CRISPR Components Research Articles

Abstract PR01: Modeling colorectal cancer in vivo through CRISPR-based genome editing

Abstract In addition to activating and inactivating mutations in protein coding genes, cancer cells commonly show gross structural abnormalities in their genomes. These include deletions, duplications, inversions, and translocations. Unfortunately, conventional gene targeting- and RNAi-based approaches are not easily applied to study chromosome rearrangements. To enable this, we developed flexible CRISPR-based in vivo models systems that drive the generation of targeted genomic breaks, and the induction of gross chromosomal aberrations, without the need for viral delivery of CRISPR components. Using this approach we have produced novel transgenic systems to recapitulate two recurrent genomic rearrangements observed in human colorectal cancer and provide the first evidence that these events are sufficient to initiate tumor development. Further, we show that these tumors, while histologically similar to Apc mutant CRC, are molecularly distinct and are exquisitely sensitive to therapies targeting the Wnt pathway. We are now using this and other genome-editing platforms to systematically investigate the role of other recurrent genetic alterations in GI malignancies. Citation Format: Teng Han, Emma M. Schatoff, Charles Murphy, Maria Paz Zafra, John Wilkinson, Olivier Elemento, Lukas E. Dow. Modeling colorectal cancer in vivo through CRISPR-based genome editing [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr PR01.

Nanoparticles for CRISPR-Cas9 delivery.

The DNA mutation that causes Duchenne muscular dystrophy in mice can be corrected, with minimal off-target effects, by gold nanoparticles carrying the CRISPR components.

The Promise and Challenge of In Vivo Delivery for Genome Therapeutics.

CRISPR-based genome editing technologies are poised to enable countless new therapies to prevent, treat, or cure diseases with a genetic basis. However, the safe and effective delivery of genome editing enzymes represents a substantial challenge that must be tackled to enable the next generation of genetic therapies. In this Review, we summarize recent progress in developing enzymatic tools to combat genetic disease and examine current efforts to deliver these enzymes to the cells in need of correction. Viral vectors already in use for traditional gene therapy are being applied to enable in vivo CRISPR-based therapeutics, as are emerging technologies such as nanoparticle-based delivery of CRISPR components and direct delivery of preassembled RNA-protein complexes. Success in these areas will allow CRISPR-based genome editing therapeutics to reach their full potential.

CASAAV: A CRISPR-Based Platform for Rapid Dissection of Gene Function In Vivo.

In vivo loss-of-function studies are currently limited by the need for appropriate conditional knockout alleles. CRISPR/Cas9 is a powerful tool commonly used to induce loss-of-function mutations in vitro. However, CRISPR components have been difficult to deploy in vivo. To address this problem, we developed the CASAAV (CRISPR/Cas9/AAV-based somatic mutagenesis) platform, in which recombinant adeno-associated virus (AAV) is used to deliver tandem guide RNAs and Cre recombinase to Cre-dependent Cas9-P2A-GFP mice. Because Cre is under the control of a tissue-specific promoter, this system allows temporally controlled, cell type-selective knockout of virtually any gene to be obtained within a month using only one mouse line. Here, we focus on gene disruption in cardiomyocytes, but the system could easily be adapted to inactivate genes in other cell types transduced by AAV. © 2017 by John Wiley & Sons, Inc.

CRISPR edits human embryos

For the first time in the U.S., researchers have used the CRISPR/Cas9 gene-editing system in human embryos to correct a harmful hereditary gene mutation. Led by Shoukhrat Mitalipov of Oregon Health & Science University, the team injected eggs from healthy donors with sperm from a donor with hypertrophic cardiomyopathy, a common condition characterized by irregular heartbeat and heart failure. People with the disease carry a mutation in one of two copies of their MYBPC3 gene. The researchers simultaneously injected CRISPR/Cas9 components with the sperm to repair the mutation and produce embryos with two healthy copies of the gene. CRISPR/Cas9 uses guide-RNA molecules to target and cut specific segments of DNA. Natural DNA-repair mechanisms in the cell follow up by filling in the missing pieces. Mitalipov and colleagues found that in embryos coinjected with sperm and CRISPR components, a highly accurate DNA repair mechanism used the eggs’ healthy copy of the

Development of a CRISPR-Cas9 System for Efficient Genome Editing of Candida lusitaniae.

Candida lusitaniae is a member of the Candida clade that includes a diverse group of fungal species relevant to both human health and biotechnology. This species exhibits a full sexual cycle to undergo interconversion between haploid and diploid forms. C.lusitaniae is also an emerging opportunistic pathogen that can cause serious bloodstream infections in the clinic and yet has often proven to be refractory to facile genetic manipulations. In this work, we develop a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (Cas9) system to enable genome editing of C.lusitaniae. We demonstrate that expression of CRISPR-Cas9 components under species-specific promoters is necessary for efficient gene targeting and can be successfully applied to multiple genes in both haploid and diploid isolates. Gene deletion efficiencies with CRISPR-Cas9 were further enhanced in C.lusitaniae strains lacking the established nonhomologous end joining (NHEJ) factors Ku70 and DNA ligase 4. These results indicate that NHEJ plays an important role in directing the repair of DNA double-strand breaks (DSBs) in C.lusitaniae and that removal of this pathway increases integration of gene deletion templates by homologous recombination. The described approaches significantly enhance the ability to perform genetic studies in, and promote understanding of, this emerging human pathogen and model sexual species. IMPORTANCE The ability to perform efficient genome editing is a key development for detailed mechanistic studies of a species. Candida lusitaniae is an important member of the Candida clade and is relevant both as an emerging human pathogen and as a model for understanding mechanisms of sexual reproduction. We highlight the development of a CRISPR-Cas9 system for efficient genome manipulation in C.lusitaniae and demonstrate the importance of species-specific promoters for expression of CRISPR components. We also demonstrate that the NHEJ pathway contributes to non-template-mediated repair of DNA DSBs and that removal of this pathway enhances efficiencies of gene targeting by CRISPR-Cas9. These results therefore establish important genetic tools for further exploration of C.lusitaniae biology.

Centromeric DNA Facilitates Nonconventional Yeast Genetic Engineering.

Many nonconventional yeast species have highly desirable features that are not possessed by model yeasts, despite that significant technology hurdles to effectively manipulate them lay in front. Scheffersomyces stipitis is one of the most important exemplary nonconventional yeasts in biorenewables industry, which has a high native xylose utilization capacity. Recent study suggested its much better potential than Saccharomyces cerevisiae as a well-suited microbial biomanufacturing platform for producing high-value compounds derived from shikimate pathway, many of which are associated with potent nutraceutical or pharmaceutical properties. However, the broad application of S.stipitis is hampered by the lack of stable episomal expression platforms and precise genome-editing tools. Here we report the success in pinpointing the centromeric DNA as the partitioning element to guarantee stable extra-chromosomal DNA segregation. The identified centromeric sequence not only stabilized episomal plasmid, enabled homogeneous gene expression, increased the titer of a commercially relevant compound by 3-fold, and also dramatically increased gene knockout efficiency from <1% to more than 80% with the expression of CRISPR components on the new stable plasmid. This study elucidated that establishment of a stable minichromosome-like expression platform is key to achieving functional modifications of nonconventional yeast species in order to expand the current collection of microbial factories.

Conserved DNA motifs in the type II-A CRISPR leader region.

The Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas) systems consist of RNA-protein complexes that provide bacteria and archaea with sequence-specific immunity against bacteriophages, plasmids, and other mobile genetic elements. Bacteria and archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA (spacer) site-specifically into the leader-repeat junction in a process called adaptation. Previous studies have shown that parts of the leader region, especially the 3′ end of the leader, are indispensable for adaptation. However, a comprehensive analysis of leader ends remains absent. Here, we have analyzed the leader, repeat, and Cas proteins from 167 type II-A CRISPR loci. Our results indicate two distinct conserved DNA motifs at the 3′ leader end: ATTTGAG (noted previously in the CRISPR1 locus of Streptococcus thermophilus DGCC7710) and a newly defined CTRCGAG, associated with the CRISPR3 locus of S. thermophilus DGCC7710. A third group with a very short CG DNA conservation at the 3′ leader end is observed mostly in lactobacilli. Analysis of the repeats and Cas proteins revealed clustering of these CRISPR components that mirrors the leader motif clustering, in agreement with the coevolution of CRISPR-Cas components. Based on our analysis of the type II-A CRISPR loci, we implicate leader end sequences that could confer site-specificity for the adaptation-machinery in the different subsets of type II-A CRISPR loci.

Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes.

Genome editing using the CRISPR/Cas9 system requires the presence of guide RNAs bound to the Cas9 endonuclease as a ribonucleoprotein (RNP) complex in cells, which cleaves the host cell genome at sites specified by the guide RNAs. New genetic material may be introduced during repair of the double-stranded break via homology dependent repair (HDR) if suitable DNA templates are delivered with the CRISPR components. Early methods used plasmid or viral vectors to make these components in the host cell, however newer approaches using recombinant Cas9 protein with synthetic guide RNAs introduced directly as an RNP complex into cells shows faster onset of action with fewer off-target effects. This approach also enables use of chemically modified synthetic guide RNAs that have improved nuclease stability and reduces the risk of triggering an innate immune response in the host cell. This article provides detailed methods for genome editing using the RNP approach with synthetic guide RNAs using lipofection or electroporation in mammalian cells or using microinjection in murine zygotes, with or without addition of a single-stranded HDR template DNA.

Organoid technologies meet genome engineering.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming.

The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular, this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells), hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here, we described a new platform which enables, rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular, we devised a protocol that, combining the expression of the CRISPR components with neurogenic factors, generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy, fast and that does not require the generation of stable isogenic clones, practice that is time consuming and for some genes not feasible.

CRISPRs for Optimal Targeting: Delivery of CRISPR Components as DNA, RNA, and Protein into Cultured Cells and Single-Cell Embryos.

The rapid development of CRISPR technology greatly impacts the field of genetic engineering. The simplicity in design and generation of highly efficient CRISPR reagents allows more and more researchers to take on genome editing in different model systems in their own labs, even for those who found it daunting before. An active CRISPR complex contains a protein component (Cas9) and an RNA component (small guide RNA [sgRNA]), which can be delivered into cells in various formats. Cas9 can be introduced as a DNA expression plasmid, in vitro transcripts, or as a recombinant protein bound to the RNA portion in a ribonucleoprotein particle (RNP), whereas the sgRNA can be delivered either expressed as a DNA plasmid or as an in vitro transcript. Here we compared the different delivery methods in cultured cell lines as well as mouse and rat single-cell embryos and view the RNPs as the most convenient and efficient to use. We also report the detection of limited off-targeting in cells and embryos and discuss approaches to lower that chance. We hope that researchers new to CRISPR find our results helpful to their adaptation of the technology for optimal gene editing.

An Introduction to CRISPR Technology for Genome Activation and Repression in Mammalian Cells.

CRISPR interference/activation (CRISPRi/a) technology provides a simple and efficient approach for targeted repression or activation of gene expression in the mammalian genome. It is highly flexible and programmable, using an RNA-guided nuclease-deficient Cas9 (dCas9) protein fused with transcriptional regulators for targeting specific genes to effect their regulation. Multiple studies have shown how this method is an effective way to achieve efficient and specific transcriptional repression or activation of single or multiple genes. Sustained transcriptional modulation can be obtained by stable expression of CRISPR components, which enables directed reprogramming of cell fate. Here, we introduce the basics of CRISPRi/a technology for genome repression or activation.

56. AAV Vector-Mediated CRISPR Attacks on Proviral HIV-1 DNA for Purging of Cellular Reservoirs

HIV, the human immunodeficiency virus, is highly successful as a viral pathogen due to its ability to persist in the infected host as an integrated proviral DNA within cellular reservoirs, particularly in resting CD4+ T cells and macrophages. Current antiretroviral combination therapy can control viral spread and viremia but cannot eradicate latent HIV genomes, raising an urgent need for alternative strategies for long-term remission or cure. We combined two powerful technologies, synthetic AAV vectors and CRISPR-mediated gene engineering, with the ultimate aim to eliminate integrated HIV from latently infected human cells. Therefore, we first screened a library of peptide display mutants based on 12 different AAV serotypes and identified novel capsids that mediate potent transduction of relevant HIV target cells, including primary human T-lymphocytes and macrophages. Our lead candidates – two peptide-modified variants of AAV9 or AAVrh.10 – were capable of transducing >65% of primary humanCD4+ T cells and were superior to all 12 wildtype AAV isolates. Concurrently, we created AAV vector genomes that express – alone or in combination – the two essential CRISPR components, i.e., the Cas9 protein and a g(uide)RNA directed against either the HIV-1 long terminal repeats (LTRs), or against viral structural genes. AAV/CRISPR vector efficiency and specificity were validated using a T7 endonuclease assay, showing successful Cas9-mediated induction of double-stranded DNA breaks at the specific HIV-1 target regions and subsequent error-prone repair in HeLaP4 cells with a stably integrated HIV-1 genome. To further potentiate the approach, we exploited a highly customizable self-complementary AAV vector backbone recently developed in our lab that permits multiplexing up to three gRNAs under control of the U6, H1 and 7SK promoters in a single vector particle. We engineered this template to co-express our best gRNAs against the two HIV-1 LTRs and the viral gag gene, and compared its potency with our different first-generation single gRNA vectors. In reporter cells carrying a GFP-encoding HIV-1 genome, simultaneous cleavage was indeed detected at three different sites (5’LTR, 3’LTR and gag), reaching an impressive 63.5% cutting efficiency (versus 36.6-50% for the single gRNA vectors) and demonstrating the power of these vectors to cleave HIV-1 proviral DNA. Finally, we will present results from our ongoing validation of proviral cleavage in human T-cell lines that are latently infected with HIV-1 as a physiologically relevant model of HIV-1 latency. The described experiments prove the possibility to target, cut and destroy integrated HIV-1 proviral DNA with AAV/CRISPR vectors in human cells, and therefore support the further development of our tools and approach into a novel clinical modality for purging of latent HIV-1 reservoirs.

Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila.

We and others recently demonstrated that the readily programmable CRISPR/Cas9 system can be used to edit the Drosophila genome. However, most applications to date have relied on aberrant DNA repair to stochastically generate frameshifting indels and adoption has been limited by a lack of tools for efficient identification of targeted events. Here we report optimized tools and techniques for expanded application of the CRISPR/Cas9 system in Drosophila through homology-directed repair (HDR) with double-stranded DNA (dsDNA) donor templates that facilitate complex genome engineering through the precise incorporation of large DNA sequences, including screenable markers. Using these donors, we demonstrate the replacement of a gene with exogenous sequences and the generation of a conditional allele. To optimize efficiency and specificity, we generated transgenic flies that express Cas9 in the germline and directly compared HDR and off-target cleavage rates of different approaches for delivering CRISPR components. We also investigated HDR efficiency in a mutant background previously demonstrated to bias DNA repair toward HDR. Finally, we developed a web-based tool that identifies CRISPR target sites and evaluates their potential for off-target cleavage using empirically rooted rules. Overall, we have found that injection of a dsDNA donor and guide RNA-encoding plasmids into vasa-Cas9 flies yields the highest efficiency HDR and that target sites can be selected to avoid off-target mutations. Efficient and specific CRISPR/Cas9-mediated HDR opens the door to a broad array of complex genome modifications and greatly expands the utility of CRISPR technology for Drosophila research.

Comparative network clustering of direct repeats (DRs) and cas genes confirms the possibility of the horizontal transfer of CRISPR locus among bacteria

CRISPRs are a diverse family of DNA repeat sequences that are widely distributed among archaea and bacteria. The CRISPR locus is usually composed of three key elements; direct repeats (DRs), spacer sequences and the cas genes. Although recent studies have suggested that spacers may be of extrachromosomal origin, the evolutionary origin of the other two elements of the CRISPR locus has remained unresolved. With the aim to elucidate the evolutionary origin and association of DRs and cas genes of the CRISPR locus, a comparative analysis of the evolutionary network clusters of DRs, cas1 and 16S rRNA genes sequences from 100 different bacteria was conducted. Significant matches of DR and cas1 gene clades imply that these CRISPR components are evolutionary closely linked and potentially evolving simultaneously as a whole locus. On the contrary, the prominent discordance between the CASS (DR and cas1) clades and the 16S rRNA clusters indicates that CRISPR locus has been transferred horizontally as a complete package. Sequence analysis also revealed that DR and cas1 genes are coevolving under analogous evolutionary pressure. This atypical evolutionary pattern also signifies the possibility of horizontal transfer event of CRISPR locus.