Regeneration of Plants from DNA-free Edited Grapevine Protoplasts

CRISPR-Cas technology has widely extended the application fields of genome editing in plant breeding, making possible specific and minimal mutations within a genetic pool. With respect to standard genome editing technologies, CRISPR-Cas machinery can be introduced in the form of ribonucleoproteins (RNPs), thus avoiding the introduction of exogenous DNA into cells. The interest on the application of DNA-free delivery into plant cells is constantly increasing, especially in the case of valuable woody plants elite varieties where CRISPR-Cas9 technology would preserve their genotype, while still resulting into targeted genetic modifications. The use of single cells fits well the requirements of New Breeding Technologies, by ensuring both the CRISPR-Cas DNA-free delivery as RNPs and, since every plant will be regenerated from a single edited cell, the absence of chimerism. However, the use of protoplasts cell culture from woody plants is generally hampered by low editing efficiencies and an unsuccessful regenerative process. We here describe a successful DNA-free methodology to obtain fully edited grapevine plants, regenerated from protoplasts obtained from V. vinifera cv. Crimson seedless L. embryogenic callus. The transfected protoplasts were edited on the Downy Mildew susceptibility gene VvDMR6-2. The regenerated edited plants exhibited homozygous deletions of 1bp or 2bp, and homozygous insertion of 1bp.

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Highly efficient generation of canker resistant sweet orange enabled by an improved CRISPR/Cas9 system

10.1101/2021.10.20.465103 ◽

2021 ◽

Author(s):

Xiaoen Huang ◽

Nian Wang

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Target Genes ◽

High Efficiency ◽

Sweet Orange ◽

Susceptibility Gene ◽

Transformation Rate ◽

Biallelic Mutation ◽

Important Species ◽

Citrus Production

Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. However, it is susceptible to many diseases including citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) that triggers devastating effects on citrus production. Conventional breeding has not met the challenge to improve disease resistance of sweet orange due to the long juvenility and other limitations. CRISPR-mediated genome editing has shown promising potentials for genetic improvements of plants. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously (dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, CsU6 to drive sgRNA expression), we further improved our CRISPR/Cas9 system by choosing superior promoters (CmYLCV or CsUbi promoter) to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility gene CsLOB1, which is required for causing canker symptoms by Xcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant to Xcc. Biallelic mutation of the EBE region abolishes the induction of CsLOB1 by Xcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease resistant varieties via CRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible.

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Concurrent Genome and Epigenome Editing by CRISPR-Mediated Sequence Replacement

10.1101/675447 ◽

2019 ◽

Author(s):

Jes Alexander ◽

Gregory M. Findlay ◽

Martin Kircher ◽

Jay Shendure

Keyword(s):

Genome Editing ◽

Cpg Island ◽

Exogenous Dna ◽

Cpg Dinucleotides ◽

Epigenome Editing ◽

Functional Consequences ◽

Cpg Dinucleotide ◽

Methylation Patterns

AbstractRecent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double stranded break (DSB), followed by NHEJ or HDR mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, i.e. to drive replacement of the DNA sequence intervening the dual cuts via NHEJ. In a proof-of-concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event, and opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.

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Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker

Plant Biotechnology Journal ◽

10.1111/pbi.12677 ◽

2017 ◽

Vol 15 (7) ◽

pp. 817-823 ◽

Cited By ~ 143

Author(s):

Hongge Jia ◽

Yunzeng Zhang ◽

Vladimir Orbović ◽

Jin Xu ◽

Frank F. White ◽

...

Keyword(s):

Genome Editing ◽

Disease Susceptibility ◽

Susceptibility Gene ◽

Citrus Canker ◽

Disease Susceptibility Gene

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Multiplex labeling and manipulation of endogenous neuronal proteins using sequential CRISPR/Cas9 gene editing

10.1101/2022.01.02.474730 ◽

2022 ◽

Author(s):

Wouter J Droogers ◽

Jelmer Willems ◽

Harold D MacGillavry ◽

Arthur PH de Jong

Keyword(s):

Genome Editing ◽

Single Molecule ◽

Time Window ◽

Single Cells ◽

Synaptic Proteins ◽

Flp Recombinase ◽

Neuronal Proteins ◽

Endogenous Proteins ◽

Living Neurons ◽

High Degree

Recent advances in CRISPR/Cas9-mediated knock-in methods enable labeling of individual endogenous proteins with fluorophores, to determine their spatiotemporal expression in intact biological preparations. However, multiplex knock-in methods remain limited, particularly in postmitotic cells, due to a high degree of crosstalk between genome editing events. We present Conditional Activation of Knock-in Expression (CAKE), which delivers efficient, flexible and accurate multiplex genome editing in neurons. CAKE is based on sequential gRNA expression operated by a Cre- or Flp-recombinase to control the time window for genomic integration of each donor sequence, which diminishes crosstalk between genome editing events. Importantly, CAKE is compatible with multiple CRISPR/Cas9 strategies, and we show the utilization of CAKE for co-localization of various endogenous proteins, including synaptic scaffolds, ion channels and neurotransmitter receptor subunits. Knock-in efficacy was highly sensitive to DNA vector amount, while knock-in crosstalk was dependent on the rate of donor DNA integration and timing of Cre activation. We applied CAKE to study the co-distribution of endogenous synaptic proteins using dual-color single-molecule localization microscopy, and we introduced dimerization modules to acutely control synaptic receptor dynamics in living neurons. Taken together, CAKE is a versatile method for multiplex protein labeling, enabling accurate detection, precise localization and acute manipulation of endogenous proteins in single cells.

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Digital-WGS: Automated, highly efficient whole-genome sequencing of single cells by digital microfluidics

Science Advances ◽

10.1126/sciadv.abd6454 ◽

2020 ◽

Vol 6 (50) ◽

pp. eabd6454

Author(s):

Qingyu Ruan ◽

Weidong Ruan ◽

Xiaoye Lin ◽

Yang Wang ◽

Fenxiang Zou ◽

...

Keyword(s):

Sample Preparation ◽

Whole Genome Sequencing ◽

Single Cell ◽

Genome Sequencing ◽

Single Cells ◽

Digital Microfluidics ◽

Dropout Rate ◽

Great Promise ◽

Whole Genome ◽

Exogenous Dna

Single-cell whole-genome sequencing (WGS) is critical for characterizing dynamic intercellular changes in DNA. Current sample preparation technologies for single-cell WGS are complex, expensive, and suffer from high amplification bias and errors. Here, we describe Digital-WGS, a sample preparation platform that streamlines high-performance single-cell WGS with automatic processing based on digital microfluidics. Using the method, we provide high single-cell capture efficiency for any amount and types of cells by a wetted hydrodynamic structure. The digital control of droplets in a closed hydrophobic interface enables the complete removal of exogenous DNA, sufficient cell lysis, and lossless amplicon recovery, achieving the low coefficient of variation and high coverage at multiple scales. The single-cell genomic variations profiling performs the excellent detection of copy number variants with the smallest bin of 150 kb and single-nucleotide variants with allele dropout rate of 5.2%, holding great promise for broader applications of single-cell genomics.

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Erratum to: Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing

Journal of Assisted Reproduction and Genetics ◽

10.1007/s10815-017-0946-y ◽

2017 ◽

Vol 34 (7) ◽

pp. 963-963 ◽

Cited By ~ 2

Author(s):

Xiangjin Kang ◽

Wenyin He ◽

Yuling Huang ◽

Qian Yu ◽

Yaoyong Chen ◽

...

Keyword(s):

Genome Editing ◽

Genetic Modifications

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Kluyveromyces marxianus as a robust synthetic biology platform host

10.1101/353680 ◽

2018 ◽

Author(s):

Paul Cernak ◽

Raissa Estrela ◽

Snigdha Poddar ◽

Jeffrey M. Skerker ◽

Ya-Fang Cheng ◽

...

Keyword(s):

Synthetic Biology ◽

Genome Editing ◽

Kluyveromyces Marxianus ◽

Complex Traits ◽

Lipid Production ◽

Carbon Sources ◽

Industrial Applications ◽

Human Society ◽

Exogenous Dna ◽

Renewable Chemicals

ABSTRACTThroughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineer the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits– thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.

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Retron Editing for Precise Genome Editing without Exogenous Donor DNA in Human Cells

10.1101/2021.05.11.443596 ◽

2021 ◽

Author(s):

Xiangfeng Kong ◽

Zikang Wang ◽

Yingsi Zhou ◽

Xing Wang ◽

Linyu Shi ◽

...

Keyword(s):

Genome Editing ◽

Ex Vivo ◽

Human Cells ◽

Exogenous Dna ◽

Guide Rna ◽

Homology Directed Repair ◽

Non Coding Rna ◽

Bacterial Genetic ◽

Low Efficiency

CRISPR-Cas9 mediated seamless genome editing can be achieved by incorporating donor DNA into the CRISPR-Cas9 target loci via homology-directed repair (HDR), albeit with relative low efficiency due to the inefficient delivery of exogenous DNA. Retrons are bacterial genetic element composed of a non-coding RNA (ncRNA) and reverse transcriptase (RT). Retrons coupled with CRISPR-Cas9 have been shown to enhance precise genome editing via HDR in yeast through fusing guide RNA (gRNA) to the 3′ end of retron ncRNA, producing multicopy single-stranded DNA (msDNA) covalently tethered to gRNA. Here, we further engineered retrons by fusing Cas9 with E.coli RT from different clades and joining gRNA at the 5′ end of retron ncRNA, and found that retron editing can achieve precise genome editing efficiently in human cells. By co- expression of Cas9-RT fusions and retron-ncRNA gRNA (rgRNA) in HEK293T cells, we demonstrated the rates of retron editing at endogenous genomic loci was up to 10 %. We expect our retron editing system could aid in advancing the ex vivo and in vivo therapeutic applications of retron.

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Converting Escherichia coli MG1655 into a chemical overproducer through inactivating defense system against exogenous DNA

10.1101/2020.08.15.251348 ◽

2020 ◽

Author(s):

Jingge Wang ◽

Chaoyong Huang ◽

Kai Guo ◽

Lianjie Ma ◽

Xiangyu Meng ◽

...

Keyword(s):

Escherichia Coli ◽

Plasmid Stability ◽

Value Added ◽

Test Tube ◽

Host Strain ◽

Exogenous Dna ◽

Genetic Modifications ◽

High Level ◽

K 12 ◽

Level Production

AbstractEscherichia coli strain K-12 MG1655 has been proposed as an appropriate host strain for industrial production. However, the direct application of this strain suffers from the transformation inefficiency and plasmid instability. Herein, we conducted genetic modifications at a serial of loci of MG1655 genome, generating a robust and universal host strain JW128 with higher transformation efficiency and plasmid stability that can be used to efficiently produce desired chemicals after introducing the corresponding synthetic pathways. Using JW128 as the host, the titer of isobutanol reached 5.76 g/L in shake-flask fermentation, and the titer of lycopene reached 1.91 g/L in test-tube fermentation, 40-fold and 5-fold higher than that of original MG1655, respectively. These results demonstrated JW128 is a promising chassis for high-level production of value-added chemicals.

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Split Selectable Markers

10.1101/452979 ◽

2018 ◽

Author(s):

Nathaniel Jillette ◽

Menghan Du ◽

Albert Cheng

Keyword(s):

Genome Editing ◽

Future Development ◽

Selectable Markers ◽

Genetic Modifications ◽

Selection Of

AbstractSelectable markers are widely adopted in transgenesis and genome editing for selecting engineered cells with desired genotype but are limited in choices. We present here split selectable markers each allowing for selection of multiple “unlinked” transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR/Cas-mediated biallelic knock-ins. Future development of split selectable markers may support enrichment or selection of “hyper-engineered” cells containing tens of transgenes or genetic modifications.

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