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

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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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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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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Characterization and repurposing of the endogenous Type I-F CRISPR-Cas system ofZymomonas mobilisfor genome engineering

10.1101/576355 ◽

2019 ◽

Cited By ~ 1

Author(s):

Yanli Zheng ◽

Jiamei Han ◽

Wenyao Liang ◽

Runxia Li ◽

Xiaoyun Hu ◽

...

Keyword(s):

Zymomonas Mobilis ◽

Genome Engineering ◽

Functional Characterization ◽

Gene Replacement ◽

Type I ◽

Nucleotide Substitutions ◽

Sustainable Resources ◽

Production Platform ◽

Facultative Anaerobic ◽

Application Scope

ABSTRACTEstablishment of production platform organisms through prokaryotic engineering represents an efficient means to generate alternatives for yielding renewable biochemicals and biofuels from sustainable resources.Zymomonas mobilis, a natural facultative anaerobic ethanologen, possesses many attractive physiological attributes, making it an important industrial microorganism. To facilitate the broad applications of this strain for biorefinery, an efficient genome engineering toolkit forZ. mobiliswas established in this study by repurposing the endogenous Type I-F CRISPR-Cas system upon its functional characterization, and further updated. This toolkit includes a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Using the updated toolkit, genome engineering purposes were achieved with efficiencies of up to 100%, including knockout ofcas3gene, replacement ofcas3with the mCherry-encodingrfpgene, nucleotide substitutions incas3, and deletion of two large genomic fragments up to 10 kb. This study established thus far the most efficient, straightforward and convenient genome engineering toolkit forZ. mobilis, and laid a foundation for further native CRISPRi studies inZ.mobilis, which extended the application scope of CRISPR-based technologies, and could also be applied to other industrial microorganisms with unexploited endogenous CRISPR-Cas systems.

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All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

10.1101/295055 ◽

2018 ◽

Cited By ~ 1

Author(s):

Raed Ibraheim ◽

Chun-Qing Song ◽

Aamir Mir ◽

Nadia Amrani ◽

Wen Xue ◽

...

Keyword(s):

Gene Therapy ◽

Neisseria Meningitidis ◽

Genome Editing ◽

Viral Vectors ◽

Genome Engineering ◽

Degradation Pathway ◽

Type I ◽

Guide Rna ◽

Hereditary Tyrosinemia

AbstractClustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: SpyCas9, SauCas9 and CjeCas9. However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors, especially those in which Cas9 and sgRNA are combined into a single vector genome. Here, we present an additional in vivo editing platform using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct PAM, making it an excellent candidate for safe gene therapy applications. We find that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogrammed the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we delivered NmeCas9 with its single-guide RNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded >35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo. Our findings indicate that NmeCas9 can facilitate future efforts to correct disease-causing mutations by expanding the targeting scope of RNA-guided nucleases.

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Exploration of the Diversity of Clustered Regularly Interspaced Short Palindromic Repeats-Cas Systems in Clostridium novyi sensu lato

Frontiers in Microbiology ◽

10.3389/fmicb.2021.711413 ◽

2021 ◽

Vol 12 ◽

Author(s):

Thibault Le Gratiet ◽

Caroline Le Marechal ◽

Marie Devaere ◽

Marianne Chemaly ◽

Cédric Woudstra

Keyword(s):

Clostridium Botulinum ◽

Type I ◽

Group Iii ◽

Clostridium Novyi ◽

Associated Proteins ◽

Partial Type ◽

Type V ◽

First Time ◽

Restriction Modification ◽

Shed Light

Classified as the genospecies Clostridium novyi sensu lato and distributed into four lineages (I–IV), Clostridium botulinum (group III), Clostridium novyi, and Clostridium haemolyticum are clostridial pathogens that cause animal diseases. Clostridium novyi sensu lato contains a large mobilome consisting of plasmids and circular bacteriophages. Here, we explored clustered regularly interspaced short palindromic repeats (CRISPR) arrays and their associated proteins (Cas) to shed light on the link between evolution of CRISPR-Cas systems and the plasmid and phage composition in a study of 58 Clostridium novyi sensu lato genomes. In 55 of these genomes, types I-B (complete or partial), I-D, II-C, III-B, III-D, or V-U CRISPR-Cas systems were detected in chromosomes as well as in mobile genetic elements (MGEs). Type I-B predominated (67.2%) and was the only CRISPR type detected in the Ia, III, and IV genomic lineages. Putative type V-U CRISPR Cas14a genes were detected in two different cases: next to partial type-IB CRISPR loci on the phage encoding the botulinum neurotoxin (BoNT) in lineage Ia and in 12 lineage II genomes, as part of a putative integrative element related to a phage-inducible chromosomal island (PICI). In the putative PICI, Cas14a was associated with CRISPR arrays and restriction modification (RM) systems as part of an accessory locus. This is the first time a PICI containing such locus has been detected in C. botulinum. Mobilome composition and dynamics were also investigated based on the contents of the CRISPR arrays and the study of spacers. A large proportion of identified protospacers (20.2%) originated from Clostridium novyi sensu lato (p1_Cst, p4_BKT015925, p6_Cst, CWou-2020a, p1_BKT015925, and p2_BKT015925), confirming active exchanges within this genospecies and the key importance of specific MGEs in Clostridium novyi sensu lato.

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Establishment and application of a CRISPR–Cas12a assisted genome-editing system in Zymomonas mobilis

Microbial Cell Factories ◽

10.1186/s12934-019-1219-5 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 10

Author(s):

Wei Shen ◽

Jun Zhang ◽

Binan Geng ◽

Mengyue Qiu ◽

Mimi Hu ◽

...

Keyword(s):

Genome Editing ◽

Dna Cleavage ◽

Zymomonas Mobilis ◽

Genome Engineering ◽

Gene Deletion ◽

Genetic Manipulation ◽

Strain Improvement ◽

Genomic Research ◽

Francisella Novicida ◽

A Genome

Abstract Background Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. Results Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR–Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR–Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR–Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. Conclusions This study applied CRISPR–Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR–Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

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Genome editing in plants using CRISPR type I-D nuclease

Communications Biology ◽

10.1038/s42003-020-01366-6 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Keishi Osakabe ◽

Naoki Wada ◽

Tomoko Miyaji ◽

Emi Murakami ◽

Kazuya Marui ◽

...

Keyword(s):

Genome Editing ◽

First Generation ◽

Genome Engineering ◽

Targeted Mutagenesis ◽

Plant Genome ◽

Type I ◽

Crispr System ◽

Target Dna ◽

Short Indels

Abstract Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I—the most abundant CRISPR system in bacteria—has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.

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Genome editing in mammals using CRISPR type I-D nuclease

10.1101/2020.03.14.991976 ◽

2020 ◽

Cited By ~ 1

Author(s):

Keishi Osakabe ◽

Naoki Wada ◽

Emi Murakami ◽

Yuriko Osakabe

Keyword(s):

Genome Editing ◽

Genomic Dna ◽

Genome Engineering ◽

Dna Degradation ◽

Targeted Mutagenesis ◽

Eukaryotic Cells ◽

Type I ◽

Effector Pathway ◽

Target Dna ◽

Target Effects

SUMMARYAdoption of the CRISPR-Cas system has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I—the most abundant CRISPR system in bacteria—has been less developed. Type I systems in which Cas3 nuclease degrades the target DNA are known; in contrast, for the sub-type CRISPR type I-D (TiD), which lacks a typical Cas3 nuclease in its cascade, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d—a nuclease in TiD—is multi-functional in PAM recognition, stabilization and target DNA degradation. TiD can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. TiD off-target effects, which were dependent on the mismatch position in the protospacer of TiD, were also identified. Our findings suggest TiD as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.

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Functional characterization of the mouse Serpina1 paralog DOM-7

Biological Chemistry ◽

10.1515/hsz-2018-0154 ◽

2018 ◽

Vol 399 (6) ◽

pp. 577-582 ◽

Cited By ~ 1

Author(s):

Karen Jülicher ◽

Annabell Wähner ◽

Kerstin Haase ◽

Karen W. Barbour ◽

Franklin G. Berger ◽

...

Keyword(s):

Genome Editing ◽

Mouse Models ◽

Mouse Strain ◽

Neutrophil Elastase ◽

Genome Engineering ◽

Functional Characterization ◽

Cdna Clones ◽

High Complexity ◽

A1at Deficiency

Abstract The generation of authentic mouse-models for human α1-antitrypsin (A1AT)-deficiency is difficult due to the high complexity of the mouse Serpina1 gene locus. Depending on the exact mouse strain, three to five paralogs are expressed, with different proteinase inhibitory properties. Nowadays with CRISPR-technology, genome editing of complex genomic loci is feasible and could be employed for the generation of A1AT-deficiency mouse models. In preparation of a CRISPR/Cas9-based genome-engineering approach we identified cDNA clones with a functional CDS for the Serpina1-paralog DOM-7. Here, we show that DOM-7 functionally inhibits neutrophil elastase (ELANE) and chymotrypsin, and therefore needs to be considered when aiming at the generation of A1AT-deficient models.

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DNA targeting by Clostridium cellulolyticum CRISPR–Cas9 Type II-C system

Nucleic Acids Research ◽

10.1093/nar/gkz1225 ◽

2020 ◽

Vol 48 (4) ◽

pp. 2026-2034 ◽

Cited By ~ 1

Author(s):

Iana Fedorova ◽

Anatolii Arseniev ◽

Polina Selkova ◽

Georgii Pobegalov ◽

Ignatiy Goryanin ◽

...

Keyword(s):

Genome Editing ◽

Streptococcus Pyogenes ◽

Genome Engineering ◽

Bacterial Species ◽

Type Ii ◽

Biotechnological Applications ◽

Clostridium Cellulolyticum ◽

Dna Targeting ◽

Biochemical Diversity

Abstract Type II CRISPR–Cas9 RNA-guided nucleases are widely used for genome engineering. Type II-A SpCas9 protein from Streptococcus pyogenes is the most investigated and highly used enzyme of its class. Nevertheless, it has some drawbacks, including a relatively big size, imperfect specificity and restriction to DNA targets flanked by an NGG PAM sequence. Cas9 orthologs from other bacterial species may provide a rich and largely untapped source of biochemical diversity, which can help to overcome the limitations of SpCas9. Here, we characterize CcCas9, a Type II-C CRISPR nuclease from Clostridium cellulolyticum H10. We show that CcCas9 is an active endonuclease of comparatively small size that recognizes a novel two-nucleotide PAM sequence. The CcCas9 can potentially broaden the existing scope of biotechnological applications of Cas9 nucleases and may be particularly advantageous for genome editing of C. cellulolyticum H10, a bacterium considered to be a promising biofuel producer.

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