Retrons and their applications in genome engineering

Abstract Precision genome editing technologies have transformed modern biology. These technologies have arisen from the redirection of natural biological machinery, such as bacteriophage lambda proteins for recombineering and CRISPR nucleases for eliciting site-specific double-strand breaks. Less well-known is a widely distributed class of bacterial retroelements, retrons, that employ specialized reverse transcriptases to produce noncoding intracellular DNAs. Retrons’ natural function and mechanism of genetic transmission have remained enigmatic. However, recent studies have harnessed their ability to produce DNA in situ for genome editing and evolution. This review describes retron biology and function in both natural and synthetic contexts. We also highlight areas that require further study to advance retron-based precision genome editing platforms.

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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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CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

10.20944/preprints202007.0578.v1 ◽

2020 ◽

Author(s):

Anindya Bandyopadhyay ◽

Nagesh Kancharla ◽

vivek javalkote ◽

santanu dasgupta ◽

Thomas Brutnell

Keyword(s):

Genome Editing ◽

Genome Engineering ◽

Temperature Stability ◽

Double Strand Breaks ◽

Plant Genome ◽

Strand Breaks ◽

Guide Rna ◽

Versatile Tool ◽

Type V ◽

Global Population

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

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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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Targeted-Antibacterial-Plasmids (TAPs) combining conjugation and CRISPR/Cas systems achieve strain-specific antibacterial activity

10.1101/2020.10.12.335968 ◽

2020 ◽

Author(s):

Audrey Reuter ◽

Cécile Hilpert ◽

Annick Dedieu-Berne ◽

Sophie Lematre ◽

Erwan Gueguen ◽

...

Keyword(s):

Antibacterial Activity ◽

Pathogenic Bacteria ◽

Bacterial Species ◽

Carbapenem Resistance ◽

Double Strand Breaks ◽

Resistant Bacteria ◽

Innovative Strategy ◽

Strand Breaks ◽

Drug Resistant Bacteria

AbstractThe global emergence of drug-resistant bacteria leads to the loss of efficacy of our antibiotics arsenal and severely limits the success of currently available treatments. Here, we developed an innovative strategy based on Targeted-Antibacterial-Plasmids (TAPs) that use bacterial conjugation to deliver CRISPR/Cas systems exerting a strain-specific antibacterial activity. TAPs are highly versatile as they can be directed against any specific genomic or plasmid DNA using the custom algorithm (CSTB) that identifies appropriate targeting spacer sequences. We demonstrate the ability of TAPs to induce strain-selective killing by introducing lethal double strand breaks (DSBs) into the targeted genomes. TAPs directed against a plasmid-born carbapenem resistance gene efficiently resensitise the strain to the drug. This work represents an essential step towards the development of an alternative to antibiotic treatments, which could be used for in situ microbiota modification to eradicate targeted resistant and/or pathogenic bacteria without affecting other non-targeted bacterial species.

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TALEN-mediated genome editing: prospects and perspectives

Biochemical Journal ◽

10.1042/bj20140295 ◽

2014 ◽

Vol 462 (1) ◽

pp. 15-24 ◽

Cited By ~ 65

Author(s):

David A. Wright ◽

Ting Li ◽

Bing Yang ◽

Martin H. Spalding

Keyword(s):

Genome Editing ◽

Double Strand Breaks ◽

Zinc Finger Nucleases ◽

Transcription Activator ◽

Strand Breaks ◽

Natural Processes ◽

Current State ◽

A Genome ◽

Dna Dsbs ◽

Repair Mechanisms

Genome editing is the practice of making predetermined and precise changes to a genome by controlling the location of DNA DSBs (double-strand breaks) and manipulating the cell's repair mechanisms. This technology results from harnessing natural processes that have taken decades and multiple lines of inquiry to understand. Through many false starts and iterative technology advances, the goal of genome editing is just now falling under the control of human hands as a routine and broadly applicable method. The present review attempts to define the technique and capture the discovery process while following its evolution from meganucleases and zinc finger nucleases to the current state of the art: TALEN (transcription-activator-like effector nuclease) technology. We also discuss factors that influence success, technical challenges and future prospects of this quickly evolving area of study and application.

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Faculty Opinions recommendation of Search-and-replace genome editing without double-strand breaks or donor DNA.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.736778482.793579890 ◽

2020 ◽

Author(s):

Yousin Suh

Keyword(s):

Genome Editing ◽

Double Strand Breaks ◽

Strand Breaks

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

10.1101/161109 ◽

2017 ◽

Cited By ~ 1

Author(s):

Alexandre Paix ◽

Andrew Folkmann ◽

Daniel H Goldman ◽

Heather Kulaga ◽

Michael Grzelak ◽

...

Keyword(s):

Genome Editing ◽

Rational Design ◽

High Efficiency ◽

Fluorescent Protein ◽

Double Strand Breaks ◽

Template Switching ◽

Dna Breaks ◽

Simple Method ◽

Strand Breaks ◽

Strand Annealing

AbstractThe RNA-guided DNA endonuclease Cas9 has emerged as a powerful new tool for genome engineering. Cas9 creates targeted double-strand breaks (DSBs) in the genome. Knock-in 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 1000 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 (SDSA). Our findings enable rational design of synthetic donor DNAs for efficient genome editing.SignificanceGenome editing, the introduction of precise changes in the genome, is revolutionizing our ability to decode the genome. Here we describe a simple method for genome editing that takes advantage of an efficient mechanism for DNA repair called synthesis-dependent strand annealing. We demonstrate that synthetic linear DNAs (ssODNs and PCR fragments) with ∼35bp homology arms function as efficient donors for SDSA repair of Cas9-induced double-strand breaks. Edits from 1 to 1000 base pairs can be introduced in the genome without cloning or selection.

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Loss of SORCS2 is associated with neuronal DNA double-strand breaks

10.1101/2021.04.28.441600 ◽

2021 ◽

Author(s):

Katerina O. Gospodinova ◽

Ditte Olsen ◽

Mathias Kaas ◽

Susan M. Anderson ◽

Jonathan Phillips ◽

...

Keyword(s):

Dna Damage ◽

Neuronal Activity ◽

Neuronal Cell ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Neuronal Viability ◽

Functional Studies ◽

Neuronal Stimulation ◽

And Function

AbstractSORCS2 is one of five proteins that constitute the Vps10p-domain receptor family. Members of this family play important roles in cellular processes linked to neuronal survival, differentiation and function. Genetic and functional studies implicate SORCS2 in cognitive function, as well as in neurodegenerative and psychiatric disorders. DNA damage and DNA repair deficits are linked to ageing and neurodegeneration, and transient neuronal DNA double-strand breaks (DSBs) also occur as a result of neuronal activity. Here, we report a novel role for SORCS2 in DSB formation. We show that SorCS2 loss is associated with elevated DSB levels in the mouse dentate gyrus and that knocking out SORCS2 in a human neuronal cell line increased Topoisomerase IIβ-dependent DSB formation and reduced neuronal viability. Neuronal stimulation had no impact on levels of DNA damage, suggesting that the observed differences are unlikely to be the result of aberrant neuronal activity. Our findings are consistent with studies linking the VPS10 receptors and DNA damage to neurodegenerative conditions.

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Application of the Modified Cytosine Base-Editing in the Cultured Cells of Bama Minipig

10.21203/rs.3.rs-473930/v1 ◽

2021 ◽

Author(s):

Jia-sheng Pan ◽

Zi-sheng Lin ◽

Jian-cong Wen ◽

Jian-feng Guo ◽

Xia-hui Wu ◽

...

Keyword(s):

Genome Editing ◽

Base Pair ◽

Double Strand Breaks ◽

Fibroblast Cells ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Single Base ◽

Lean Meat ◽

Base Editing ◽

Target Effects

Abstract Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 efficiently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modification of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modified CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed “all-in-one”-modified CBE plasmid achieved directional conversion of a single C·G base pair to a T·A base pair of the MSTN target in Bama miniature pig fibroblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs fibroblast cells carrying the MSTN premature termination and verified that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.

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