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

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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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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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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Retrons and their applications in genome engineering

Nucleic Acids Research ◽

10.1093/nar/gkz865 ◽

2019 ◽

Vol 47 (21) ◽

pp. 11007-11019 ◽

Cited By ~ 14

Author(s):

Anna J Simon ◽

Andrew D Ellington ◽

Ilya J Finkelstein

Keyword(s):

Genome Editing ◽

Genome Engineering ◽

Double Strand Breaks ◽

Modern Biology ◽

Genetic Transmission ◽

Strand Breaks ◽

Reverse Transcriptases ◽

Natural Function ◽

And Function

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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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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P53 toxicity is a hurdle to CRISPR/CAS9 screening and engineering in human pluripotent stem cells

10.1101/168443 ◽

2017 ◽

Cited By ~ 2

Author(s):

Robert J. Ihry ◽

Kathleen A. Worringer ◽

Max R. Salick ◽

Elizabeth Frias ◽

Daniel Ho ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Genome Engineering ◽

High Efficiency ◽

Cell Types ◽

Human Pluripotent Stem Cells ◽

Double Strand Breaks ◽

Toxic Response ◽

Strand Breaks ◽

Genome Wide

SUMMARYCRISPR/Cas9 has revolutionized our ability to engineer genomes and to conduct genome-wide screens in human cells. While some cell types are easily modified with Cas9, human pluripotent stem cells (hPSCs) poorly tolerate Cas9 and are difficult to engineer. Using a stable Cas9 cell line or transient delivery of ribonucleoproteins (RNPs) we achieved an average insertion or deletion efficiency greater than 80%. This high efficiency made it apparent that double strand breaks (DSBs) induced by Cas9 are toxic and kill most treated hPSCs. Cas9 toxicity creates an obstacle to the high-throughput use CRISPR/Cas9 for genome-engineering and screening in hPSCs. We demonstrated the toxic response istp53-dependent and the toxic effect oftp53severely reduces the efficiency of precise genome-engineering in hPSCs. Our results highlight that CRISPR-based therapies derived from hPSCs should proceed with caution. Following engineering, it is critical to monitor fortp53function, especially in hPSCs which spontaneously acquiretp53mutations.

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Enabling large-scale genome editing by reducing DNA nicking

10.1101/574020 ◽

2019 ◽

Cited By ~ 9

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.

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CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks

eLife ◽

10.7554/elife.55143 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 5

Author(s):

Joshua C Cofsky ◽

Deepti Karandur ◽

Carolyn J Huang ◽

Isaac P Witte ◽

John Kuriyan ◽

...

Keyword(s):

Genome Editing ◽

Active Site ◽

Strand Break ◽

Double Strand Breaks ◽

Loop Structure ◽

Strand Breaks ◽

Guide Rna ◽

Double Stranded Dna ◽

Type V ◽

Dna Substrate

Type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one strand of a double-helical DNA substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we detect intrinsic instability in DNA flanking the RNA-3′ side of R-loops, which Cas12a can exploit to expose second-strand DNA for cutting. Interestingly, DNA flanking the RNA-5′ side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

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

Nucleic Acids Research ◽

10.1093/nar/gkaa239 ◽

2020 ◽

Vol 48 (9) ◽

pp. 5183-5195 ◽

Cited By ~ 3

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.

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Characterization and repurposing of the endogenous Type I-F CRISPR–Cas system of Zymomonas mobilis for genome engineering

Nucleic Acids Research ◽

10.1093/nar/gkz940 ◽

2019 ◽

Vol 47 (21) ◽

pp. 11461-11475 ◽

Cited By ~ 16

Author(s):

Yanli Zheng ◽

Jiamei Han ◽

Baiyang Wang ◽

Xiaoyun Hu ◽

Runxia Li ◽

...

Keyword(s):

Genome Editing ◽

Zymomonas Mobilis ◽

Genome Engineering ◽

Functional Characterization ◽

Type I ◽

Dna Targeting ◽

Engineering Practices ◽

Restriction Modification ◽

Shed Light

Abstract Application of CRISPR-based technologies in non-model microorganisms is currently very limited. Here, we reported efficient genome engineering of an important industrial microorganism, Zymomonas mobilis, by repurposing the endogenous Type I-F CRISPR–Cas system upon its functional characterization. This toolkit included a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Through this toolkit, various genome engineering purposes were efficiently achieved, including knockout of ZMO0038 (100% efficiency), cas2/3 (100%), and a genomic fragment of >10 kb (50%), replacement of cas2/3 with mCherry gene (100%), in situ nucleotide substitution (100%) and His-tagging of ZMO0038 (100%), and multiplex gene deletion (18.75%) upon optimal donor size determination. Additionally, the Type I-F system was further applied for CRISPRi upon Cas2/3 depletion, which has been demonstrated to successfully silence the chromosomally integrated mCherry gene with its fluorescence intensity reduced by up to 88%. Moreover, we demonstrated that genome engineering efficiency could be improved under a restriction–modification (R–M) deficient background, suggesting the perturbance of genome editing by other co-existing DNA targeting modules such as the R–M system. This study might shed light on exploiting and improving CRISPR–Cas systems in other microorganisms for genome editing and metabolic engineering practices.

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

10.1101/2021.09.08.459484 ◽

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.

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