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CRISPR-based modular assembly of a UAS-cDNA/ORF plasmid library for more than 5500 Drosophila genes conserved in humans.
Construction of a genome-wide transgenic UAS-cDNA/ORF library in Drosophila based on the binary GAL4/UAS system has been severely hampered by technical difficulties, although genome-wide cDNA or ORF resources of Drosophila, human, and mouse have been publicly available for more than a decade. Here, we developed a new method named CRISPR-based modular assembly (CRISPRmass) for the high-throughput construction of a genome-wide UAS-cDNA/ORF library from publicly available cDNA/ORF resources. Through cleavage of shared vector sequences of cDNA/ORF plasmids by CRISPR/Cas9 and subsequent insertion of UAS modules by Gibson assembly, the procedure of construction of such a library by CRISPRmass is standardized as massively parallel two-step test tube reactions before bacterial transformation. Using CRISPRmass, we generated 5551 UAS-cDNA/ORF constructs covering 83% of the Drosophila genes conserved in humans in the Drosophila Genomics Resource Center (DGRC) Gold Collection, and among them, 5518 were generated within 3 mo by three people. Our results show that CRISPRmass allows modulization, simplicity, efficiency, and adaptability in the generation of a genome-wide UAS-cDNA/ORF plasmid library by using publicly available cDNA/ORF resources. CRISPRmass can be applied to editing various genome-wide libraries in general and is an alternative to Gateway technology in high-throughput plasmid library editing. Furthermore, the more than 5500 UAS-cDNA/ORF plasmids of Drosophila genes serve as a powerful resource for gain-of-function (GOF) screening in cultured cells and for generation of a transgenic UAS-cDNA/ORF library in Drosophila.
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Genome Editing VEGFA Prevents Corneal Neovascularization In Vivo.
Corneal neovascularization (CNV) is a common clinical finding seen in a range of eye diseases. Current therapeutic approaches to treat corneal angiogenesis, in which vascular endothelial growth factor (VEGF) A plays a central role, can cause a variety of adverse side effects. The technology of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 can edit VEGFA gene to suppress its expression. CRISPR offers a novel opportunity to treat CNV. This study shows that depletion of VEGFA with a novel CRISPR/Cas9 system inhibits proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Importantly, subconjunctival injection of this dual AAV-SpCas9/sgRNA-VEGFA system is demonstrated which blocks suture-induced expression of VEGFA, CD31, and α-smooth muscle actin as well as corneal neovascularization in mice. This study has established a strong foundation for the treatment of corneal neovascularization via a gene editing approach for the first time.
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Transgene integration into the human AAVS1 locus enhances myosin II-dependent contractile force by reducing expression of myosin binding subunit 85.
The adeno-associated virus site 1 (AAVS1) locus in the human genome is a strong candidate for gene therapy by insertion of an exogenous gene into the locus. The AAVS1 locus includes the coding region for myosin binding subunit 85 (MBS85). Although the function of MBS85 is not well understood, myosin II-dependent contractile force may be affected by altered expression of MBS85. The effect of altered expression of MBS85 on cellular contractile force should be examined prior to the application of gene therapy. In this study, we show that transgene integration into AAVS1 and consequent reduction of MBS85 expression changes myosin II-dependent cellular contractile force. We established a human fibroblast cell line with exogenous DNA knocked-in to AAVS1 (KI cells) using the CRISPR/Cas9 genome editing system. Western blotting analysis showed that KI cells had significantly reduced MBS85 expression. KI cells also showed greater cellular contractile force than control cells. The increased contractile force was associated with phosphorylation of the myosin II regulatory light chain (MRLC). Transfection of KI cells with an MBS85 expression plasmid restored cellular contractile force and phosphorylation of MRLC to the levels in control cells. These data suggest that transgene integration into the human AAVS1 locus induces an increase in cellular contractile force and thus should be considered as a gene therapy to effect changes in cellular contractile force.
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Application of genome engineering for treatment of retinal diseases.
Genome engineering with clustered regularly interspaced short palindromic repeats (CRISPR) system can be used as a tool to correct pathological mutations or modulate gene expression levels associated with pathogenesis of human diseases. Owing to well-established local administration methods including intravitreal and subretinal injection, it is relatively easy to administer therapeutic genome engineering machinery to ocular tissues for treating retinal diseases. In this context, we have investigated the potential of in vivo genome engineering as a therapeutic approach in the form of ribonucleoprotein or CRISPR packaged in viral vectors. Major issues in therapeutic application of genome engineering include specificity and efficacy according to types of CRISPR system. In addition to previous platforms based on ribonucleoprotein and CRISPRassociated protein 9 derived from Campylobacter jejuni, we evaluated the therapeutic effects of a CRISPR RNA-guided endonuclease derived from Lachnospiraceae bacterium ND2006 (LbCpf1) in regulating pathological angiogenesis in an animal model of wet-type age-related macular degeneration. LbCpf1 targeting Vegfa or Hif1a effectively disrupted the expression of genes in ocular tissues, resulting in suppression of choroidal neovascularization. It was also notable that there were no significant off-target effects in vivo. [BMB Reports 2018; 51(7): 315-316].
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Catecholaminergic cell type-specific expression of Cre recombinase in knock-in transgenic rats generated by the Combi-CRISPR technology.
BACKGROUND: Cell groups containing catecholamines provide a useful model to study the molecular and cellular mechanisms underlying the morphogenesis, physiology, and pathology of the central nervous system. For this purpose, it is necessary to establish a system to induce catecholaminergic group-specific expression of Cre recombinase. Recently, we introduced a gene cassette encoding 2A peptide fused to Cre recombinase into the site between the C-terminus and translational termination codons of the rat tyrosine hydroxylase (TH) open reading frame by the Combi-CRISPR technology, which is a genomic editing method to enable an efficient knock-in (KI) of long DNA sequence into a target site. However, the expression patterns of the transgene and its function as well as the effect of the mutation on the biochemical and behavioral phenotypes in the KI strains have not been characterized yet. NEW METHOD: We aimed to evaluate the usefulness of TH-Cre KI rats as an experimental model for investigating the structure and function of catecholaminergic neurons in the brain. RESULTS: We detected cell type-specific expression of Cre recombinase and site-specific recombination activity in the representative catecholaminergic groups in the TH-Cre KI rat strains. In addition, we measured TH protein levels and catecholamine accumulation in the brain regions, as well as motor, reward-related, and anxiety-like behaviors, indicating that catecholamine metabolism and general behavior are apparently normal in these KI rats. CONCLUSIONS: TH-Cre KI rat strains produced by the Combi-CRISPR system offer a beneficial model to study the molecular and cellular mechanics for the morphogenesis, physiology, and pathology of catecholamine-containing neurons in the brain.
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RNA-guided gene activation by CRISPR-Cas9-based transcription factors.
Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.
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AcrIIA5 Suppresses Base Editors and Reduces Their Off-Target Effects.
The CRISPR/nCas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) are capable of catalyzing C•G to T•A or A•T to G•C conversions, respectively, and have become new, powerful tools for achieving precise genetic changes in a wide range of organisms. These base editors hold great promise for correcting pathogenic mutations and for being used for therapeutic applications. However, the recognition of cognate DNA sequences near their target sites can cause severe off-target effects that greatly limit their clinical applications, and this is an urgent problem that needs to be resolved for base editing systems. The recently discovered phage-derived proteins, anti-CRISPRs, which can suppress the natural CRISPR nuclease activity, may be able to ameliorate the off-target effects of base editing systems. Here, we confirm for the first time that AcrIIA2, AcrIIA4, and AcrIIA5 efficiently inhibit base editing systems in human cells. In particular, AcrIIA5 has a significant inhibitory effect on all base editing variant systems tested in our study. We further show that the off-target effects of BE3 and ABE7.10 were significantly reduced in AcrIIA5 treated cells. This study suggests that AcrIIA5 should be widely used for the precise control of base editing and to thoroughly "shut off" nuclease activity of both CBE and ABE systems.
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Single-step generation of gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9.
Specific gene knockout and rescue experiments are powerful tools in developmental and stem cell biology. Nevertheless, the experiments require multiple steps of molecular manipulation for gene knockout and subsequent rescue procedures. Here we report an efficient and single step strategy to generate gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9 genome editing technology. We inserted a tetracycline-regulated inducible gene promoter (tet-OFF/TRE-CMV) upstream of the endogenous promoter region of vascular endothelial growth factor receptor 2 (VEGFR2/Flk1) gene, an essential gene for endothelial cell (EC) differentiation, in mouse embryonic stem cells (ESCs) with homologous recombination. Both homo- and hetero-inserted clones were efficiently obtained through a simple selection with a drug-resistant gene. The insertion of TRE-CMV promoter disrupted endogenous Flk1 expression, resulting in null mutation in homo-inserted clones. When the inserted TRE-CMV promoter was activated with doxycycline (Dox) depletion, Flk1 expression was sufficiently recovered from the downstream genomic Flk1 gene. Whereas EC differentiation was almost completely perturbed in homo-inserted clones, Flk1 rescue with TRE-CMV promoter activation restored EC appearance, indicating that phenotypic changes in EC differentiation can be successfully reproduced with this knockout-rescue system. Thus, this promoter insertion strategy with CRISPR/Cas9 would be a novel attractive method for knockout-rescue experiments.
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Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants.
BACKGROUND: Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing have led to the use of long single-stranded DNA (lssDNA) molecules for generating conditional mutations. However, there is still limited available data on the efficiency and reliability of this method. RESULTS: We generated conditional mouse alleles using lssDNA donor templates and performed extensive characterization of the resulting mutations. We observed that the use of lssDNA molecules as donors efficiently yielded founders bearing the conditional allele, with seven out of nine projects giving rise to modified alleles. However, rearranged alleles including nucleotide changes, indels, local rearrangements and additional integrations were also frequently generated by this method. Specifically, we found that alleles containing unexpected point mutations were found in three of the nine projects analyzed. Alleles originating from illegitimate repairs or partial integration of the donor were detected in eight projects. Furthermore, additional integrations of donor molecules were identified in four out of the seven projects analyzed by copy counting. This highlighted the requirement for a thorough allele validation by polymerase chain reaction, sequencing and copy counting of the mice generated through this method. We also demonstrated the feasibility of using lssDNA donors to generate thus far problematic point mutations distant from active CRISPR cutting sites by targeting two distinct genes (Gckr and Rims1). We propose a strategy to perform extensive quality control and validation of both types of mouse models generated using lssDNA donors. CONCLUSION: lssDNA donors reproducibly generate conditional alleles and can be used to introduce point mutations away from CRISPR/Cas9 cutting sites in mice. However, our work demonstrates that thorough quality control of new models is essential prior to reliably experimenting with mice generated by this method. These advances in genome editing techniques shift the challenge of mutagenesis from generation to the validation of new mutant models.
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Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability.
Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activity in vitro for 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.
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