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Efficient in vivo labeling of endogenous proteins with SMART delineates retina cellular and synaptic organization.
A key application of CRISPR/Cas9-based genomic editing is modification of genes to introduce engineered sequences. However, the editing flexibility is severely constrained by the requirement for targeting sites in proximity to the desired modification site, which makes many modifications intractable. Here, we develop a strategy that overcomes this key limitation to allow CRISPR-based editing at any position with high efficiency. It relies on reconstructing the targeted gene using Silently Mutate And Repair Template (SMART) where we mutate the gap sequence in the repair template to prevent its base pairing with the target DNA while maintaining the same amino acid coding. Using vertebrate retina as a neuronal model system we document the application of SMART editing for labeling endogenous proteins in vivo with high efficiency. We show that SMART editing allows us to access numerous cell types in the retina and address fundamental cell biological questions pertaining to its organization. We propose that this approach will facilitate functional genomic studies in a wide range of systems and increase the precision of corrective gene therapies.
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Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates.
Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle cell disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the β-globin locus (HBB). We compared the activity of zinc-finger nucleases (ZFNs) and CRISPR/Cas9 for editing, and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODNs), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLVs), and adenovirus 5/35 serotype (Ad5/35) to transvert the base pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. In vitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base pair transversions were significantly reduced when analyzing cells in vivo in immunodeficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODNs. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high-efficiency gene correction in HSPCs.
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CRISPR-Cas9 recognition of enzymatically synthesized base-modified nucleic acids.
An enzymatic method has been successfully established enabling the generation of partially base-modified RNA (previously named RZA) constructs, in which all G residues were replaced by isomorphic fluorescent thienoguanosine (thG) analogs, as well as fully modified RZA featuring thG, 5-bromocytosine, 7-deazaadenine and 5-chlorouracil. The transcriptional efficiency of emissive fully modified RZA was found to benefit from the use of various T7 RNA polymerase variants. Moreover, dthG could be incorporated into PCR products by Taq DNA polymerase together with the other three base-modified nucleotides. Notably, the obtained RNA products containing thG as well as thG together with 5-bromocytosine could function as effectively as natural sgRNAs in an in vitro CRISPR-Cas9 cleavage assay. N1-Methylpseudouridine was also demonstrated to be a faithful non-canonical substitute of uridine to direct Cas9 nuclease cleavage when incorporated in sgRNA. The Cas9 inactivation by 7-deazapurines indicated the importance of the 7-nitrogen atom of purines in both sgRNA and PAM site for achieving efficient Cas9 cleavage. Additional aspects of this study are discussed in relation to the significance of sgRNA-protein and PAM--protein interactions that were not highlighted by the Cas9-sgRNA-DNA complex crystal structure. These findings could expand the impact and therapeutic value of CRISPR-Cas9 and other RNA-based technologies.
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Application of the CRISPR/Cas9 system for modification of flower color in Torenia fournieri.
BACKGROUND: CRISPR/Cas9 technology is one of the most powerful and useful tools for genome editing in various living organisms. In higher plants, the system has been widely exploited not only for basic research, such as gene functional analysis, but also for applied research such as crop breeding. Although the CRISPR/Cas9 system has been used to induce mutations in genes involved in various plant developmental processes, few studies have been performed to modify the color of ornamental flowers. We therefore attempted to use this system to modify flower color in the model plant torenia (Torenia fournieri L.). RESULTS: We attempted to induce mutations in the torenia flavanone 3-hydroxylase (F3H) gene, which encodes a key enzyme involved in flavonoid biosynthesis. Application of the CRISPR/Cas9 system successfully generated pale blue (almost white) flowers at a high frequency (ca. 80% of regenerated lines) in transgenic torenia T0 plants. Sequence analysis of PCR amplicons by Sanger and next-generation sequencing revealed the occurrence of mutations such as base substitutions and insertions/deletions in the F3H target sequence, thus indicating that the obtained phenotype was induced by the targeted mutagenesis of the endogenous F3H gene. CONCLUSIONS: These results clearly demonstrate that flower color modification by genome editing with the CRISPR/Cas9 system is easily and efficiently achievable. Our findings further indicate that this system may be useful for future research on flower pigmentation and/or functional analyses of additional genes in torenia.
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Core Hairpin Structure of SpCas9 sgRNA Functions in a Sequence- and Spatial Conformation-Dependent Manner.
The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is a widely used genome-editing tool with great clinical potential. However, its application is limited because of low editing efficiency of some target sequences and off-target effects. As this system contains only the Cas9 protein and a single-guide RNA (sgRNA; engineered from crRNA and tracrRNA), the structure and function of these components should be studied in detail to address the current clinical needs. Consequently, we investigated the structural and sequence features of the core hairpin (the first stem loop of sgRNA) of SpCas9 sgRNA. We showed that the core hairpin structure of sgRNA is essential for SpCas9/sgRNA-mediated DNA cleavage and that the internal loop structure in the core hairpin plays a vital role in target DNA cleavage. We observed that the root stem structure within the core hairpin preferentially forms Watson-Crick base pairs and should be of a specific length to maintain an appropriate spatial conformation for Cas9 binding. However, the length of the leaf stem structure of the core hairpin is flexible, having a variable nucleotide composition. Furthermore, extension of the leaf stem structure enhances the DNA cleavage activity of the Cas9/sgRNA complex, and this could be used to enhance the efficiency of gene editing. These observations provide insight into the sgRNA/Cas9 interaction, indicating that sgRNA modification could be a strategy for improved DNA editing efficiency, and optimized sgRNA can be further used for genome-wide functional screening and clinical application.
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Adenovirus vectors in hematopoietic stem cell genome editing.
Genome editing of hematopoietic stem cells (HSCs) represents a therapeutic option for a number of hematological genetic diseases, as HSCs have the potential for self-renewal and differentiation into all blood cell lineages. This review presents advances of genome editing in HSCs utilizing adenovirus vectors as delivery vehicles. We focus on capsid-modified, helper-dependent adenovirus vectors that are devoid of all viral genes and therefore exhibit an improved safety profile. We discuss HSC genome engineering for several inherited disorders and infectious diseases including hemoglobinopathies, Fanconi anemia, hemophilia, and HIV-1 infection by ex vivo and in vivo editing in transgenic mice, nonhuman primates, as well as in human CD34+ cells. Mechanisms of therapeutic gene transfer including episomal expression of designer nucleases and base editors, transposase-mediated random integration, and targeted homology-directed repair triggered integration into selected genomic safe harbor loci are also reviewed.
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Directed evolution of adenine base editors with increased activity and therapeutic application.
The foundational adenine base editors (for example, ABE7.10) enable programmable A•T to G•C point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG protospacer adjacent motif (PAM) sites, ABE8s result in ~1.5× higher editing at protospacer positions A5-A7 and ~3.2× higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ~4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2 with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification, which is maintained when multiplexed across three loci. Delivered as messenger RNA, ABE8s induce no significant levels of single guide RNA (sgRNA)-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.
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Construct design for CRISPR/Cas-based genome editing in plants.
CRISPR construct design is a key step in the practice of genome editing, which includes identification of appropriate Cas proteins, design and selection of guide RNAs (gRNAs), and selection of regulatory elements to express gRNAs and Cas proteins. Here, we review the choices of CRISPR-based genome editors suited for different needs in plant genome editing applications. We consider the technical aspects of gRNA design and the associated computational tools. We also discuss strategies for the design of multiplex CRISPR constructs for high-throughput manipulation of complex biological processes or polygenic traits. We provide recommendations for different elements of CRISPR constructs and discuss the remaining challenges of CRISPR construct optimization in plant genome editing.
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An overview and potential of CRISPR-Cas systems for genome editing.
Genome editing involves altering of the DNA in organisms including bacteria, plants, and animals using molecular scissors that helps in treatment and diagnosis of various diseases. Genome editing technology is exponentially growing and have been developed for enabling precise genomic alterations and the addition, removal, and correction of genes. These modifications begin with the creation of double-stranded breaks (DSBs) that is generated by nucleases and can be joined through homology-directed repair (HDR) or non-homologous end-joining (NHEJ). NHEJ is quick but increases mutation chances due to deletions and insertions of nucleotides at the break site, while HDR uses homologous templates for precise repair and targeted DNA specific to the gene or sequence. Other methods such as zinc-finger protein is a transcription factor that binds with DNA and binds specific to that sequence, which uniquely recognise 3-base pairs of DNA. TALENs consists of two domains: TALE domain, a transcription activator and FokI that is a restriction endonuclease that cuts the DNA at specific sites. CRISPR-Cas systems are clustered regularly interspersed short palindromic repeats present in various bacterial species. These sequences activate RNA-guided DNA cleavage, aiding in the development of an adaptive immune defence against foreign DNA. CRISPR-Cas9 is widely used for genome editing, regulation, diagnostic and many.
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Re-engineered RNA-Guided FokI-Nucleases for Improved Genome Editing in Human Cells.
Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 enables us to generate targeted sequence changes in the genomes of cells and organisms. However, off-target effects have been a persistent problem hampering the development of therapeutics based on CRISPR/Cas9 and potentially confounding research results. Efforts to improve Cas9 specificity, like the development of RNA-guided FokI-nucleases (RFNs), usually come at the cost of editing efficiency and/or genome targetability. To overcome these limitations, we engineered improved chimeras of RFNs that enable higher cleavage efficiency and provide broader genome targetability, while retaining high fidelity for genome editing in human cells. Furthermore, we developed a new RFN ortholog derived from Staphylococcus aureus Cas9 and characterize its utility for efficient genome engineering. Finally, we demonstrate the feasibility of RFN orthologs to functionally hetero-dimerize to modify endogenous genes, unveiling a new dimension of RFN target design opportunities.
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