN	1097-4172 ; 0092-8674
ISSN (online)	1097-4172
ISSN	0092-8674
DOI	10.1016/j.cell.2023.08.031
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Zs.A 1167: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
Zs.MG 58: Show issues

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Article ; Online: Base-pairing probability in the microRNA stem region affects the binding and editing specificity of human A-to-I editing enzymes ADAR1-p110 and ADAR2.

Ishiguro, Soh / Galipon, Josephine / Ishii, Rintaro / Suzuki, Yutaka / Kondo, Shinji / Okada-Hatakeyama, Mariko / Tomita, Masaru / Ui-Tei, Kumiko

RNA biology

2018 Volume 15, Issue 7, Page(s) 976–989

Abstract: Adenosine deaminases acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I). A-to-I RNA editing targets double-stranded RNA (dsRNA), and increases the complexity of gene regulation by modulating base pairing-dependent processes ... ...

Abstract	Adenosine deaminases acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I). A-to-I RNA editing targets double-stranded RNA (dsRNA), and increases the complexity of gene regulation by modulating base pairing-dependent processes such as splicing, translation, and microRNA (miRNA)-mediated gene silencing. This study investigates the genome-wide binding preferences of the nuclear constitutive isoforms ADAR1-p110 and ADAR2 on human miRNA species by RNA immunoprecipitation of ADAR-bound small RNAs (RIP-seq). Our results suggest that secondary structure predicted by base-pairing probability in the mainly double-stranded region of a pre-miRNA or mature miRNA duplex may determine ADAR isoform preference for binding distinct subpopulations of miRNAs. Furthermore, we identify 31 unique editing sites with statistical significance, 19 sites of which are novel editing sites. Editing sites are enriched in the seed region responsible for target recognition by miRNAs, and isoform-specific nucleotide motifs in the immediate vicinity and opposite of editing sites are consistent with previous studies, and further reveal that ADAR2 may edit A/C bulges more frequently than ADAR1-p110 in the context of miRNA.
MeSH term(s)	Adenosine/genetics ; Adenosine Deaminase/chemistry ; Adenosine Deaminase/genetics ; Adenosine Deaminase/metabolism ; Base Pairing ; Deamination ; Genome-Wide Association Study ; HeLa Cells ; Humans ; Inosine/genetics ; Isoenzymes/chemistry ; Isoenzymes/genetics ; Isoenzymes/metabolism ; MicroRNAs/chemistry ; MicroRNAs/genetics ; MicroRNAs/metabolism ; Nucleotide Motifs ; Protein Structure, Secondary ; RNA Editing ; RNA-Binding Proteins/chemistry ; RNA-Binding Proteins/genetics ; RNA-Binding Proteins/metabolism
Chemical Substances	Isoenzymes ; MicroRNAs ; RNA-Binding Proteins ; Inosine (5A614L51CT) ; ADAR protein, human (EC 3.5.4.37) ; ADARB1 protein, human (EC 3.5.4.4) ; Adenosine Deaminase (EC 3.5.4.4) ; Adenosine (K72T3FS567)
Language	English
Publishing date	2018-07-24
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1555-8584
ISSN (online)	1555-8584
DOI	10.1080/15476286.2018.1486658
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