Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo

AbstractAdeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.

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Precision Cas9 Genome Editing in vivo with All-in-one, Self-targeting AAV Vectors

10.1101/2020.10.09.333997 ◽

2020 ◽

Author(s):

Raed Ibraheim ◽

Phillip W. L. Tai ◽

Aamir Mir ◽

Nida Javeed ◽

Jiaming Wang ◽

...

Keyword(s):

Genome Editing ◽

Type I ◽

End Joining ◽

Adeno Associated Virus ◽

Homology Directed Repair ◽

Safety Risks ◽

Target Sites ◽

Hereditary Tyrosinemia ◽

Mps I

AbstractAdeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits, especially for large effectors like Cas9s. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. The use of compact effectors has enabled single-AAV delivery of Cas9s with 1-3 guides for edits that use end-joining repair pathways, but many precise edits that correct disease-causing mutations in vivo require homology-directed repair (HDR) templates. Here, we describe single-vector, ∼4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs to produce segmental deletions, or a single sgRNA with an HDR template. We also examine the utility of Nme2Cas9 target sites in the vector for self-inactivation. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice. These results will enable single-vector AAVs to achieve diverse therapeutic genome editing outcomes.

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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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Using the Endogenous CRISPR-Cas System of Heliobacterium modesticaldum To Delete the Photochemical Reaction Center Core Subunit Gene

Applied and Environmental Microbiology ◽

10.1128/aem.01644-19 ◽

2019 ◽

Vol 85 (23) ◽

Cited By ~ 3

Author(s):

Patricia L. Baker ◽

Gregory S. Orf ◽

Kimberly Kevershan ◽

Michael E. Pyne ◽

Taner Bicer ◽

...

Keyword(s):

Homologous Recombination ◽

Genome Editing ◽

Reaction Center ◽

Photochemical Reaction ◽

Genetic Locus ◽

Gene Replacement ◽

Type I ◽

Content Type ◽

Heliobacterium Modesticaldum

ABSTRACT In Heliobacterium modesticaldum, as in many Firmicutes, deleting genes by homologous recombination using standard techniques has been extremely difficult. The cells tend to integrate the introduced plasmid into the chromosome by a single recombination event rather than perform the double recombination required to replace the targeted locus. Transformation with a vector containing only a homologous recombination template for replacement of the photochemical reaction center gene pshA produced colonies with multiple genotypes, rather than a clean gene replacement. To address this issue, we required an additional means of selection to force a clean gene replacement. In this study, we report the genetic structure of the type I-A and I-E CRISPR-Cas systems from H. modesticaldum, as well as methods to leverage the type I-A system for genome editing. In silico analysis of the CRISPR spacers revealed a potential consensus protospacer adjacent motif (PAM) required for Cas3 recognition, which was then tested using an in vivo interference assay. Introduction of a homologous recombination plasmid that carried a miniature CRISPR array targeting sequences in pshA (downstream of a naturally occurring PAM sequence) produced nonphototrophic transformants with clean replacements of the pshA gene with ∼80% efficiency. Mutants were confirmed by PCR, sequencing, optical spectroscopy, and growth characteristics. This methodology should be applicable to any genetic locus in the H. modesticaldum genome. IMPORTANCE The heliobacteria are the only phototrophic members of the largely Gram-positive phylum Firmicutes, which contains medically and industrially important members, such as Clostridium difficile and Clostridium acetobutylicum. Heliobacteria are of interest in the study of photosynthesis because their photosynthetic system is unique and the simplest known. Since their discovery in the early 1980s, work on the heliobacteria has been hindered by the lack of a genetic transformation system. The problem of introducing foreign DNA into these bacteria has been recently rectified by our group; however, issues still remained for efficient genome editing. The significance of this work is that we have characterized the endogenous type I CRISPR-Cas system in the heliobacteria and leveraged it to assist in genome editing. Using the CRISPR-Cas system allowed us to isolate transformants with precise replacement of the pshA gene encoding the main subunit of the photochemical reaction center.

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Supramolecular nanosubstrate–mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies

Science Advances ◽

10.1126/sciadv.abb7107 ◽

2020 ◽

Vol 6 (43) ◽

pp. eabb7107

Author(s):

Peng Yang ◽

Shih-Jie Chou ◽

Jindian Li ◽

Wenqiao Hui ◽

Wenfei Liu ◽

...

Keyword(s):

Fluorescent Protein ◽

Integration Site ◽

Genetic Diseases ◽

Proliferative Potential ◽

Adeno Associated Virus ◽

Guide Rna ◽

Homology Directed Repair ◽

Green Fluorescent ◽

Virus Integration

Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate–mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)–encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies.

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Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo

Hepatology ◽

10.1002/hep.23481 ◽

2009 ◽

Vol 51 (4) ◽

pp. 1200-1208 ◽

Cited By ~ 78

Author(s):

Nicole K. Paulk ◽

Karsten Wursthorn ◽

Zhongya Wang ◽

Milton J. Finegold ◽

Mark A. Kay ◽

...

Keyword(s):

Mouse Model ◽

Gene Repair ◽

Adeno Associated Virus ◽

Virus Gene ◽

Hereditary Tyrosinemia

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Enhanced CRISPR-Cas9 correction of Duchenne muscular dystrophy in mice by a self-complementary AAV delivery system

Science Advances ◽

10.1126/sciadv.aay6812 ◽

2020 ◽

Vol 6 (8) ◽

pp. eaay6812 ◽

Cited By ~ 18

Author(s):

Yu Zhang ◽

Hui Li ◽

Yi-Li Min ◽

Efrain Sanchez-Ortiz ◽

Jian Huang ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Genome Editing ◽

Dystrophin Gene ◽

Adeno Associated Virus ◽

Guide Rnas ◽

Dystrophin Expression ◽

Cas9 Nuclease ◽

High Doses

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the dystrophin gene (DMD). Previously, we applied CRISPR-Cas9–mediated “single-cut” genome editing to correct diverse genetic mutations in animal models of DMD. However, high doses of adeno-associated virus (AAV) are required for efficient in vivo genome editing, posing challenges for clinical application. In this study, we packaged Cas9 nuclease in single-stranded AAV (ssAAV) and CRISPR single guide RNAs in self-complementary AAV (scAAV) and delivered this dual AAV system into a mouse model of DMD. The dose of scAAV required for efficient genome editing were at least 20-fold lower than with ssAAV. Mice receiving systemic treatment showed restoration of dystrophin expression and improved muscle contractility. These findings show that the efficiency of CRISPR-Cas9–mediated genome editing can be substantially improved by using the scAAV system. This represents an important advancement toward therapeutic translation of genome editing for DMD.

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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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In vivo suppressor mutations correct a murine model of hereditary tyrosinemia type I

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.96.21.11928 ◽

1999 ◽

Vol 96 (21) ◽

pp. 11928-11933 ◽

Cited By ~ 37

Author(s):

K. Manning ◽

M. Al-Dhalimy ◽

M. Finegold ◽

M. Grompe

Keyword(s):

Murine Model ◽

Type I ◽

Suppressor Mutations ◽

Tyrosinemia Type I ◽

Hereditary Tyrosinemia

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Assessing the efficacy of CRISPR/Cas9 genome editing in the wheat pathogen Parastagonspora nodorum

10.21203/rs.2.23248/v1 ◽

2020 ◽

Author(s):

Haseena Khan ◽

Megan C McDonald ◽

Simon J Willams ◽

Peter Solomon

Keyword(s):

Homologous Recombination ◽

Genome Editing ◽

Pathogenic Fungus ◽

Point Mutations ◽

Dna Breaks ◽

Gene Manipulation ◽

Homology Directed Repair ◽

Rnp Complex ◽

Parastagonospora Nodorum

Abstract Background: The genome-editing tool CRISPR/Cas9 has revolutionized gene manipulation by providing an efficient method to generate targeted mutations. This technique deploys the Cas9 endonuclease and a guide RNA (gRNA) which interact to form a Cas9-gRNA complex that initiates gene editing through the introduction of double stranded DNA breaks. We tested the efficacy of the CRISPR/Cas9 approach as a means of facilitating a variety of reverse genetic approaches in the wheat pathogenic fungus Parastagonospora nodorum . Results: Parastagonospora nodorum protoplasts were transformed with the Cas9 protein and gRNA in the form of a preassembled ribonuclear protein (RNP) complex targeting the Tox3 effector gene. Subsequent screening of the P. nodorum transformants revealed 100% editing of those mutants screened. We further tested the efficacy of RNP complex when co-transformed with a Tox3 -Homology Directed Repair cassette harbouring 1 kb of homologous flanking DNA. Subsequent screening of resulting transformants demonstrated homologous recombination efficiencies exceeding 70%. A further transformation with a Tox3 -Homology Directed Repair cassette harbouring a selectable marker with 50 bp micro-homology flanks was also achieved 25% homologous recombination efficiency. The success of these homology directed repair approaches demonstrate that CRISPR/Cas9 is amenable to other in vivo DNA manipulation approaches such as the insertion of DNA and generating point mutations. Conclusion: These data highlight the significant potential that CRISPR/Cas9 has in expediting gene transgene-free knockouts in Parastagonospora nodorum and also in facilitating other gene manipulation approaches. Access to these tools will significantly decrease the time required to assess the requirement of gene for disease and to undertake functional studies to determine its role.

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Retron Editing for Precise Genome Editing without Exogenous Donor DNA in Human Cells

10.1101/2021.05.11.443596 ◽

2021 ◽

Author(s):

Xiangfeng Kong ◽

Zikang Wang ◽

Yingsi Zhou ◽

Xing Wang ◽

Linyu Shi ◽

...

Keyword(s):

Genome Editing ◽

Ex Vivo ◽

Human Cells ◽

Exogenous Dna ◽

Guide Rna ◽

Homology Directed Repair ◽

Non Coding Rna ◽

Bacterial Genetic ◽

Low Efficiency

CRISPR-Cas9 mediated seamless genome editing can be achieved by incorporating donor DNA into the CRISPR-Cas9 target loci via homology-directed repair (HDR), albeit with relative low efficiency due to the inefficient delivery of exogenous DNA. Retrons are bacterial genetic element composed of a non-coding RNA (ncRNA) and reverse transcriptase (RT). Retrons coupled with CRISPR-Cas9 have been shown to enhance precise genome editing via HDR in yeast through fusing guide RNA (gRNA) to the 3′ end of retron ncRNA, producing multicopy single-stranded DNA (msDNA) covalently tethered to gRNA. Here, we further engineered retrons by fusing Cas9 with E.coli RT from different clades and joining gRNA at the 5′ end of retron ncRNA, and found that retron editing can achieve precise genome editing efficiently in human cells. By co- expression of Cas9-RT fusions and retron-ncRNA gRNA (rgRNA) in HEK293T cells, we demonstrated the rates of retron editing at endogenous genomic loci was up to 10 %. We expect our retron editing system could aid in advancing the ex vivo and in vivo therapeutic applications of retron.

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