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Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope
Here the authors report that a thermophilic archaeal TnpB enables efficient gene editing in the natural host: they see that the TnpB has different TAM requirements for eliciting cell death and for facilitating gene editing. They show that TnpB can be harnessed for flexible single-nucleotide editing with templated repair.
- Xu Feng
- , Ruyi Xu
- & Qunxin She
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Article
| Open Access
CoCas9 is a compact nuclease from the human microbiome for efficient and precise genome editing
Cas9 nucleases hold clinical significance for genome editing therapies. Here the authors characterize CoCas9, a compact, efficient and precise Cas9 from the human microbiome, and show that delivery via AAV vectors enables efficient editing in the mouse retina, expanding the genome editing toolbox.
- Eleonora Pedrazzoli
- , Michele Demozzi
- & Anna Cereseto
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Article
| Open Access
RNA targeting and cleavage by the type III-Dv CRISPR effector complex
Here, Schwartz, Bravo, and Ahsan et al. show how multi-subunit fusion proteins are arranged around a crRNA in a type III CRISPR-Cas effector to cleave target RNA. Structures and molecular dynamics of this complex show three distinct active sites that can be used for programmable RNA cleavage.
- Evan A. Schwartz
- , Jack P. K. Bravo
- & David W. Taylor
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| Open Access
Insights into the inhibition of protospacer integration via direct interaction between Cas2 and AcrVA5
Here, the authors characterize an anti-CRISPR protein that prevents protospacer integration by Cas1-Cas2, providing structural insights that may benefit CRISPR-Cas systems research.
- Mingfang Bi
- , Wenjing Su
- & Xiaobing Mo
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Article
| Open Access
Ultrasensitive single-step CRISPR detection of monkeypox virus in minutes with a vest-pocket diagnostic device
The recent monkeypox outbreak highlighted the need for rapid and accurate diagnosis of this disease. Here, authors develop an ultrasensitive and streamlined CRISPR assay using miniaturized device, which can detect monkeypox virus in rash fluid swab, oral swab, saliva, and urine within 15 minutes.
- Yunxiang Wang
- , Hong Chen
- & Shengqi Wang
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Article
| Open Access
Splice modulators target PMS1 to reduce somatic expansion of the Huntington’s disease-associated CAG repeat
Somatic expansion of a CAG repeat in HTT drives onset of Huntington’s disease. Using a human cell line model and splice modulators, here the authors show that PMS1 is an enhancer of CAG repeat expansion, making it a target for therapeutic intervention.
- Zachariah L. McLean
- , Dadi Gao
- & James F. Gusella
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Article
| Open Access
The CRISPR-Cas13a Gemini System for noncontiguous target RNA activation
CRISPR-Cas13a based methods currently use contiguous target RNA activation, which only enables single-target detection or editing. Here the authors propose a noncontiguous target RNA activation approach which can provide rapid, simultaneous and sensitive detection of two RNAs in a single readout, as well as parallel dual transgene knockdown.
- Hongrui Zhao
- , Yan Sheng
- & Jiaming Hu
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Article
| Open Access
Developmental progression of DNA double-strand break repair deciphered by a single-allele resolution mutation classifier
DNA double-strand breaks (DSBs) are repaired by a hierarchically regulated network of pathways. Here, authors develop ICP for deciphering somatic DSB repair patterns in multicellular organisms and discover developmental regulation in flies and mosquitoes, enabling tracking of mutant alleles and interhomolog copying of gene cassettes.
- Zhiqian Li
- , Lang You
- & Ethan Bier
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Article
| Open Access
CRISPR-powered quantitative keyword search engine in DNA data storage
Targeting the files containing content-of-interest is a challenge in DNA data storage. Here, the authors develop a CRISPR-powered search engine to quantitatively identify the keyword in files stored in DNA.
- Jiongyu Zhang
- , Chengyu Hou
- & Changchun Liu
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Article
| Open Access
Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing
The ability to edit large stretches of mRNA transcripts remains a significant challenge. Here, the authors demonstrate that CRISPR-Cas13 systems can be repurposed to assist trans-splicing of exogenous RNA fragments into an endogenous pre-mRNA transcript, a method termed CRISPR Assisted mRNA Fragment Trans-splicing (CRAFT).
- David N. Fiflis
- , Nicolas A. Rey
- & Aravind Asokan
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Article
| Open Access
Identifying regulators of aberrant stem cell and differentiation activity in colorectal cancer using a dual endogenous reporter system
Aberrant stem cell-like activity and impaired differentiation are central to the development of colorectal cancer. Here, authors develop a dual endogenous reporter system to identify functional regulators of aberrant stem cell and differentiation programs, showing that SMARCB1 restricts differentiation, and nominating other regulators with therapeutic potential.
- Sandor Spisak
- , David Chen
- & Nilay S. Sethi
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Article
| Open Access
dCas13-mediated translational repression for accurate gene silencing in mammalian cells
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
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Article
| Open Access
Enhancing prime editor activity by directed protein evolution in yeast
Compared to traditional Cas9 nucleases prime editors (PEs) are less active. Here the authors use OrthoRep, a yeast-based platform for directed protein evolution to enhance the editing efficiency of PEs: they identify mutations that have a positive effect on kinetics and use this knowledge to generate an efficient in vivo PE.
- Yanik Weber
- , Desirée Böck
- & Gerald Schwank
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Article
| Open Access
Cas9-assisted biological containment of a genetically engineered human commensal bacterium and genetic elements
Engineered biosensing bacteria can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. Here the authors present a robust biocontainment assisted by Cas9 and an engineered gene expression control combined in a genetically engineered human commensal bacterium that successfully functioned in a mouse intestinal tract as well as cell culture condition.
- Naoki Hayashi
- , Yong Lai
- & Timothy K. Lu
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Article
| Open Access
Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis and transforms DNA/RNA sensing
The authors find that small circular DNA nanostructures which partially match gRNA sequences only minimally activate Cas12a. They report AutoCAR (Autocatalytic Cas12a Circular DNA Amplification Reaction) which allows a single nucleic acid target to activate multiple ribonucleoproteins, and increases reporter cleavage rates.
- Fei Deng
- , Yi Li
- & Ewa M. Goldys
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Article
| Open Access
An anti-CRISPR that represses its own transcription while blocking Cas9-target DNA binding
Anti-CRISPR proteins can help to evade bacterial immunity. Here, the authors elucidate the mechanism of AcrIIA15, which inhibits CRISPR-Cas9 system and also functions as a transcriptional repressor of itself as a fusion protein.
- Xieshuting Deng
- , Wei Sun
- & Yanli Wang
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Article
| Open Access
Development of pathophysiologically relevant models of sickle cell disease and β-thalassemia for therapeutic studies
Sickle cell disease (SCD) and β-thalassemia (BT) are globally prevalent inherited blood disorders but, despite extensive research, no ex vivo system exists for SCD and BT. Here, the authors generate pathophysiologically relevant erythroid progenitor models of SCD and BT.
- Pragya Gupta
- , Sangam Giri Goswami
- & Sivaprakash Ramalingam
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Article
| Open Access
Cbp1 and Cren7 form chromatin-like structures that ensure efficient transcription of long CRISPR arrays
CRISPR arrays form the physical memory of prokaryotic adaptive immune systems by incorporating viral DNA sequences as spacers. Here, Blombach et al. show that transcription factor Cbp1 recruits chromatin protein Cren7 at CRISPR arrays, forming ‘chimeric’ chromatin-like structures that regulate expression of long CRISPR arrays in Sulfolobales archaea.
- Fabian Blombach
- , Michal Sýkora
- & Finn Werner
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Article
| Open Access
Orthogonal inducible control of Cas13 circuits enables programmable RNA regulation in mammalian cells
The lack of control over Cas13 activity has limited its utility. Here the authors report Control of RNA with Inducible SpliT CAs13 Orthologs and Exogenous Ligands (CRISTAL), controlled by orthogonal split inducible Cas13 effectors that can be turned ON or OFF, providing precise temporal control.
- Yage Ding
- , Cristina Tous
- & Wilson W. Wong
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| Open Access
Domain-inlaid Nme2Cas9 adenine base editors with improved activity and targeting scope
Nme2Cas9 has been well established as a genome editing platform. Here the authors engineer Nme2Cas9 to further increase the activity and targeting scope of compact Nme2Cas9 base editors and validate domain-inlaid Nme2-ABEs for single-AAV delivery in vivo.
- Nathan Bamidele
- , Han Zhang
- & Erik J. Sontheimer
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Article
| Open Access
Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells
The most recent class of base editors utilize DddAtox, a deaminase domain that can act upon double-stranded DNA. Here the authors target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers; they also identify a variety of DddAtox orthologues.
- Friedrich Fauser
- , Bhakti N. Kadam
- & Jeffrey C. Miller
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Article
| Open Access
Logical design of synthetic cis-regulatory DNA for genetic tracing of cell identities and state changes
Descriptive data in biomedical research are expanding rapidly, but functional validation methods lag behind. Here, authors present Logical Synthetic cis-regulatory DNA, a framework to design reporters that mark cellular states and pathways, showcasing its applicability to complex phenotypic states.
- Carlos Company
- , Matthias Jürgen Schmitt
- & Gaetano Gargiulo
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| Open Access
Anti-CRISPR Anopheles mosquitoes inhibit gene drive spread under challenging behavioural conditions in large cages
CRISPR-based gene drives have the potential to spread within populations and are considered as promising vector control tools. Here the authors show an anti-drive mosquito strain that prevents the spread and collapse of a population suppression gene drive in laboratory Anopheles mosquito large cage trials in complex ecological and behavioral conditions.
- Rocco D’Amato
- , Chrysanthi Taxiarchi
- & Ruth Müller
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Article
| Open Access
BacPE: a versatile prime-editing platform in bacteria by inhibiting DNA exonucleases
Prime editing in bacteria is currently inefficient. Here the authors report BacPE, a versatile prime editing platform in Escherichia coli that works by inhibiting 3′→5′ DNA exonucleases, highlighting the intrinsic genetic factors that are adverse to efficient prime editing.
- Hongyuan Zhang
- , Jiacheng Ma
- & Quanjiang Ji
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Article
| Open Access
A multiplexed, confinable CRISPR/Cas9 gene drive can propagate in caged Aedes aegypti populations
Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Here the authors find that a CRISPR/Cas9 based split gene drive in Aedes aegypti could successfully bias inheritance up to 89% over successive generations in a multi-cage trial with further deep sequencing suggesting that the multiplexing design could mitigate resistance allele formation.
- Michelle A. E. Anderson
- , Estela Gonzalez
- & Luke Alphey
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Article
| Open Access
Programmable RNA base editing with photoactivatable CRISPR-Cas13
Cas13 systems suffer from a lack of spatiotemporal control. Here the authors report paCas13, a light-inducible Cas13 system created by fusing Magnet with fragment pairs; they also report padCas13, a light-inducible base-editing system by fusing ADAR2 to catalytically inactive paCas13 fragments.
- Jeonghye Yu
- , Jongpil Shin
- & Won Do Heo
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Article
| Open Access
Massively parallel profiling of RNA-targeting CRISPR-Cas13d
Systematic understanding of CRISPR enzyme RNA binding specificity and cleavage is lacking. Here the authors report RNA chip-hybridised association-mapping platform (RNA-CHAMP), a workflow that repurposes next generation DNA sequencing chips to measure the binding affinity for RNA targets.
- Hung-Che Kuo
- , Joshua Prupes
- & Ilya J. Finkelstein
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Article
| Open Access
The low-density lipoprotein receptor promotes infection of multiple encephalitic alphaviruses
Ma et al. identify LDLR as an entry receptor for Eastern equine encephalitis virus (EEEV) and other alphaviruses. Soluble decoy proteins with multiple LA domain 3 repeats of LDLR inhibit EEEV infection in cell culture and mice.
- Hongming Ma
- , Lucas J. Adams
- & Michael S. Diamond
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Article
| Open Access
CRISPR-Cas-based identification of a sialylated human milk oligosaccharides utilization cluster in the infant gut commensal Bacteroides dorei
Human milk oligosaccharides (HMOs) utilization by Bacteroides species remains poorly understood. Here, the authors describe a single specific gene cluster responsible for sialylated HMOs utilization in a B. dorei natural isolate and prove its functionality in vivo using CRISPR-Cas12a.
- Sivan Kijner
- , Dena Ennis
- & Moran Yassour
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Article
| Open Access
Transient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells
Here the authors demonstrate that the frequency of HDR in human hematopoietic stem and progenitor cells is increased by the delivery of an inhibitor of 53BP1 as a recombinant peptide. This approach is applicable for a variety of therapeutically relevant loci in HSPCs as well in other primary human cell types.
- Ron Baik
- , M. Kyle Cromer
- & Matthew H. Porteus
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Article
| Open Access
Dissecting the genetic landscape of GPCR signaling through phenotypic profiling in C. elegans
To overcome challenges posted by vast number of GPCR genes and redundancy, the authors disrupted nearly all GPCR-encoding genes in C. elegans, enabling effective examination of GPCR signaling and offering a valuable resource for the research community.
- Longjun Pu
- , Jing Wang
- & Changchun Chen
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Article
| Open Access
Shuttle peptide delivers base editor RNPs to rhesus monkey airway epithelial cells in vivo
Gene editing strategies for cystic fibrosis are challenging. Here the authors improve on their previously reported shuttle peptide noncovalently combined with Cas ribonucleoprotein (RNP), and derive the S315 peptide for delivery: they show base editing in the respiratory tract of the rhesus macaques.
- Katarina Kulhankova
- , Soumba Traore
- & Paul B. McCray Jr.
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Article
| Open Access
In vivo imaging of mitochondrial DNA mutations using an integrated nano Cas12a sensor
Mutations in mitochondrial DNA (mtDNA) play critical roles in human diseases. Here, the authors describe an integrated Cas12a sensor for sensing mtDNA mutations in vivo, showing potential for diagnostic and therapeutic purposes.
- Yanan Li
- , Yonghua Wu
- & Kaixiang Zhang
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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
Genome-wide CRISPR off-target prediction and optimization using RNA-DNA interaction fingerprints
Analysis of CRISPR-Cas off-targets is important. Here the authors incorporate molecular dynamics simulations in the computational analysis of CRISPR editing and report the CRISOT tool suite and apply this to genome-wide CRISPR off-target prediction and sgRNA optimisation.
- Qinchang Chen
- , Guohui Chuai
- & Qi Liu
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Article
| Open Access
Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA
New strategies are being developed to simplify CRISPR-based nucleic acid detection. By investigating the competitive reaction between a full-sized crRNA and split crRNA for CRISPR-Cas12a, the authors develop an asymmetric CRISPR assay for amplification-free, cascade signal amplification detection of nucleic acids.
- Jeong Moon
- & Changchun Liu
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Article
| Open Access
Lung SORT LNPs enable precise homology-directed repair mediated CRISPR/Cas genome correction in cystic fibrosis models
Roughly 10% of Cystic Fibrosis (CF) patients still have no effective medicine to take. Lung Selective Organ Targeting (SORT) Lipid Nanoparticles can efficiently deliver Cas9 mRNA, sgRNA, and donor ssDNA templates for precise homology-directed repair-mediated gene correction in ex vivo and in vivo CF models.
- Tuo Wei
- , Yehui Sun
- & Daniel J. Siegwart
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Article
| Open Access
Specific heterozygous variants in MGP lead to endoplasmic reticulum stress and cause spondyloepiphyseal dysplasia
Biallelic loss-of-function variants in the gene encoding Matrix Gla Protein (MGP) are known to cause a recessive disorder called Keutel syndrome. Here, the authors report that heterozygous missense variants affecting one particular cysteine residue of MGP can cause a clinically distinct, dominant disorder, likely via impaired signal peptide processing leading to cellular stress and apoptosis.
- Ophélie Gourgas
- , Gabrielle Lemire
- & Monzur Murshed
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Article
| Open Access
Hidden prevalence of deletion-inversion bi-alleles in CRISPR-mediated deletions of tandemly arrayed genes in plants
The multiplex CRISPR system is the tool of choice for creating targeted tandemly arrayed genes (TAGs) deletions in plants. Here, the authors show that up to 80% of CRISPR-mediated TAG knockout alleles in Arabidopsis and rice are deletion-inversion bi-alleles, an unwanted products of targeted TAG deletions.
- Jiuer Liu
- , Feng-Zhu Wang
- & Jian-Feng Li
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Article
| Open Access
Sonogenetic control of multiplexed genome regulation and base editing
Exogenous control of genes in vivo is important. Here the authors report a system that can be inducibly activated through thermal energy produced by ultrasound absorption and use this to control induction of gene activation and base editing: they apply this in cell lines and in a mouse model.
- Pei Liu
- , Josquin Foiret
- & Lei S. Qi
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Article
| Open Access
mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy
Large genes require dual adeno-associated viral (AAV) vectors for in vivo delivery/expression, but current methods have limitations. Here the authors develop and functionally evaluate REVeRT, an efficient and flexible dual AAV vector technology based on reconstitution via mRNA trans-splicing.
- Lisa Maria Riedmayr
- , Klara Sonnie Hinrichsmeyer
- & Elvir Becirovic
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Article
| Open Access
Mitigating a TDP-43 proteinopathy by targeting ataxin-2 using RNA-targeting CRISPR effector proteins
TDP43 proteinopathies are a devastating group of neurodegenerative disorders. Here the authors show that RNA-targeting CRISPR effector proteins can be used to mitigate TDP-43 pathology when targeting ataxin-2, a modifier of TDP-43-associated toxicity, and apply this to a mouse model.
- M. Alejandra Zeballos C.
- , Hayden J. Moore
- & Thomas Gaj
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Article
| Open Access
Digital data storage on DNA tape using CRISPR base editors
DNA is an alternative to data storage materials for its durability, density, and energetics. Here the authors demonstrate the storage of digital information on DNA molecules using base-editing.
- Afsaneh Sadremomtaz
- , Robert F. Glass
- & Reza Zadegan
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Article
| Open Access
Lipid nanoparticles with PEG-variant surface modifications mediate genome editing in the mouse retina
There is a need for development of efficient delivery vehicles for the treatment of inherited retinal degeneration with gene therapy. Here, Gautam et al., show that surface modifications of lipid nanoparticles with PEG variants alters their cellular tropism allowing gene editing in diverse retinal cell types in mice.
- Milan Gautam
- , Antony Jozic
- & Gaurav Sahay
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Article
| Open Access
Next-generation CRISPR gene-drive systems using Cas12a nuclease
One method for reducing the impact of vector-borne diseases is through the use of CRISPR-based gene drives, which manipulate insect populations due to their ability to rapidly propagate desired genetic traits into a target population. Here the authors describe a Cas12a gene drive system whose activity can be finetuned in a temperature-dependent manner.
- Sara Sanz Juste
- , Emily M. Okamoto
- & Víctor López Del Amo
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Article
| Open Access
A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering
Thermophilic genetic manipulation tools are limited. Here the authors report a thermophilic type I-B CRISPR-Cas system and show it displays efficient transcriptional repression or DNA cleavage activity: they develop a tool for genome editing and transcriptional repression in both thermophile and mesophile hosts.
- Zhiheng Yang
- , Zilong Li
- & Weishan Wang
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Article
| Open Access
PAM-flexible genome editing with an engineered chimeric Cas9
CRISPR enzymes require a defined protospacer adjacent motif (PAM) which can be limiting for editing applications. Here the authors recombine the PAM-interacting domain of SpRY with the N-terminus of Sc + + to generate a chimeric enzyme with highly flexible PAM preference: SpRYc.
- Lin Zhao
- , Sabrina R. T. Koseki
- & Pranam Chatterjee
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Article
| Open Access
Fast, multiplexable and efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9
Methods for somatic gene perturbation would offer advantages for screening multiple muscle gene candidates. Here the authors couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery and report a system for effective somatic gene deletions in mice.
- Marco Thürkauf
- , Shuo Lin
- & Markus A. Rüegg
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Article
| Open Access
Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system
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
Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform