Imaging Method Using CRISPR/dCas9 and Engineered gRNA Scaffolds Can Perturb Replication Timing at the HSPA1 Locus.

Fluorescence microscopy imaging of specific chromosomal sites is essential for genome architecture research. To enable visualization of endogenous loci in mammalian cells, programmable DNA-binding proteins such as TAL effectors and CRISPR/dCas9 are commonly utilized. In addition, site-specific insertion of a TetO repeat array, coupled with TetR-enhanced green fluorescent protein fusion protein expression, can be used for labeling nonrepetitive endogenous loci. Here, we performed a comparison of several live-cell chromosome tagging methods, including their effect on subnuclear positioning, expression of adjacent genes, and DNA replication timing. Our results showed that the CRISPR-based imaging method can delay DNA replication timing and sister chromatid resolution at certain region. However, subnuclear localization of the labeled locus and gene expression from adjacent loci were unaffected by either TetO/TetR or CRISPR-based methods, suggesting that CRISPR-based imaging could be used for applications that do not require DNA replication analysis.

Engineering inducible biomolecular assemblies for genome imaging and manipulation in living cells.

Genome architecture and organization play critical roles in cell life. However, it remains largely unknown how genomic loci are dynamically coordinated to regulate gene expression and determine cell fate at the single cell level. We have developed an inducible system which allows Simultaneous Imaging and Manipulation of genomic loci by Biomolecular Assemblies (SIMBA) in living cells. In SIMBA, the human heterochromatin protein 1α (HP1α) is fused to mCherry and FRB, which can be induced to form biomolecular assemblies (BAs) with FKBP-scFv, guided to specific genomic loci by a nuclease-defective Cas9 (dCas9) or a transcriptional factor (TF) carrying tandem repeats of SunTag. The induced BAs can not only enhance the imaging signals at target genomic loci using a single sgRNA, either at repetitive or non-repetitive sequences, but also recruit epigenetic modulators such as histone methyltransferase SUV39H1 to locally repress transcription. As such, SIMBA can be applied to simultaneously visualize and manipulate, in principle, any genomic locus with controllable timing in living cells.

Precise Regulation of Cas9-Mediated Genome Engineering by Anti-CRISPR-Based Inducible CRISPR Controllers.

CRISPR/Cas9 is a powerful genome editing tool, but its off-target cleavage activity can result in unintended adverse outcomes for therapeutic applications. Here we report the design of a simple tunable CRISPR controller in which a chemically inducible anti-CRISPR protein AcrIIA4 is engineered to disable Cas9 DNA binding upon the addition of trimethoprim. Dose-dependent control over Cas9 editing and dCas9 induction was achieved, which drastically improved the specificity and biosafety of the CRISPR/Cas9 system. We utilized the anti-CRISPR protein AcrIIA4 as a means to interfere with Cas9 DNA binding activity. By fusing AcrIIA4 to a ligand-inducible destabilization domain DHFR(DD), we show significantly reduced off-target activity in mammalian cells. Furthermore, we describe a new inducible promoter system Acr-OFF based on CRISPR controllers, which is regulated by an FDA-approved ligand trimethoprim.

Replication timing maintains the global epigenetic state in human cells.

The temporal order of DNA replication [replication timing (RT)] is correlated with chromatin modifications and three-dimensional genome architecture; however, causal links have not been established, largely because of an inability to manipulate the global RT program. We show that loss of RIF1 causes near-complete elimination of the RT program by increasing heterogeneity between individual cells. RT changes are coupled with widespread alterations in chromatin modifications and genome compartmentalization. Conditional depletion of RIF1 causes replication-dependent disruption of histone modifications and alterations in genome architecture. These effects were magnified with successive cycles of altered RT. These results support models in which the timing of chromatin replication and thus assembly plays a key role in maintaining the global epigenetic state.

Expanding the Potential of Mammalian Genome Engineering via Targeted DNA Integration.

Inserting custom designed DNA sequences into the mammalian genome plays an essential role in synthetic biology. In particular, the ability to introduce foreign DNA in a site-specific manner offers numerous advantages over random DNA integration. In this review, we focus on two mechanistically distinct systems that have been widely adopted for targeted DNA insertion in mammalian cells, the CRISPR/Cas9 system and site-specific recombinases. The CRISPR/Cas9 system has revolutionized the genome engineering field thanks to its high programmability and ease of use. However, due to its dependence on linearized DNA donor and endogenous cellular pathways to repair the induced double-strand break, CRISPR/Cas9-mediated DNA insertion still faces limitations such as small insert size, and undesired editing outcomes via error-prone repair pathways. In contrast, site-specific recombinases, in particular the Serine integrases, demonstrate large-cargo capability and no dependence on cellular repair pathways for DNA integration. Here we first describe recent advances in improving the overall efficacy of CRISPR/Cas9-based methods for DNA insertion. Moreover, we highlight the advantages of site-specific recombinases over CRISPR/Cas9 in the context of targeted DNA integration, with a special focus on the recent development of programmable recombinases. We conclude by discussing the importance of protein engineering to further expand the current toolkit for targeted DNA insertion in mammalian cells.

Two-Color Imaging of Nonrepetitive Endogenous Loci in Human Cells.

Tools for live cell imaging of multiple nonrepetitive genomic loci in mammalian cells are necessary to study chromatin dynamics. Here, we report a new system based on two chromosomally integrated orthogonal irregular repeat arrays and particularly a new general strategy to construct irregular repeat arrays. Briefly, we utilized a "bridge oligonucleotide-mediated ligation" protocol to assemble 8-mer repeats de novo which were then combined into a final 96-mer repeat array using Golden Gate cloning. This strategy was used for assembling a new mutant TetO irregular repeat array, which worked orthogonally to the wild type TetO repeat. Single copy integration of the new repeat array did not cause replication deficiencies at the tagged locus. Moreover, the mutant TetO irregular repeat could also be visualized by CRISPR imaging. Our new irregular repeat assembly method demonstrates a generally applicable strategy that can be used for assembling additional orthogonal repeat arrays for imaging genomic loci and irregular repeats to visualize RNA or proteins via signal amplification.

Publisher Correction: An efficient gene knock-in strategy using 5'-modified double-stranded DNA donors with short homology arms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An efficient gene knock-in strategy using 5'-modified double-stranded DNA donors with short homology arms.

Here, we report a rapid CRISPR-Cas9-mediated gene knock-in strategy that uses Cas9 ribonucleoprotein and 5'-modified double-stranded DNA donors with 50-base-pair homology arms and achieved unprecedented 65/40% knock-in rates for 0.7/2.5 kilobase inserts, respectively, in human embryonic kidney 293T cells. The identified 5'-end modification led to up to a fivefold increase in gene knock-in rates at various genomic loci in human cancer and stem cells.

CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci.

Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a method for tracking any locus of interest is desirable. Recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or transcription activator-like effectors were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions without the need for a pre-existing repetitive sequence. SHACKTeR requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by polymerase chain reaction, was successful at labeling 10 different loci in HCT116 cells. We also showed the feasibility of knock-in into lamina-associated, heterochromatin regions, demonstrating that these regions prefer non-homologous end joining for knock-in. Using SHACKTeR, we were able to observe DNA replication at a specific locus by long-term live cell imaging. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision.

We developed a CRISPR-Cas9- and homology-directed-repair-assisted genome-scale engineering method named CHAnGE that can rapidly output tens of thousands of specific genetic variants in yeast. More than 98% of target sequences were efficiently edited with an average frequency of 82%. We validate the single-nucleotide resolution genome-editing capability of this technology by creating a genome-wide gene disruption collection and apply our method to improve tolerance to growth inhibitors.

Targeting Specificity of the CRISPR/Cas9 System.

CRISPR/Cas9 system has accelerated research across many fields since its demonstration for genome editing. CRISPR also offers vast therapeutic potential, but an important hurdle of this technology is the off-target mutations it can induce. In this viewpoint, we will discuss recent strategies for improving CRISPR specificity, emphasizing how a complete mechanistic understanding of CRISPR/Cas9 can benefit such efforts. We also propose that agreeing upon a consensus protocol with the highest specificity could benefit researchers working on CRISPR-based therapies. In addition to improving CRISPR/Cas9 specificity, accurate detection of off-target events is also crucial, and we will discuss various unbiased off-target detection methods in terms of their advantages and disadvantages. We suggest that using a combination of cell-based and cell-free methods can prove more useful. In addition, we point out that improving predictive algorithms for off-target sites would require pooling of the available off-target analysis data and standardization of the protocols used for obtaining the data. Moreover, we highlight the risk of insertional mutagenesis for gene correction applications requiring the use of donor DNA. We conclude by discussing future prospects for the field, as well as steps that can be taken to overcome the aforementioned challenges.

Fully Automated One-Step Synthesis of Single-Transcript TALEN Pairs Using a Biological Foundry.

Transcription activator-like effector nuclease (TALEN) is a programmable genome editing tool with wide applications. Since TALENs perform cleavage of DNA as heterodimers, a pair of TALENs must be synthesized for each target genome locus. Conventionally, TALEN pairs are either expressed on separate vectors or synthesized separately and then subcloned to the same vector. Neither approach allows high-throughput construction of TALEN libraries for large-scale applications. Here we present a single-step assembly scheme to synthesize and express a pair of TALENs in a single-transcript format with the help of a P2A self-cleavage sequence. Furthermore, we developed a fully automated platform to custom manufacture TALENs in a versatile biological foundry. 400 pairs of TALENs can be synthesized with over 96.2% success rate at a material cost of $2.1/pair. This platform opens the door to TALEN-based genome-wide studies.

Use of genome-editing tools to treat sickle cell disease.

Recent advances in genome-editing techniques have made it possible to modify any desired DNA sequence by employing programmable nucleases. These next-generation genome-modifying tools are the ideal candidates for therapeutic applications, especially for the treatment of genetic disorders like sickle cell disease (SCD). SCD is an inheritable monogenic disorder which is caused by a point mutation in the ß-globin gene. Substantial success has been achieved in the development of supportive therapeutic strategies for SCD, but unfortunately there is still a lack of long-term universal cure. The only existing curative treatment is based on allogeneic stem cell transplantation from healthy donors; however, this treatment is applicable to a limited number of patients only. Hence, a universally applicable therapy is highly desirable. In this review, we will discuss the three programmable nucleases that are commonly used for genome-editing purposes: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). We will continue by exemplifying uses of these methods to correct the sickle cell mutation. Additionally, we will present induction of fetal globin expression as an alternative approach to cure sickle cell disease. We will conclude by comparing the three methods and explaining the concerns about their use in therapy.

Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-α.

BACKGROUND: One-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-α (TNF-α). METHODS: Mice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-α, were tested for their effects on breast cancer cell proliferation. RESULTS: We produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-α, antibodies acted synergistically on SK-BR-3 cells, but antagonistically on BT-474 cells. A representative anti-HER2 antibody inhibited Akt and ERK1/2 phosphorylation leading to cyclin D1 accumulation and growth arrest in SK-BR-3 cells, independently from TNF-α. CONCLUSIONS: Novel antibodies against extracellular domain of HER2 may serve as potent anti-cancer bioactive molecules. Cell-dependent synergy and antagonism between anti-HER2 antibodies and TNF-α provide evidence for a complex interplay between HER2 and TNF-α signaling pathways. Such complexity may drastically affect the outcome of HER2-directed therapeutic interventions.