Genetic techniques articles within Nature Communications

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  • Article
    | Open Access

    Current gene silencing tools can have drawbacks. Here the authors report CRISPRδ, an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13): they also show that fusion of a translational repressor to dCas13 further improved the performance.

    • Antonios Apostolopoulos
    • , Naohiro Kawamoto
    •  & Shintaro Iwasaki

  • Article
    | Open Access

    The delivery of CRISPR RNPs has potential advantages over other genome editing approaches, including reduced off-target editing and reduced immunogenicity. Here the authors report self-deliverable Cas9 RNPs capable of robustly editing cultured cells in vitro and the mouse brain upon direct injections.

    • Kai Chen
    • , Elizabeth C. Stahl
    •  & Jennifer A. Doudna

  • Editorial
    | Open Access

    Orphan crops hold the potential to diversify our food systems. Considering their unique characteristics, our deep understanding of major crops, and the availability of modern genomic tools, taking a different research path from what major crops have gone through could accelerate the genetic improvement of orphan crops.

  • Article
    | Open Access

    Ddd-Aderived cytosine base editors (DdCBEs) are important for research of mitochondrial DNA mutation diseases. Here the authors report a strategy for screening and characterising dsDNA cytidine deaminases, and identify 7 DddA homologs which they optimise to minimise nuclear and mitochondrial off-target editing.

    • Haifeng Sun
    • , Zhaojun Wang
    •  & Bin Shen

  • Article
    | Open Access

    In the field of plant genome engineering, new nucleases with improved editing efficiency and alterative PAM requirements are needed. Here, the authors report a probiotic sourced CRISPR-LrCas9 system with similar PAM requirement to Cas12a and show its high efficiencies in various genome editing applications.

    • Zhaohui Zhong
    • , Guanqing Liu
    •  & Yong Zhang

  • Article
    | Open Access

    CRISPRi is used for gene silencing in mammalian cells. Here the authors report a gene-suppression/activation strategy using active Cas9 complexed with truncated gRNAs (tgCRISPRi/a) without causing DNA cleavage: they use this to repress or activate expression of several target genes throughout somatic tissues in Drosophila melanogaster.

    • Ankush Auradkar
    • , Annabel Guichard
    •  & Ethan Bier

  • Article
    | Open Access

    TadA deaminases widely used in many base editors lack post-translational control in cells. Here the authors report a split adenine base editor (sABE) using chemically induced dimerisation (CID) to control the catalytic activity of TadA8e and show this can be used for PCSK9 gene editing in the mouse liver.

    • Hongzhi Zeng
    • , Qichen Yuan
    •  & Xue Gao

  • Article
    | Open Access

    Cas12a is widely used in diagnostic platforms. Here the authors show that Cas12a can be programmed to directly detect RNA substrates, this is due to the 3’-end of the crRNA tolerating both RNA and DNA substrates: they use this to report a method, SAHARA, to detect RNA sequences.

    • Santosh R. Rananaware
    • , Emma K. Vesco
    •  & Piyush K. Jain

  • Article
    | Open Access

    The lack of large-scale QTL cloning method hampers systematic dissection of genetic base of quantitative traits. Here, the authors develop a multi-omics data-based technique for large-scale and rapid cloning of quantitative genes of tassel branch number and discovery of selection signatures in maize breeding.

    • Xi Wang
    • , Juan Li
    •  & Lin Li

  • Article
    | Open Access

    The safety of CRISPR-Cas9 editing is a concern. Here the authors use whole genomic analysis by 10x linked-read sequencing and optical genome mapping to interrogate the genome integrity after editing: they see large structural variants at on-target sites and unexpected large chromosomal deletions.

    • Hsiu-Hui Tsai
    • , Hsiao-Jung Kao
    •  & John Yu

  • Article
    | Open Access

    Analysis of DNA methylation usually requires a chemical or an enzymatic pretreatment step. Here, the authors report a PCR-based technology for the detection of DNA methylation in untreated DNA, and present analytical and clinical results from methylation analysis of the MGMT promoter.

    • Kamilla Kolding Bendixen
    • , Maria Mindegaard
    •  & Rasmus Koefoed Petersen

  • Article
    | Open Access

    Limited work has been done on concurrent C-to-G and A-to-G base editing. Here the authors test how a number of chromatin-associated factors affect base editing and show that HMGN1 enhanced the efficiency; by fusing HMGN1 to GBE and ABE they develop a CRISPR-based dual-function A-to-G and C-to-G base editor (GGBE).

    • Chao Yang
    • , Zhenzhen Ma
    •  & Xueli Zhang

  • Article
    | Open Access

    Rapid and facile detection of specific nucleic acid modifications could have numerous applications. Here the authors present Specific Terminal Mediated Polymerase Chain Reaction (STEM-PCR) as a generic and accessible approach, and demonstrate proof-of-principle cancer biomarker detection.

    • Gaolian Xu
    • , Hao Yang
    •  & Hongchen Gu

  • Article
    | Open Access

    The generation of CRISPR-mediated transcriptional activation (CRISPRa)-competent cell lines pose significant technical challenges. Here the authors report a platform for production of CRISPRa-ready cell populations which they combine with optimised expressed and synthetic gRNA scaffolds to enhance functionality.

    • Amy J. Heidersbach
    • , Kristel M. Dorighi
    •  & Benjamin Haley

  • Article
    | Open Access

    Application of CRISPR-Cas13d is limited by the inability to predict on- and off-targets. Here the authors perform CRISPR-Cas13d proliferation screens followed by modeling of Cas13d on- and off-targets; they design a deep learning model, DeepCas13, to predict the on-target activity of a gRNA.

    • Xiaolong Cheng
    • , Zexu Li
    •  & Wei Li

  • Article
    | Open Access

    Properties of cytidine and adenosine deaminases lead to off-target effects for cytosine base editors (CBEs) and adenine base editors (ABEs). Here the authors report that 25 TadA orthologs could be engineered to generate functional ABEs, CBEs or ACBEs via single/double mutations with minimised off-targets.

    • Shuqian Zhang
    • , Bo Yuan
    •  & Tian-Lin Cheng

  • Article
    | Open Access

    Hypercompact CRISPR-Cas12f systems have been engineered to generate miniABEs but these have limitations. Here the authors generate Cas12f-derived miniCBEs and develop miniABEs with improved editing and targeting scopes; they use these to correct pathogenic mutations in cell lines and introduce mutations in vivo.

    • Shuqian Zhang
    • , Liting Song
    •  & Tian-Lin Cheng

  • Article
    | Open Access

    Strategies to improve the specificity of nuclease-based prime editor (PEn) are needed. Here the authors report a 53BP1-inhibitory ubiquitin variant-assisted PEn platform (uPEn) to inhibit NHEJ and enable precise prime editing for generation of insertions, deletions and replacements.

    • Xiangyang Li
    • , Guiquan Zhang
    •  & Xingxu Huang

  • Review Article
    | Open Access

    CRISPR-Cas tools have shown exceptional promise in genome engineering over the past decade. Here the authors review the development of CRISPR-Cas9/Cas12/Cas13 nucleases, DNA base editors, prime editors, and RNA base editors, as well as their editing precision, off-target effects, and clinical considerations.

    • Jianli Tao
    • , Daniel E. Bauer
    •  & Roberto Chiarle

  • Article
    | Open Access

    Low-cost targeted approach to construct haplotype-resolved assemblies is needed to facilitate population genetic studies. Here, the authors demonstrate assembling high-quality MHC haplotypes with CRISPR-based enrichment and long-read sequencings.

    • Taotao Li
    • , Duo Du
    •  & Yun Liu

  • Article
    | Open Access

    Visualisation of point mutations in situ is informative for studying genetic diseases. Here the authors report single guide genome oligopaint via local denaturation fluorescence in situ hybridisation, sgGOLDFISH, a direct hybridisation genome imaging method with single-nucleotide sensitivity.

    • Yanbo Wang
    • , W. Taylor Cottle
    •  & Taekjip Ha

  • Article
    | Open Access

    Gene activation methods are valuable for studying gene functions and may have potential applications in bioengineering and medicine. Here the authors developed Narta technology to achieve gene activation by recruiting artificial transcription factors to transcription sites through nascent RNAs of the target gene.

    • Ying Liang
    • , Haiyue Xu
    •  & Baohui Chen

  • Article
    | Open Access

    Plasma cells are terminally differentiated B cells that are specialized for antibody secretion. Authors show here that genomic deletion of the p38α mitogen activated protein kinase specifically in the B cell lineage leads to diminished plasma cell differentiation via impairment of a transcriptional regulatory program by BLIMP1.

    • Jianfeng Wu
    • , Kang Yang
    •  & Jiahuai Han

  • Article
    | Open Access

    Reductive stress, reflected by the elevated intracellular NADH/NAD+ ratio, is associated with multiple human diseases. Here, the authors develop a genetic tool to manipulate the ratios of cellular NADH/NAD+ and NADPH/NADP+, and identify purine biosynthesis as an NADH-sensing pathway to mediate reductive stress.

    • Ronghui Yang
    • , Chuanzhen Yang
    •  & Binghui Li

  • Article
    | Open Access

    The success of CRISPR experiments relies on the choice of gRNA. Here the authors report crisprVerse, which enables efficient gRNA design and annotation for methods including CRISPRko, CRISPRa, CRISPRi, CRISPRbe and CRISPRkd, enabled for RNA- and DNA-targeting nucleases, including Cas9, Cas12 and Cas13.

    • Luke Hoberecht
    • , Pirunthan Perampalam
    •  & Jean-Philippe Fortin

  • Article
    | Open Access

    Existing methods for generating sgRNA predictions do not account for the tracrRNA sequence. Here the authors report an on-target model, Rule Set 3, to generate optimal predictions for multiple tracrRNA variants, and validate this on a new dataset of sgRNAs showing improvement over prior prediction models.

    • Peter C. DeWeirdt
    • , Abby V. McGee
    •  & John G. Doench

  • Article
    | Open Access

    CRISPR-Cas induced HDR methods tend to have a low efficiency. Here the authors report an HDR improvement strategy, Recursive Editing, that selectively retargets undesired indel outcomes to create additional opportunities for HDR; they introduce REtarget, a tool for Recursive Editing experimental design.

    • Lukas Möller
    • , Eric J. Aird
    •  & Jacob E. Corn

  • Article
    | Open Access

    Rare tumour specific mutations in patient samples act as markers to monitor the course of disease. Here the authors report superRCA assays for rapid, highly specific detection of DNA sequence variants present at very low frequencies in DNA samples with flow cytometry readout; they use this on AML patients.

    • Lei Chen
    • , Anna Eriksson
    •  & Ulf Landegren

  • Article
    | Open Access

    The prime editors (PEs) have shown great promise for precise genome modification. Here the authors place a stabilizing viral xrRNA motif to the 3′ of pegRNAs to enhance editing efficiencies.

    • Guiquan Zhang
    • , Yao Liu
    •  & Jianghuai Liu

  • Article
    | Open Access

    Base editors are genome engineering tools that can generate nucleotide substitutions without introducing double-stranded breaks. Here the authors show that a phage-derived peptidyl CRISPR inhibitor can be employed to modulate the activity and targeting scope of CRISPR base editor for precision base editing applications.

    • Kun Jia
    • , Yan-ru Cui
    •  & Jia Liu

  • Article
    | Open Access

    Ultrasound can be used to non-invasively control neuronal functions. Here the authors report the use of human Transient receptor potential ankyrin 1 (hsTRPA1) to achieve ultrasound sensitivity in mammalian cells, and show that it can be used to manipulate neurons in the mammalian brain.

    • Marc Duque
    • , Corinne A. Lee-Kubli
    •  & Sreekanth H. Chalasani

  • Article
    | Open Access

    The causal relationship between DNA demethylation and gene expression regulation has not yet been fully resolved. Here the authors develop a nuclease-dead Cas9 (dCas9) and gRNA site-specific targeting approach to physically block DNA methylation at specific promoters to cause DNA demethylation in cells and tackle this question.

    • Daniel M. Sapozhnikov
    •  & Moshe Szyf

  • Article
    | Open Access

    Mobile element insertions (MEIs) are a source of repetitive genetic variation and can lead to genetic disorders. Here the authors use Cas9-targeted nanopore sequencing to efficiently saturate enrichment for known and non-reference MEIs.

    • Torrin L. McDonald
    • , Weichen Zhou
    •  & Alan P. Boyle

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