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

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

Nature Communications ◽

10.1038/s41467-021-26518-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Raed Ibraheim ◽

Phillip W. L. Tai ◽

Aamir Mir ◽

Nida Javeed ◽

Jiaming Wang ◽

...

Keyword(s):

Genome Editing ◽

Control Measure ◽

Type I ◽

Adeno Associated Virus ◽

Sequencing Platform ◽

Homology Directed Repair ◽

Safety Risks ◽

Quality Control Measure ◽

Hereditary Tyrosinemia

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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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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Using Transcriptomic Analysis to Assess Double-Strand Break Repair Activity: Towards Precise in Vivo Genome Editing

International Journal of Molecular Sciences ◽

10.3390/ijms21041380 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1380 ◽

Cited By ~ 1

Author(s):

Giovanni Pasquini ◽

Virginia Cora ◽

Anka Swiersy ◽

Kevin Achberger ◽

Lena Antkowiak ◽

...

Keyword(s):

Genome Editing ◽

Mammalian Cells ◽

Time Course ◽

Sequencing Analysis ◽

End Joining ◽

Dsb Repair ◽

Repair Gene ◽

Repair Pathways

Mutations in more than 200 retina-specific genes have been associated with inherited retinal diseases. Genome editing represents a promising emerging field in the treatment of monogenic disorders, as it aims to correct disease-causing mutations within the genome. Genome editing relies on highly specific endonucleases and the capacity of the cells to repair double-strand breaks (DSBs). As DSB pathways are cell-cycle dependent, their activity in postmitotic retinal neurons, with a focus on photoreceptors, needs to be assessed in order to develop therapeutic in vivo genome editing. Three DSB-repair pathways are found in mammalian cells: Non-homologous end joining (NHEJ); microhomology-mediated end joining (MMEJ); and homology-directed repair (HDR). While NHEJ can be used to knock out mutant alleles in dominant disorders, HDR and MMEJ are better suited for precise genome editing, or for replacing entire mutation hotspots in genomic regions. Here, we analyzed transcriptomic in vivo and in vitro data and revealed that HDR is indeed downregulated in postmitotic neurons, whereas MMEJ and NHEJ are active. Using single-cell RNA sequencing analysis, we characterized the dynamics of DSB repair pathways in the transition from dividing cells to postmitotic retinal cells. Time-course bulk RNA-seq data confirmed DSB repair gene expression in both in vivo and in vitro samples. Transcriptomic DSB repair pathway profiles are very similar in adult human, macaque, and mouse retinas, but not in ground squirrel retinas. Moreover, human-induced pluripotent stem-cell-derived neurons and retinal organoids can serve as well suited in vitro testbeds for developing genomic engineering approaches in photoreceptors. Our study provides additional support for designing precise in vivo genome-editing approaches via MMEJ, which is active in mature photoreceptors.

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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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Microhomologies are prevalent at Cas9-induced larger deletions

Nucleic Acids Research ◽

10.1093/nar/gkz459 ◽

2019 ◽

Vol 47 (14) ◽

pp. 7402-7417 ◽

Cited By ~ 20

Author(s):

Dominic D G Owens ◽

Adam Caulder ◽

Vincent Frontera ◽

Joe R Harman ◽

Alasdair J Allan ◽

...

Keyword(s):

Genome Editing ◽

Biomedical Research ◽

High Frequency ◽

Cultured Cells ◽

Mouse Embryos ◽

Deletion Breakpoint ◽

End Joining ◽

Crispr System ◽

Cas9 Nuclease ◽

Target Sites

Abstract The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.

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Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

10.1101/358424 ◽

2018 ◽

Cited By ~ 2

Author(s):

Wannaporn Ittiprasert ◽

Victoria H. Mann ◽

Shannon E. Karinshak ◽

Avril Coghlan ◽

Gabriel Rinaldi ◽

...

Keyword(s):

Schistosoma Mansoni ◽

Genome Editing ◽

Human Macrophage ◽

Wild Type ◽

End Joining ◽

Chromosomal Breakage ◽

Knock Out ◽

Guide Rna ◽

Homology Directed Repair ◽

Non Homologous End Joining

AbstractCRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 (ω1) ofSchistosoma mansonias a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5’-and 3’-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed ∼4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding ω1 were reduced >80% and lysates of ω1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of ω1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

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Highly efficient homology-directed repair using transient CRISPR/Cpf1-geminiviral replicon in tomato

10.1101/521419 ◽

2019 ◽

Cited By ~ 4

Author(s):

Tien Van Vu ◽

Velu Sivankalyani ◽

Eun-Jung Kim ◽

Duong Thi Hai Doan ◽

Mil Thi Tran ◽

...

Keyword(s):

Genome Editing ◽

De Novo ◽

Phenotypic Selection ◽

Culture Conditions ◽

Nonhomologous End Joining ◽

Physical Culture ◽

End Joining ◽

Dark Cycle ◽

Homology Directed Repair ◽

Error Prone Repair

ABSTRACTGenome editing via the homology-directed repair (HDR) pathway in somatic plant cells is very inefficient compared to error-prone repair by nonhomologous end joining (NHEJ). Here, we increased HDR-based genome editing efficiency approximately 3-fold compared to a Cas9-based single-replicon system via the use of de novo multi-replicon systems equipped with CRISPR/LbCpf1 in tomato and obtained replicon-free but stable HDR alleles. The efficiency of CRISPR/LbCpf1-based HDR was significantly modulated by physical culture conditions such as temperature and light. Ten days of incubation at 31°C under a light/dark cycle after Agrobacterium-mediated transformation resulted in the best performance among the tested conditions. Furthermore, we developed our single-replicon system into a multi-replicon system that effectively increased HDR efficiency. Although this approach is still challenging, we showed the feasibility of HDR-based genome editing of a salt-tolerant SlHKT1;2 allele without genomic integration of antibiotic markers or any phenotypic selection. Self-pollinated offspring plants carrying the HKT1;2 HDR allele showed stable inheritance and germination tolerance in the presence of 100 mM NaCl. Our work may pave the way for transgene-free editing of alleles of interest in asexually as well as sexually reproducing plants.

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