Featured
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Article
| Open Access
Engineering self-deliverable ribonucleoproteins for genome editing in the brain
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
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Article
| Open Access
Phage-assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference
Existing TadA-derived CBEs exhibit residual A•T-to-G•C editing activity and suffer from lower activity at several sequence contexts and with non-SpCas9 targeting domains. Here, the authors use phage-assisted evolution to evolve CBE6 variants that address these limitations.
- Emily Zhang
- , Monica E. Neugebauer
- & David R. Liu
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Article
| Open Access
CRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus
Culex mosquitoes are carriers of major diseases like West Nile virus and are a public health concern. Here the authors present a CRISPR-Cas9 gene drive as a control technology in the Culex quinquefasciatus mosquito species.
- Tim Harvey-Samuel
- , Xuechun Feng
- & Valentino M. Gantz
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Article
| Open Access
Developing mitochondrial base editors with diverse context compatibility and high fidelity via saturated spacer library
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
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Article
| Open Access
A split and inducible adenine base editor for precise in vivo base editing
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
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Article
| Open Access
Whole genomic analysis reveals atypical non-homologous off-target large structural variants induced by CRISPR-Cas9-mediated genome editing
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
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Article
| Open Access
HMGN1 enhances CRISPR-directed dual-function A-to-G and C-to-G base editing
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
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Article
| Open Access
A versatile, high-efficiency platform for CRISPR-based gene activation
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
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Article
| Open Access
Modeling CRISPR-Cas13d on-target and off-target effects using machine learning approaches
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
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Article
| Open Access
TadA orthologs enable both cytosine and adenine editing of base editors
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
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Article
| Open Access
TadA reprogramming to generate potent miniature base editors with high precision
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
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Article
| Open Access
Development of a versatile nuclease prime editor with upgraded precision
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
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Review Article
| Open Access
Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing
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
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Article
| Open Access
TAPE-seq is a cell-based method for predicting genome-wide off-target effects of prime editor
Methods to predict genome-wide off-target activities of prime editors (PEs) are currently lacking. Here the authors report a cell-based assay, TAgmentation of Prime Editor sequencing (TAPE-seq), that provides genome-wide off-target candidates for PEs.
- Jeonghun Kwon
- , Minyoung Kim
- & Jungjoon K. Lee
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Article
| Open Access
Achieving single nucleotide sensitivity in direct hybridization genome imaging
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
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Article
| Open Access
Automated high-throughput genome editing platform with an AI learning in situ prediction model
A large number of cell disease models with pathogenic SNVs are needed. Here the authors report an automated high-throughput platform to perform the genome editing process from gRNA design to the analysis of the editing results; they characterise in situ base editing outcomes.
- Siwei Li
- , Jingjing An
- & Meng Wang
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Article
| Open Access
A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation
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
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Article
| Open Access
SuperFi-Cas9 exhibits remarkable fidelity but severely reduced activity yet works effectively with ABE8e
Increased-fidelity SpCas9 variants have been developed, but often show proportionally reduced activity. Here the authors characterise the on-target activity and off-target propensity of SuperFi-Cas9 in mammalian cells and also see strongly reduced activity but with high fidelity features.
- Péter István Kulcsár
- , András Tálas
- & Ervin Welker
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Article
| Open Access
A comprehensive Bioconductor ecosystem for the design of CRISPR guide RNAs across nucleases and technologies
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
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Article
| Open Access
Accounting for small variations in the tracrRNA sequence improves sgRNA activity predictions for CRISPR screening
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
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Article
| Open Access
Recursive Editing improves homology-directed repair through retargeting of undesired outcomes
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
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Article
| Open Access
Enhancement of prime editing via xrRNA motif-joined pegRNA
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
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Article
| Open Access
Phage peptides mediate precision base editing with focused targeting window
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
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Article
| Open Access
Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9
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
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Article
| Open Access
Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain
While prime editing is a promising technology, PE2 systems often have low efficiency. Here the authors fuse a Rad51 DNA-binding domain to create hyPE2 with improved editing efficiency.
- Myungjae Song
- , Jung Min Lim
- & Hyongbum Henry Kim
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Article
| Open Access
Small molecule inhibition of ATM kinase increases CRISPR-Cas9 1-bp insertion frequency
The mutational outcome of CRISPR-Cas9 editing can be both predictable and targeted. Here the authors show that ATM inhibitor KU-60019 increases 1 bp insertions at the targeted locus.
- Heysol C. Bermudez-Cabrera
- , Sannie Culbertson
- & Richard I. Sherwood
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Article
| Open Access
Enhancing CRISPR-Cas9 gRNA efficiency prediction by data integration and deep learning
High-quality gRNA activity data is needed for accurate on-target efficiency predictions. Here the authors generate activity data for over 10,000 gRNA and build a deep learning model CRISPRon for improved performance predictions.
- Xi Xiang
- , Giulia I. Corsi
- & Yonglun Luo
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Article
| Open Access
Genetic fate-mapping reveals surface accumulation but not deep organ invasion of pleural and peritoneal cavity macrophages following injury
Body cavity macrophages reside on the serous surfaces of organs and believed to participate in organ repair following injury. Here the authors show with a fate-mapping reporter system that these cells, although accumulate at the surfaces of injured liver or lung, don’t penetrate deeply into the tissue.
- Hengwei Jin
- , Kuo Liu
- & Bin Zhou
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Article
| Open Access
A Cas-embedding strategy for minimizing off-target effects of DNA base editors
DNA base editors can display off-target effects on DNA and RNA. Here the authors embed the base editing enzymes in the middle of nCas9 to reduce these without impacting on-target editing.
- Yajing Liu
- , Changyang Zhou
- & Tian Chi
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Article
| Open Access
MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits
Cas9 fused to DNA damage repair proteins can improve rates of gene knock-in but the chimeric protein is often large. Here the authors fuse Cas9 to a minimal Brex27 motif from BRCA2 consisting of thirty-six amino acids to enhance the efficacy and safety of gene editing.
- Linyuan Ma
- , Jinxue Ruan
- & Jie Xu
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Article
| Open Access
Docking sites inside Cas9 for adenine base editing diversification and RNA off-target elimination
Current SpCas9 adenine base editors that minimise RNA off-target activities have constrained editing windows. Here the authors use domain insertion of TadA into Cas9 to narrow, expand or shift the editing window with RNA off-target minimization simultaneously
- Shuo Li
- , Bo Yuan
- & Tian-Lin Cheng
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Article
| Open Access
Synthesizing AND gate minigene circuits based on CRISPReader for identification of bladder cancer cells
Synthetic biology logic gates can be used to distinguish healthy cells from cancer cells. Here the authors design minigene circuits that show more robust identification of cancer cells compared to traditional genetic circuits.
- Yuchen Liu
- , Weiren Huang
- & Zhiming Cai
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Article
| Open Access
Prime editing for functional repair in patient-derived disease models
Prime editing uses Cas9 nickase fused to a reverse transcriptase to edit genetic information. Here, the authors prime edit primary adult stem cells in 3D organoid cultures to show functional correction of pathogenic mutations without genome-wide off-target effects.
- Imre F. Schene
- , Indi P. Joore
- & Sabine A. Fuchs
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Article
| Open Access
Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection
CRISPR-Cas12a based detection systems can be sensitive down to the picomolar range. Here the authors modify the 3′- and 5′-ends of the crRNA and show this enhances trans-cleavage for improved sensitivity.
- Long T. Nguyen
- , Brianna M. Smith
- & Piyush K. Jain
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Article
| Open Access
Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs
Numerous strategies exist to limit the off-target activity of CRISPR-Cas9 nucleases. Here the authors co-administer truncated gRNAs that block both Cas9 and high-fidelity Cas9 variants from cleaving at off-target sites.
- John C. Rose
- , Nicholas A. Popp
- & Douglas M. Fowler
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Article
| Open Access
Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells
Previous gene editing in haematopoietic stem cells (HSCs) has focussed on a heterogeneous CD34+ population. Here, the authors demonstrate high efficiency CRISPR/Cas9-based editing of purified long-term HSCs using non-homologous end joining and homology-directed repair, by directing isoform-specific expression of GATA1.
- Elvin Wagenblast
- , Maria Azkanaz
- & Eric R. Lechman
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Article
| Open Access
Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning
Application of highly specific Cas9 variants can be restricted by the design of the guide RNA. Here the authors present DeepHF, a gRNA activity prediction tool built from genome-scale screens of 50,000 guides covering 20,000 genes.
- Daqi Wang
- , Chengdong Zhang
- & Yongming Wang
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Article
| Open Access
Expanding C–T base editing toolkit with diversified cytidine deaminases
Cytosine base editors are limited by editing scope and potential off-target effects. Here the authors screen diversified lamprey cytidine deaminases along with different protein fusion architectures and present base editors with improved fidelity.
- Tian-Lin Cheng
- , Shuo Li
- & Zilong Qiu
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Article
| Open Access
Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks
Here the authors fuse hRad51 and variants thereof to Cas9 nickase to facilitate homology-directed repair without generating double strand breaks, minimizing indel formation and off-target editing. This tool represents progress towards the goal of performing HDR without an excess of undesired side products.
- Holly A. Rees
- , Wei-Hsi Yeh
- & David R. Liu
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Article
| Open Access
High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases
The development of site-specific recombinases as genome editing tools is limited by the difficulty of altering their DNA sequence specificity. Here the authors present Rec-seq, a method for identifying specificity determinants and off-target substrates of recombinases in an unbiased manner.
- Jeffrey L. Bessen
- , Lena K. Afeyan
- & David R. Liu
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Article
| Open Access
Multiplexed Cas9 targeting reveals genomic location effects and gRNA-based staggered breaks influencing mutation efficiency
Designing effective genome engineering strategies requires an understanding of the impact that genomic locus has on CRISPR-Cas9 activity. Here the authors use TRIP integrations to profile editing outcomes genome-wide and observe that gRNA sequence influences the structure of the double strand break.
- Santiago Gisler
- , Joana P. Gonçalves
- & Maarten van Lohuizen
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Article
| Open Access
Diversifying the structure of zinc finger nucleases for high-precision genome editing
Genome editing often requires cleavage within a narrow sequence window. Here the authors develop an expanded set of zinc finger nuclease architectures that increase the available configurations by a factor of 64 and can target almost every base at loci of therapeutic significance.
- David E. Paschon
- , Stephanie Lussier
- & Edward J. Rebar
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Article
| Open Access
Engineering of high-precision base editors for site-specific single nucleotide replacement
Base editors can target multiple bases within a window around the target site, reducing their specificity. Here the authors engineer the connection between the deaminase and Cas domain to narrow the window of activity.
- Junjie Tan
- , Fei Zhang
- & Ralph Bock
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Article
| Open Access
Highly efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a uridinylate-rich 3′-overhang
Cpf1 is a promising alternative to Cas9 though indel generation efficiency is target dependent. Here the authors show that the addition of a polyU 3′ overhang can improve the efficiency of low efficiency guide RNAs.
- Su Bin Moon
- , Jeong Mi Lee
- & Yong-Sam Kim
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Article
| Open Access
In vivo base editing of post-mitotic sensory cells
Base editing allows the precise introduction of point mutations into cellular DNA without requiring double-stranded DNA breaks or homology-directed repair, which is inefficient in postmitotic cells. Here the authors demonstrate in vivo base editing of post-mitotic somatic cells in the postnatal mouse inner ear with physiological outcomes.
- Wei-Hsi Yeh
- , Hao Chiang
- & David R. Liu
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Article
| Open Access
Incomplete prophage tolerance by type III-A CRISPR-Cas systems reduces the fitness of lysogenic hosts
CRISPR-Cas systems, such as type III-A CRISPR-Cas, provide an immune mechanism for prokaryotic hosts to resist parasites, including phages. Here, the authors show that maintenance of conditionally tolerant type III-A systems can affect the fitness of Staphylococcus aureus lysogens.
- Gregory W. Goldberg
- , Elizabeth A. McMillan
- & Luciano A. Marraffini
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Article
| Open Access
Engineering cell signaling using tunable CRISPR–Cpf1-based transcription factors
Cpf1 has been repurposed as a transcriptional repressor in bacteria and plants. Here, the authors construct activators and repressors in human cells using Cpf1 coupled to riboswitches and GPCRs.
- Yuchen Liu
- , Jinghong Han
- & Guohui Nie
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Article
| Open Access
Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing
Using CRISPR to write specific genetic sequences can sometimes be difficult due to the preference of mammalian cells to repair breaks using NHEJ. Here the authors form nanoparticles to localize the template sequence to the nuclease, shifting repair in favor of HDR.
- Jared Carlson-Stevermer
- , Amr A. Abdeen
- & Krishanu Saha
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Article
| Open Access
In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni
The amount of genetic material that can be packaged in AAV vectors used for genome editing is limited. Here the authors show that the smallest known Cas9 orthologue, cjCas9, can be packaged in a single AAV vector along with sgRNA and a marker gene, and demonstrate efficient gene editing in mice.
- Eunji Kim
- , Taeyoung Koo
- & Jin-Soo Kim
In vivo genome editing via CRISPR/Cas9-mediated homology-independent targeted integration for Bietti crystalline corneoretinal dystrophy treatment