A minimal CRISPR-Cas3 system for genome engineering

AbstractCRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions. Here, we utilized a Cas3-based system featuring a processive nuclease, expressed endogenously or heterologously, for genome engineering purposes. Using an optimized and minimal CRISPR-Cas3 system (Type I-C) programmed with a single crRNA, large deletions ranging from 7 - 424 kb were generated in Pseudomonas aeruginosa with high efficiency and speed. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated deletion boundaries were variable in the absence of a homology-directed repair (HDR) template, and successfully and efficiently specified when present. The minimal Cas3 system is also portable; large deletions were induced with high efficiency in Pseudomonas syringae and Escherichia coli using an “all-in-one” vector. Notably, Cas3 generated bi-directional deletions originating from the programmed cut site, which was exploited to iteratively reduce a P. aeruginosa genome by 837 kb (13.5%) using 10 distinct crRNAs. We also demonstrate the utility of endogenous Cas3 systems (Type I-C and I-F) and develop an “anti-anti-CRISPR” strategy to circumvent endogenous CRISPR-Cas inhibitor proteins. CRISPR-Cas3 could facilitate rapid strain manipulation for synthetic biological and metabolic engineering purposes, genome minimization, and the analysis of large regions of unknown function.

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First Report of CRISPR/Cas9 Gene Editing in Castanea sativa Mill

Frontiers in Plant Science ◽

10.3389/fpls.2021.728516 ◽

2021 ◽

Vol 12 ◽

Author(s):

Vera Pavese ◽

Andrea Moglia ◽

Elena Corredoira ◽

Mª Teresa Martínez ◽

Daniela Torello Marinoni ◽

...

Keyword(s):

Somatic Embryos ◽

Gene Editing ◽

Genome Engineering ◽

High Efficiency ◽

Chlorophyll Biosynthesis ◽

Point Mutations ◽

Castanea Sativa ◽

Visual Assessment ◽

European Chestnut ◽

Selection Medium

CRISPR/Cas9 has emerged as the most important tool for genome engineering due to its simplicity, design flexibility, and high efficiency. This technology makes it possible to induce point mutations in one or some target sequences simultaneously, as well as to introduce new genetic variants by homology-directed recombination. However, this approach remains largely unexplored in forest species. In this study, we reported the first example of CRISPR/Cas9-mediated gene editing in Castanea genus. As a proof of concept, we targeted the gene encoding phytoene desaturase (pds), whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Globular and early torpedo-stage somatic embryos of Castanea sativa (European chestnut) were cocultured for 5 days with a CRISPR/Cas9 construct targeting two conserved gene regions of pds and subsequently cultured on a selection medium with kanamycin. After 8 weeks of subculture on selection medium, four kanamycin-resistant embryogenetic lines were isolated. Genotyping of these lines through target Sanger sequencing of amplicons revealed successful gene editing. Cotyledonary somatic embryos were maturated on maltose 3% and cold-stored at 4°C for 2 months. Subsequently, embryos were subjected to the germination process to produce albino plants. This study opens the way to the use of the CRISPR/Cas9 system in European chestnut for biotechnological applications

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Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages

mBio ◽

10.1128/mbio.00308-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 69

Author(s):

Ian R. Monk ◽

Jai J. Tree ◽

Benjamin P. Howden ◽

Timothy P. Stinear ◽

Timothy J. Foster

Keyword(s):

Staphylococcus Aureus ◽

Plasmid Dna ◽

High Efficiency ◽

Recombinant Dna ◽

Genetic Manipulation ◽

Bacterial Pathogen ◽

Type I ◽

Content Type ◽

E Coli ◽

Adenine Methylation

ABSTRACTStaphylococcus aureusis a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates ofS. aureus. Here, we describe the construction and characterization of the IMXXB series ofEscherichia colistrains that mimic the type I adenine methylation profiles ofS. aureusclonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of majorS. aureuslineages.IMPORTANCEThe genetic manipulation of clinicalS. aureusisolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset ofS. aureusstrains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite ofE. colistrains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer intoS. aureus. The IMXXB series ofE. colistrains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation ofS. aureus.

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Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering

Open Biology ◽

10.1098/rsob.210241 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Yile Hao ◽

Qinhua Wang ◽

Jie Li ◽

Shihui Yang ◽

Yanli Zheng ◽

...

Keyword(s):

Genome Editing ◽

Large Scale ◽

Genome Engineering ◽

High Efficiency ◽

Helicase Domain ◽

Type I ◽

Strand Breaks ◽

Gene Insertion ◽

Type V

New CRISPR-based genome editing technologies are developed to continually drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon a Type I-F system. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity, an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing without visible cell killing. By harnessing this CRISPR-nCas3 in situ gene insertion, nucleotide substitution and deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, yet useful approach to convert the naturally most abundantly occurring Type I systems into advanced genome editing tools to facilitate high-throughput prokaryotic engineering.

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High efficiency generalized transduction in Escherichia coli O157:H7

F1000Research ◽

10.12688/f1000research.2-7.v1 ◽

2013 ◽

Vol 2 ◽

pp. 7 ◽

Cited By ~ 9

Author(s):

Martin G Marinus ◽

Anthony R Poteete

Keyword(s):

High Efficiency ◽

Genetic Manipulation ◽

Point Mutations ◽

Ultra Violet ◽

E Coli ◽

Recombination System ◽

Kanamycin Cassette ◽

Uremic Syndrome ◽

K 12 ◽

Generalized Transduction

Genetic manipulation in enterohemorrhagicE. coliO157:H7 is currently restricted to recombineering, a method that utilizes the recombination system of bacteriophage lambda, to introduce gene replacements and base changesinter aliainto the genome. Bacteriophage 933W is a prophage inE. coliO157:H7 strain EDL933, which encodes the genes (stx2AB) for the production of Shiga toxin which is the basis for the potentially fatal Hemolytic Uremic Syndrome in infected humans. We replaced thestx2ABgenes with a kanamycin cassette using recombineering. After induction of the prophage by ultra-violet light, we found that bacteriophage lysates were capable of transducing to wildtype, point mutations in the lactose, arabinose and maltose genes. The lysates could also transduce tetracycline resistant cassettes. Bacteriophage 933W is also efficient at transducing markers inE. coliK-12. Co-transduction experiments indicated that the maximal amount of transferred DNA was likely the size of the bacteriophage genome, 61 kB. All tested transductants, in bothE. coliK-12 and O157:H7, were kanamycin-sensitive indicating that the transducing particles contained host DNA.

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Transforming the Untransformable: Application of Direct Transformation To Manipulate Genetically Staphylococcus aureus and Staphylococcus epidermidis

mBio ◽

10.1128/mbio.00277-11 ◽

2012 ◽

Vol 3 (2) ◽

Cited By ~ 242

Author(s):

Ian R. Monk ◽

Ishita M. Shah ◽

Min Xu ◽

Man-Wah Tan ◽

Timothy J. Foster

Keyword(s):

Staphylococcus Aureus ◽

Staphylococcus Epidermidis ◽

High Efficiency ◽

Genetic Manipulation ◽

Laboratory Strain ◽

Small Subset ◽

Type I ◽

Major Barrier ◽

Content Type ◽

Functional Genomic Analysis

ABSTRACTThe strong restriction barrier present inStaphylococcus aureusandStaphylococcus epidermidishas limited functional genomic analysis to a small subset of strains that are amenable to genetic manipulation. Recently, a conserved type IV restriction system termed SauUSI (which specifically recognizes cytosine methylated DNA) was identified as the major barrier to transformation with foreign DNA. Here we have independently corroborated these findings in a widely used laboratory strain ofS. aureus. Additionally, we have constructed a DNA cytosine methyltransferase mutant in the high-efficiencyEscherichia colicloning strain DH10B (called DC10B). Plasmids isolated from DC10B can be directly transformed into clinical isolates ofS. aureusandS. epidermidis. We also show that the loss of restriction (both type I and IV) in anS. aureusUSA300 strain does not have an impact on virulence. Circumventing the SauUSI restriction barrier, combined with an improved deletion and transformation protocol, has allowed the genetic manipulation of previously untransformable strains of these important opportunistic pathogens.IMPORTANCEStaphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding ofStaphylococcus aureusandStaphylococcus epidermidisinfections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated.

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Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery

eLife ◽

10.7554/elife.04766 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 554

Author(s):

Steven Lin ◽

Brett T Staahl ◽

Ravi K Alla ◽

Jennifer A Doudna

Keyword(s):

Genome Engineering ◽

High Efficiency ◽

Embryonic Stem ◽

Human Cells ◽

Dna Breaks ◽

Site Specific ◽

Guide Rna ◽

Homology Directed Repair ◽

Double Strand Dna Breaks ◽

Cell Mortality

The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (<xref ref-type="bibr" rid="bib13">Jinek et al., 2013</xref>), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

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Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering

10.1101/2021.07.12.451994 ◽

2021 ◽

Author(s):

Yile Hao ◽

Qinhua Wang ◽

Jie Li ◽

Shihui Yang ◽

Lixin Ma ◽

...

Keyword(s):

Genome Editing ◽

Large Scale ◽

Genome Engineering ◽

High Efficiency ◽

Double Strand Breaks ◽

Helicase Domain ◽

Type I ◽

Strand Breaks ◽

Type V

New CRISPR-based genome editing technologies are developed to continuedly drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon an endogenous Type I system of Zymomonas mobilis. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity; an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing, without visible cell killing. By harnessing this CRISPR-nCas3, deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, easy, yet useful approach to convert many endogenous Type I systems into advanced genome editing tools. We envision that many CRISPR-nCas3-based toolkits would be soon available for various industrially important non-model bacteria that carry active Type I systems to facilitate high-throughput prokaryotic engineering.

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High-efficiency retron-mediated single-stranded DNA production in plants

10.22541/au.163717150.01158451/v1 ◽

2021 ◽

Author(s):

Wenjun Jiang ◽

Gundra Sivakrishna Rao ◽

Rashid Aman ◽

Haroon Butt ◽

Radwa Kamel ◽

...

Keyword(s):

Mammalian Cells ◽

Genome Engineering ◽

High Efficiency ◽

Plant Biotechnology ◽

Fold Increase ◽

Gene Encoding ◽

Single Stranded Dna ◽

Homology Directed Repair ◽

Potential Applications

ABSTRACT Background: Retrons are a class of retroelements that produce multicopy single-stranded DNA (msDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the over-production of single-stranded DNA (ssDNA), genome engineering, and directed evolution in bacteria, yeast, and mammalian cells. However, no studies have shown retron-mediated ssDNA production in plants, which could unlock potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Main methods and major results: Here, we demonstrated retron-mediated over-production of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated CRISPR-retron-coupled ssDNA over-production and targeted HDR in N. benthamiana. Conclusion: We present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications.

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Genome editing reveals that pSCL4 is required for chromosome linearity in Streptomyces clavuligerus

Microbial Genomics ◽

10.1099/mgen.0.000669 ◽

2021 ◽

Vol 7 (11) ◽

Cited By ~ 1

Author(s):

Juan Pablo Gomez-Escribano ◽

Lis Algora Gallardo ◽

Kenan A. J. Bozhüyük ◽

Steven G. Kendrew ◽

Benjamin D. Huckle ◽

...

Keyword(s):

Genome Editing ◽

Type Strain ◽

High Efficiency ◽

Genetic Manipulation ◽

Sequence Data ◽

Streptomyces Clavuligerus ◽

Sequence Coverage ◽

Content Type ◽

Large Deletions ◽

Low Levels

Streptomyces clavuligerus is an industrially important actinomycete whose genetic manipulation is limited by low transformation and conjugation efficiencies, low levels of recombination of introduced DNA, and difficulty in obtaining consistent sporulation. We describe the construction and application of versatile vectors for Cas9-mediated genome editing of this strain. To design spacer sequences with confidence, we derived a highly accurate genome assembly for an isolate of the type strain (ATCC 27064). This yielded a chromosome assembly (6.75 Mb) plus assemblies for pSCL4 (1795 kb) and pSCL2 (149 kb). The strain also carries pSCL1 (12 kb), but its small size resulted in only partial sequence coverage. The previously described pSCL3 (444 kb) is not present in this isolate. Using our Cas9 vectors, we cured pSCL4 with high efficiency by targeting the plasmid’s parB gene. Five of the resulting pSCL4-cured isolates were characterized and all showed impaired sporulation. Shotgun genome sequencing of each of these derivatives revealed large deletions at the ends of the chromosomes in all of them, and for two clones sufficient sequence data was obtained to show that the chromosome had circularized. Taken together, these data indicate that pSCL4 is essential for the structural stability of the linear chromosome.

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Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1711979114 ◽

2017 ◽

Vol 114 (50) ◽

pp. E10745-E10754 ◽

Cited By ~ 63

Author(s):

Alexandre Paix ◽

Andrew Folkmann ◽

Daniel H. Goldman ◽

Heather Kulaga ◽

Michael J. Grzelak ◽

...

Keyword(s):

Genome Editing ◽

Rational Design ◽

Genome Engineering ◽

High Efficiency ◽

Fluorescent Protein ◽

Template Switching ◽

Dna Breaks ◽

Homology Directed Repair ◽

Switching Characteristics ◽

Strand Annealing

The RNA-guided DNA endonuclease Cas9 has emerged as a powerful tool for genome engineering. Cas9 creates targeted double-stranded breaks (DSBs) in the genome. Knockin of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double stranded) engage in a high-efficiency HDR mechanism that requires only ∼35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1,000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand annealing. Our findings enable rational design of synthetic donor DNAs for efficient genome editing.

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