4.7 Review

Construct design for CRISPR/Cas-based genome editing in plants

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Article Biotechnology & Applied Microbiology

CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells

Ibrahim C. Kurt et al.

Summary: This study presents two base editor architectures that can efficiently induce targeted C-to-G base transversions, with reduced levels of unwanted mutations. These new base editors hold promise for optimizing C-to-G base editors for both research and therapeutic applications.

NATURE BIOTECHNOLOGY (2021)

Article Biotechnology & Applied Microbiology

Glycosylase base editors enable C-to-A and C-to-G base changes

Dongdong Zhao et al.

Summary: The study introduces novel glycosylase base editors (GBEs) that can induce C-to-A and C-to-G transversions in Escherichia coli and mammalian cells, respectively. The new editors exhibit high editing specificity and efficiency, making them potential tools for targeting G/C disease-causing mutations.

NATURE BIOTECHNOLOGY (2021)

Article Biotechnology & Applied Microbiology

Predicting the efficiency of prime editing guide RNAs in human cells

Hui Kwon Kim et al.

Summary: This study identified factors affecting PE2 efficiency through high-throughput evaluation and developed three computational models to predict pegRNA efficiency, which can be applied to edits of various types and positions. Spearman's correlations between 0.47 and 0.81 were found when testing the accuracy of the predictions using independent test data sets.

NATURE BIOTECHNOLOGY (2021)

Letter Biotechnology & Applied Microbiology

Precise genome modification in tomato using an improved prime editing system

Yuming Lu et al.

PLANT BIOTECHNOLOGY JOURNAL (2021)

Article Engineering, Biomedical

A web tool for the design of prime-editing guide RNAs

Ryan D. Chow et al.

Summary: Prime editing allows diverse genomic alterations to be made at target sites without double-strand breaks or donor templates. However, designing pegRNAs can be complex and time-consuming, as they require customization for each edit. The web tool pegFinder facilitates the rapid design of pegRNAs for downstream experimentation, with the incorporation of sgRNA predictions and nomination of secondary nicking sgRNAs for improved editing efficiency.

NATURE BIOMEDICAL ENGINEERING (2021)

Article Biochemistry & Molecular Biology

Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants

Juan Li et al.

Summary: This study developed a range of SpCas9 variants for genome editing in plants, as well as base editors capable of generating conversions at non-canonical PAM sites.

MOLECULAR PLANT (2021)

Article Biochemical Research Methods

PnB Designer: a web application to design prime and base editor guide RNAs for animals and plants

Sebastian M. Siegner et al.

Summary: PnB Designer is a web-based application that simplifies the design of pegRNAs for PEs and guide RNAs for BEs, allowing for easy design of targeting guide RNAs for single or multiple targets on variant or reference genomes. It can also be used to design guide RNAs to install or revert SNVs, scanning the genome with different ABE PAM variants and returning the best BE to use.

BMC BIOINFORMATICS (2021)

Review Biochemistry & Molecular Biology

Genome engineering for crop improvement and future agriculture

Caixia Gao

Summary: Genome editing has the potential to revolutionize plant breeding and address the challenge of feeding a growing population in the face of climate change. Newly developed techniques and strategies offer significant opportunities for plant improvement and crop production, but challenges must be overcome to realize the full potential of this technology.
Article Biochemistry & Molecular Biology

High-efficiency andmultiplex adenine base editing in plants using new TadA variants

Daqi Yan et al.

Summary: The study investigated the activities of three evolved E. coli adenosine deaminase TadA variants in transgenic rice, revealing that TadA8e outperformed TadA8.17 and TadA8.20. The newly generated variant TadA9, with V82S/Q154R mutations, showed broad compatibility and efficient editing in various CRISPR systems. These novel ABEs have great potential in the development of plant base editors and crop molecular breeding.

MOLECULAR PLANT (2021)

Article Biotechnology & Applied Microbiology

Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a

Teng-Kuei Huang et al.

Summary: Nicotiana tabacum, a non-food herb, shows potential as a bio-factory for producing medicines, vaccines, or valuable metabolites, but improving genetic tools for targeted genome modifications is crucial. By replacing SpCas9 with other CRISPR/Cas-based nucleases like SaCas9 and ttLbCas12a, targeting the tobacco SuRB gene showed improved efficiencies, suggesting ttLbCas12a as a superior alternative for gene editing in tobacco and other crops.

PLANT BIOTECHNOLOGY JOURNAL (2021)

Article Plant Sciences

Highly efficient multiplex editing: one-shot generation of 8x Nicotiana benthamiana and 12x Arabidopsis mutants

Johannes Stuttmann et al.

Summary: Genome editing using RNA-guided nucleases like SpCas9 has been successful in various plant species. A toolkit based on an optimized zCas9i gene allows targeting multiple independent loci simultaneously. Efficient multiplexing was achieved in Nicotiana benthamiana and Arabidopsis thaliana, with the latter showing lower efficiency due to Cas9 availability limitations. This study opens new possibilities for generating complex genotypes through multiplexing.

PLANT JOURNAL (2021)

Article Biochemical Research Methods

Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering

Teng-Kuei Huang et al.

Summary: In recent years, significant progress has been made in the field of plant genome editing using CRISPR/Cas technology. This includes the application of both natural and engineered Cas proteins, as well as the development of various gene editing techniques in plants.

TRANSGENIC RESEARCH (2021)

Article Multidisciplinary Sciences

PrimeDesign software for rapid and simplified design of prime editing guide RNAs

Jonathan Y. Hsu et al.

Summary: Prime editing guide RNA design is more complex than for standard CRISPR-based nucleases or base editors. Here the authors present PrimeDesign and PrimeVar for the rapid and simplified design of pegRNA and ngRNA combinations. PrimeVar is a comprehensive and searchable database that includes candidate pegRNA and ngRNA combinations for installing or correcting >68,500 pathogenic human genetic variants.

NATURE COMMUNICATIONS (2021)

Article Multidisciplinary Sciences

Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins

Liwei Chen et al.

Summary: A new class of base editors, C:G to G:C Base Editors, have been developed in this study to create single-base genomic transversions in human cells, targeting specific genes related to genetic diseases. These editors predominantly perform C:G to G:C editing with high purity and efficiency, showing therapeutic potential for interrogating and correcting human genetic diseases.

NATURE COMMUNICATIONS (2021)

Review Plant Sciences

CRISPR/dCas-mediated transcriptional and epigenetic regulation in plants

Changtian Pan et al.

Summary: The precise genome editing mediated by the CRISPR/Cas system has revolutionized genome engineering, with the use of dCas variants expanding its applications to genetic and translational research without inducing DNA double-strand breaks. Fusion of effector domains to dCas proteins empowers the CRISPR/dCas system as a multifunctional platform, enabling gene expression regulation, epigenetic regulation, and sequence-specific imaging. This review highlights recent advances in transcriptional activation and repression, as well as epigenetic modifications, and discusses the future directions and broader applications of the CRISPR/dCas systems in plants.

CURRENT OPINION IN PLANT BIOLOGY (2021)

Article Biotechnology & Applied Microbiology

High-efficiency prime editing with optimized, paired pegRNAs in plants

Qiupeng Lin et al.

Summary: Designing prime binding sites with a certain melting temperature and using two pegRNAs can substantially enhance the efficiency of prime editing.

NATURE BIOTECHNOLOGY (2021)

Article Multidisciplinary Sciences

Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems

Yingxiao Zhang et al.

Summary: This study expands the targeting scope of Cas12a in crop genome engineering by screening Cas12a orthologs and developing a highly multiplexable editing system, with Mb2Cas12a showing outstanding editing efficiency and tolerance to low temperature. Additionally, two compact single transcript unit CRISPR-Cas12a interference systems were developed for multi-gene repression in rice and Arabidopsis.

NATURE COMMUNICATIONS (2021)

Article Biotechnology & Applied Microbiology

Improved plant cytosine base editors with high editing activity, purity, and specificity

Qiurong Ren et al.

Summary: In this study, the A3A/Y130F-CBE_V01 was identified as the most efficient C-to-T base editor in plants, showing applications in generating microRNA-resistant mRNA transcripts and herbicide-resistant allele evolution in rice. Additionally, the development and testing of multiple CBE_V02, CBE_V03, and CBE_V04 systems in rice protoplasts demonstrated improved editing activity and purity, with the CBE_V04 systems showing enhanced editing efficiency through focal recruitment of uracil DNA glycosylase inhibitors by engineered sgRNA scaffolds. Whole-genome sequencing revealed varying levels of off-target effects among different CBE systems, with PmCDA1-CBE_V04 and A3A/Y130F-CBE_V04 identified as high-efficiency, high-purity, and highly specific next-generation plant CBEs.

PLANT BIOTECHNOLOGY JOURNAL (2021)

Article Biochemistry & Molecular Biology

PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing

Gue-Ho Hwang et al.

Summary: Prime editing technology allows targeted insertions, deletions, and base conversions, but designing pegRNAs and evaluating high-throughput sequencing data can be complex. Researchers have developed two user-friendly web-based tools, PE-Designer for pegRNA selection and PE-Analyzer for outcome analysis.

NUCLEIC ACIDS RESEARCH (2021)

Article Biochemistry & Molecular Biology

Expanding plant genome-editing scope by an engineered iSpyMacCas9 system that targets A-rich PAM sequences

Simon Sretenovic et al.

Summary: This study presents a new plant genome-editing system based on the hybrid iSpyMacCas9 platform, allowing for targeted mutagenesis and base editing at A-rich PAMs. It fills a significant technology gap in the CRISPR-Cas9 system for editing NAAR PAMs in plants, greatly expanding the targeting scope of CRISPR-Cas9. The vector systems are fully compatible with Gateway cloning and existing single-guide RNA expression systems, making it easy for others to adopt.

PLANT COMMUNICATIONS (2021)

Article Biochemistry & Molecular Biology

High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns

Ramona Gruetzner et al.

Summary: The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In this study, optimizing the efficiency of the Cas9 nuclease in generating mutations in target genes by investigating its nucleotide and/or amino acid sequence features led to the finding that adding introns to the Cas9 coding sequence significantly improved editing efficiency. The intronized Cas9 gene was shown to be effective in generating mutant phenotypes in various plants, highlighting the importance of sequence modifications for enhancing CRISPR/Cas9 genome editing.

PLANT COMMUNICATIONS (2021)

Article Plant Sciences

PAM-less plant genome editing using a CRISPR-SpRY toolbox

Qiurong Ren et al.

Summary: The engineered SpRY Cas9 variant enables efficient gene editing without PAM requirement in rice transgenic lines and Dahurian larch protoplasts, and its derived base editors can edit the rice genome efficiently in a PAM-less fashion too.

NATURE PLANTS (2021)

Article Biotechnology & Applied Microbiology

SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition

Ziyan Xu et al.

Summary: This study thoroughly investigated the nuclease activity and PAM preference of two structurally engineered SpCas9 variants, SpG and SpRY, in transgenic rice, with SpRY showing broad PAM compatibility and expanding the targeting scope of CRISPR-based tools in plant genome engineering.

GENOME BIOLOGY (2021)

Article Biotechnology & Applied Microbiology

Glycosylase base editors enable C-to-A and C-to-G base changes

Dongdong Zhao et al.

Summary: This study presents new glycosylase base editors (GBEs) that can induce C-to-A and C-to-G transversions in bacteria and mammalian cells, respectively. The GBEs can serve as a complement to existing adenine and cytidine base editors (ABE and CBE) and have the potential to target G/C disease-causing mutations.

NATURE BIOTECHNOLOGY (2021)

Review Biotechnology & Applied Microbiology

Base editing in crops: current advances, limitations and future implications

Rukmini Mishra et al.

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CRISPR/dCas9 platforms in plants: strategies and applications beyond genome editing

Mahdi Moradpour et al.

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Expanding the scope of CRISPR/Cas9-mediated genome editing in plants using an xCas9 and Cas9-NG hybrid

Qingfeng Niu et al.

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Engineered Cas9 variant tools expand targeting scope of genome and base editing in rice

Dongchang Zeng et al.

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Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors

Jordan L. Doman et al.

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Plant gene editing through de novo induction of meristems

Michael F. Maher et al.

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Targeted base editing in rice with CRISPR/ScCas9 system

Meixia Wang et al.

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Gene editing in plants: assessing the variables through a simplified case study

Jay Shockey

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Engineering herbicide resistance via prime editing in rice

Haroon Butt et al.

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The application of a heat-inducible CRISPR/Cas12b (C2c1) genome editing system in tetraploid cotton (G. hirsutum) plants

Qiongqiong Wang et al.

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Fan Wu et al.

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Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants

Russell T. Walton et al.

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Nicholas S. McCarty et al.

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Plant Prime Editors Enable Precise Gene Editing in Rice Cells

Xu Tang et al.

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Precise Modifications of Both Exogenous and Endogenous Genes in Rice by Prime Editing

Huiyuan Li et al.

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Versatile Nucleotides Substitution in Plant Using an Improved Prime Editing System

Wen Xu et al.

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Na Zheng et al.

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Base Editing Landscape Extends to Perform Transversion Mutation

Kutubuddin A. Molla et al.

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Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize

Yuan-Yuan Jiang et al.

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Plant gene expression control using genome- and epigenome-editing technologies

Gurbachan S. Miglani et al.

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Sutar Suhas Bharat et al.

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Development of Plant Prime-Editing Systems for Precise Genome Editing

Rongfang Xu et al.

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SpCas9-NG self-targets the sgRNA sequence in plant genome editing

Ruiying Qin et al.

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CRISPR-Cas12b enables efficient plant genome engineering

Meiling Ming et al.

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Genome Engineering in Rice Using Cas9 Variants that Recognize NG PAM Sequences

Kai Hua et al.

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Improving Plant Genome Editing with High-Fidelity xCas9 and Non-canonical PAM-Targeting Cas9-NG

Zhaohui Zhong et al.

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Single and multiple gene knockouts by CRISPR-Cas9 in maize

Nicolas M. Doll et al.

PLANT CELL REPORTS (2019)

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Target-dependent nickase activities of the CRISPR-Cas nucleases Cpf1 and Cas9

Becky Xu Hua Fu et al.

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Plant genome editing using xCas9 with expanded PAM compatibility

Jingying Li et al.

JOURNAL OF GENETICS AND GENOMICS (2019)

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CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing

Kornel Labun et al.

NUCLEIC ACIDS RESEARCH (2019)

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Assessment of Cas12a-mediated gene editing efficiency in plants

Joan Miquel Bernabe-Orts et al.

PLANT BIOTECHNOLOGY JOURNAL (2019)

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Engineered xCas9 and SpCas9-NG variants broaden PAM recognition sites to generate mutations in Arabidopsis plants

Zengxiang Ge et al.

PLANT BIOTECHNOLOGY JOURNAL (2019)

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An adenine base editor with expanded targeting scope using SpCas9-NGv1 in rice

Katsuya Negishi et al.

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Optimizing base editors for improved efficiency and expanded editing scope in rice

Mugui Wang et al.

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Unified energetics analysis unravels SpCas9 cleavage activity for optimal gRNA design

Dong Zhang et al.

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Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants

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Jacob Bradford et al.

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Qiurong Ren et al.

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Search-and-replace genome editing without double-strand breaks or donor DNA

Andrew V. Anzalone et al.

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Patrick Schindele et al.

PLANT BIOTECHNOLOGY JOURNAL (2019)

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High efficacy full allelic CRISPR/Cas9 gene editing in tetraploid potato

Ida Elisabeth Johansen et al.

SCIENTIFIC REPORTS (2019)

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CRISPR/Cas9 searches for a protospacer adjacent motif by lateral diffusion

Viktorija Globyte et al.

EMBO JOURNAL (2019)

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xCas9 expands the scope of genome editing with reduced efficiency in rice

Junjie Wang et al.

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Ruiying Qin et al.

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Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis

Baptiste Castel et al.

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Masaki Endo et al.

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Yingxiao Zhang et al.

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Xixun Hu et al.

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Christopher A. Lino et al.

DRUG DELIVERY (2018)

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Dan Ding et al.

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Plant Genome Editing Using FnCpf1 and LbCpf1 Nucleases at Redefined and Altered PAM Sites

Zhaohui Zhong et al.

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Shaoya Li et al.

MOLECULAR PLANT (2018)

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Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction

Luke W. Koblan et al.

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Adam J. Bogdanove et al.

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CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens

Jean-Paul Concordet et al.

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High-efficiency genome editing using a dmc1 promoter-controlled CRISPR/Cas9 system in maize

Chao Feng et al.

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Genotyping genome-edited mutations in plants using CRISPR ribonucleoprotein complexes

Zhen Liang et al.

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Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention

Qiang Zhang et al.

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CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

Janice S. Chen et al.

SCIENCE (2018)

Review Plant Sciences

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Deepa Jaganathan et al.

FRONTIERS IN PLANT SCIENCE (2018)

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Gue-Ho Hwang et al.

BMC BIOINFORMATICS (2018)

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Efficient generation of stable, heritable gene edits in wheat using CRISPR/Cas9

Rhian M. Howells et al.

BMC PLANT BIOLOGY (2018)

Article Biotechnology & Applied Microbiology

Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A

Yuan Zong et al.

NATURE BIOTECHNOLOGY (2018)

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Base editing: precision chemistry on the genome and transcriptome of living cells

Holly A. Rees et al.

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Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression

Lu Long et al.

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Repurposing CRISPR-Cas12b for mammalian genome engineering

Fei Teng et al.

CELL DISCOVERY (2018)

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Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells

Yuanming Wang et al.

GENOME BIOLOGY (2018)

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Chromatin accessibility and guide sequence secondary structure affect CRISPR-Cas9 gene editing efficiency

Kristopher Torp Jensen et al.

FEBS LETTERS (2017)

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Programmable base editing of A.T to G.C in genomic DNA without DNA cleavage

Nicole M. Gaudelli et al.

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Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion

Zenpei Shimatani et al.

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A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

Tomas Cermak et al.

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Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy

Mikihiro Shibata et al.

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Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence

Ting Zheng et al.

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Potential pitfalls of CRISPR/Cas9-mediated genome editing

Rongxue Peng et al.

FEBS JOURNAL (2016)

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Leveraging Rules of Nonsense-Mediated mRNA Decay for Genome Engineering and Personalized Medicine

Maximilian W. Popp et al.

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Xixun Hu et al.

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Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

Alexis C. Komor et al.

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Keith Lowe et al.

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Iman Farasat et al.

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Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9

Hidetaka Kaya et al.

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In vivo genome editing using Staphylococcus aureus Cas9

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Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice

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Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice

Masafumi Mikami et al.

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Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

Kabin Xie et al.

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Targeted mutagenesis in soybean using the CRISPR-Cas9 system

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Comparative assessments of CRISPR-Cas nucleases' cleavage efficiency in planta

Ross A. Johnson et al.

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Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency

Ying Dang et al.

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Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease

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WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system

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A CRISPR/Cas9 toolkit for multiplex genome editing in plants

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Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice

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RNA-Guided Human Genome Engineering via Cas9

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RNA-programmed genome editing in human cells

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