scholarly journals Enabling large-scale genome editing at repetitive elements by reducing DNA nicking

2020 ◽  
Vol 48 (9) ◽  
pp. 5183-5195 ◽  
Author(s):  
Cory J Smith ◽  
Oscar Castanon ◽  
Khaled Said ◽  
Verena Volf ◽  
Parastoo Khoshakhlagh ◽  
...  
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Genome Engineering ◽  
Repetitive Elements ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Base Editing ◽  
Single Strand Breaks ◽  
Dna Nicking ◽  
Induced Pluripotent

Abstract To extend the frontier of genome editing and enable editing of repetitive elements of mammalian genomes, we made use of a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks and single-strand breaks. We used a set of gRNAs targeting repetitive elements—ranging in target copy number from about 32 to 161 000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ∼13 200 and ∼12 200 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.

scholarly journals Enabling large-scale genome editing by reducing DNA nicking

2019 ◽  
Author(s):  
Cory J. Smith ◽  
Oscar Castanon ◽  
Khaled Said ◽  
Verena Volf ◽  
Parastoo Khoshakhlagh ◽  
...  
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Genome Engineering ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Base Editing ◽  
Single Strand Breaks ◽  
Dna Nicking ◽  
Mammalian Genomes ◽  
Induced Pluripotent

AbstractTo extend the frontier of genome editing and enable the radical redesign of mammalian genomes, we developed a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks (DSBs) and single-strand breaks (SSBs). We used a set of gRNAs targeting repetitive elements – ranging in target copy number from about 31 to 124,000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ~13,200 and ~2610 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.One Sentence SummaryBase editing with reduced DNA nicking allows for the simultaneous editing of >10,000 loci in human cells.

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

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.

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

Open Biology ◽  
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.

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

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.

scholarly journals CRISPR-Cas12a induced DNA double-strand breaks are repaired by locus-dependent and error-prone pathways in a fungal pathogen

2021 ◽  
Author(s):  
Jun Huang ◽  
David Rowe ◽  
Wei Zhang ◽  
Tyler Suelter ◽  
Barbara Valent ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Cleavage ◽  
Large Scale ◽  
Genome Engineering ◽  
Natural Populations ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Long Read ◽  
Repair Pathways

AbstractCRISPR-Cas mediated genome engineering has revolutionized functional genomics. However, basic questions remain regarding the mechanisms of DNA repair following Cas-mediated DNA cleavage. We developed CRISPR-Cas12a ribonucleoprotein genome editing in the fungal plant pathogen, Magnaporthe oryzae, and found frequent donor DNA integration despite the absence of long sequence homology. Interestingly, genotyping from hundreds of transformants showed that frequent non-canonical DNA repair outcomes predominated the recovered genome edited strains. Detailed analysis using sanger and nanopore long-read sequencing revealed five classes of DNA repair mutations, including single donor DNA insertions, concatemer donor DNA insertions, large DNA deletions, deletions plus donor DNA insertions, and infrequently we observed INDELs. Our results show that different error-prone DNA repair pathways resolved the Cas12a-mediated double-strand breaks (DSBs) based on the DNA sequence of edited strains. Furthermore, we found that the frequency of the different DNA repair outcomes varied across the genome, with some tested loci resulting in more frequent large-scale mutations. These results suggest that DNA repair pathways provide preferential repair across the genome that could create biased genome variation, which has significant implications for genome engineering and the genome evolution in natural populations.

scholarly journals Recent advances in CRISPR/Cas9 and applications for wheat functional genomics and breeding

aBIOTECH ◽  
2021 ◽  
Author(s):  
Jun Li ◽  
Yan Li ◽  
Ligeng Ma
Keyword(s):  
Functional Genomics ◽  
Genome Editing ◽  
Functional Annotation ◽  
Genome Engineering ◽  
Agronomic Traits ◽  
Wheat Breeding ◽  
Breeding Programs ◽  
Base Editing ◽  
The World ◽  
World Food Security

AbstractCommon wheat (Triticum aestivum L.) is one of the three major food crops in the world; thus, wheat breeding programs are important for world food security. Characterizing the genes that control important agronomic traits and finding new ways to alter them are necessary to improve wheat breeding. Functional genomics and breeding in polyploid wheat has been greatly accelerated by the advent of several powerful tools, especially CRISPR/Cas9 genome editing technology, which allows multiplex genome engineering. Here, we describe the development of CRISPR/Cas9, which has revolutionized the field of genome editing. In addition, we emphasize technological breakthroughs (e.g., base editing and prime editing) based on CRISPR/Cas9. We also summarize recent applications and advances in the functional annotation and breeding of wheat, and we introduce the production of CRISPR-edited DNA-free wheat. Combined with other achievements, CRISPR and CRISPR-based genome editing will speed progress in wheat biology and promote sustainable agriculture.

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

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.

scholarly journals Emerging Technologies for Genome-Wide Profiling of DNA Breakage

2021 ◽  
Vol 11 ◽  
Author(s):  
Matthew J. Rybin ◽  
Melina Ramic ◽  
Natalie R. Ricciardi ◽  
Philipp Kapranov ◽  
Claes Wahlestedt ◽  
...  
Keyword(s):  
Genome Instability ◽  
Dna Double Strand Breaks ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Nucleotide Resolution ◽  
Genomic Regions

Genome instability is associated with myriad human diseases and is a well-known feature of both cancer and neurodegenerative disease. Until recently, the ability to assess DNA damage—the principal driver of genome instability—was limited to relatively imprecise methods or restricted to studying predefined genomic regions. Recently, new techniques for detecting DNA double strand breaks (DSBs) and single strand breaks (SSBs) with next-generation sequencing on a genome-wide scale with single nucleotide resolution have emerged. With these new tools, efforts are underway to define the “breakome” in normal aging and disease. Here, we compare the relative strengths and weaknesses of these technologies and their potential application to studying neurodegenerative diseases.

scholarly journals CRISPR base editors: genome editing without double-stranded breaks

2018 ◽  
Vol 475 (11) ◽  
pp. 1955-1964 ◽  
Author(s):  
Ayman Eid ◽  
Sahar Alshareef ◽  
Magdy M. Mahfouz
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Genetic Diseases ◽  
Great Promise ◽  
Cytidine Deaminases ◽  
Strand Breaks ◽  
Base Editing ◽  
Potential Applications ◽  
Short Palindromic Repeat ◽  
Basic Biology

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 adaptive immunity system has been harnessed for genome editing applications across eukaryotic species, but major drawbacks, such as the inefficiency of precise base editing and off-target activities, remain. A catalytically inactive Cas9 variant (dead Cas9, dCas9) has been fused to diverse functional domains for targeting genetic and epigenetic modifications, including base editing, to specific DNA sequences. As base editing does not require the generation of double-strand breaks, dCas9 and Cas9 nickase have been used to target deaminase domains to edit specific loci. Adenine and cytidine deaminases convert their respective nucleotides into other DNA bases, thereby offering many possibilities for DNA editing. Such base-editing enzymes hold great promise for applications in basic biology, trait development in crops, and treatment of genetic diseases. Here, we discuss recent advances in precise gene editing using different platforms as well as their potential applications in basic biology and biotechnology.

scholarly journals A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
William H. Gittens ◽  
Dominic J. Johnson ◽  
Rachal M. Allison ◽  
Tim J. Cooper ◽  
Holly Thomas ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Genome Stability ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Human Genomes ◽  
Nucleotide Resolution

Abstract DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

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